UNIVERSITY OF CALIFORNIA MEDICAL CENTER LIBRARY SAN FRANCISCO PHYSIOLOGY AND BIOCHEMISTRY IN MODERN MEDICINE BY J. J. E. MACLEOD, M.B. PROFESSOR OF PHYSIOLOGY IN THE UNIVERSITY OF TORONTO, TORONTO, CANADA; FORMERLY PROFESSOR OF PHYSIOLOGY IN THE WESTERN RESERVE UNIVERSITY, CLEVELAND, OHIO ASSISTED BY ROY G. PEARCE, A. C. REDFIELD, AND N. B. TAYLOR AND BY OTHERS FOURTH EDITION WITH 243 ILLUSTRATIONS, INCLUDING 9 PLATES IN COLORS ST. LOUIS C. V. MOSBY COMPANY 1922 COPYRIGHT, 1918, 1919, 1920, 1922, BY C. V. MOSBY COMPANY (All Rights Strictly Reserved) (Printed in U. S. A.) Press C. V. Mosby ( St. Louis, i TO .M. W. M. PREFACE TO FOURTH EDITION The opportunity has been taken in this edition to revise each chapter so as to incorporate as much as possible of what has been added to physi- ological knowledge during the past two years. Certain chapters have been rewritten, such as those on the output of the heart, the conditions causing alterations in the acid base equilibrium of the blood, the normal electrocardiogram and the movements and emptying of the stomach. Considerable alterations have also been made in portions of other chap- ters so as to permit of the incorporation of the recent work on intra- cardiac pressure, the, capillary circulation, the mechanism of adaptation to mountain sickness, pancreatic diabetes, etc. Several of the figures appearing in earlier editions have been replaced by others of greater usefulness. These alterations have been made without changing the paging of the book. The author has again to thank Dr. N. B. Taylor for valuable assist- ance in rewriting two of the chapters and in reading the proof. J. J. R. MACLEOD. Toronto, Canada, 1922. PREFACE TO THIRD EDITION Many changes have been made in the present (third) edition of the book. The section on the nervous system has been entirely recast and re- written by my colleague, A. C. Redfield, who, besides bringing this part of the subject up to date, has incorporated with it an account of the fundamental principles of neuromuscular physiology. Although no ap- plication of this subject may at present be apparent in the investigation of disease it is certain that such exists; but it can be made only after the clinical researcher has become familiar with the brilliant work which has been done in the field in recent years by Keith Lucas, Adrian, and others. It is the function of a volume of this nature to describe not merely what already has been achieved in the clinical applications of physiology, but also to anticipate where this application is likely soon to be made and to prepare the way by describing the physiological prin- ciples that may be involved. Another section in which complete changes have been called for, is that relating to the chemistry of respiration. This has been rewritten VI PREFACE and rearranged so as to incorporate the recent work on the effects of deficiency oi; oxygen on the respiratory center, as well as the interesting and important clinical applications of the subject. Several new chapters have been added dealing with such practical problems as the measure- ment of the functional capacity of the heart, the principles of ventilation and the therapeutic value of oxygen, and the chapters on vitamines, on the capillary circulation, on surgical shock, and on the interpretation of polysphygmograms have been rewritten. In practically every other section of the book many additions have been made, particularly in that which deals with the endocrine organs, and several new figures and tables have been added. To make room for these changes some of the more technical details, appearing in the pre- vious editions, have been put in small print and some of the figures removed. This has been done in order to keep the volume as near to its original bulk as possible. I wish to take this opportunity to thank my colleagues here and else- where for many valuable suggestions and for their encouraging com- ments on the book. I am also greatly indebted to Dr. N. B. Taylor for his assistance in the preparation of this edition and for reading the proofs. J. J. E. MACLEOD. Toronto, Canada, 1920. PREFACE TO FIRST EDITION The necessity of allotting the various subjects of the medical curric- ulum to different periods, so that the more strictly scientific subjects are completed in the earlier years, has the great disadvantage that the student, being no longer in touch with laboratory work, fails to employ the scientific knowledge with full advantage in the solution of his clin- ical problems. He is apt to regard the first two or three years in the laboratory departments as inconsequential in comparison with the sup- posedly more practical instruction offered during the subsequent clinical years. He is taught by his laboratory instructors to observe accurately, and to correlate the observed facts, so that he may be enabled to draw conclusions as to the manner of working of the various functions of the animal body in health, and before proceeding to his clinical studies, he is required to show a proficiency in scientific knowledge, because it is recognized that this must serve as the basis upon which his knowledge of disease is to be built. When the clinic is reached, however, the meth- ods of the scientist are not infrequently cast aside and an understanding PREFACE Vll of disease is sought for largely by the empirical method ; namely, by the endeavor to see and examine innumerable patients, to diagnose the case according to the grouping of the signs and symptoms, and to treat it by the prescribed methods of experience. So much has to be learned and so much has to be seen during the clinical years, that the student gives little thought to the nature of the functional disturbance which is responsible for the symptoms; he fails to realize that after all, there is no essen- tial difference between the condition brought about in his patient by some pathological lesion, and that which may be produced in the labora- tory by experimental procedures, by drugs or by toxins. It must of course be recognized that just as the science of medicine originated by the grouping of symptoms into more or less characteristic diseases for which the most favorable method of treatment had to be discovered by experience, so must a certain part of the medical training be more or less empirical but it should at the same time be realized that such a method is only a means to an end, and that the real understanding of disease can be acquired only when every abnormal condition is inter- preted as a primary or secondary consequence of some perverted bodily function, and when the training in observation and the inductive method is carried from the laboratory into the clinic. It is a constant experience of clinical instructors who would employ scientific methods of instruction, that they find the students not only indifferent to an analysis of their cases from the functional standpoint, but also that they are too inadequately prepared in fundamental phys- iological knowledge, to make the analysis possible. The student may have a superficial acquaintance with the main facts of physiological science but have failed to acquire the enquiring habit of mind which will en- able him, through reflection, comparison, and personal research, to ap- ply the knowledge in practical medicine and surgery. For this lack of correlation between the laboratory and clinical stud- ies, the clinical instructors are not alone responsible. The laboratory courses are frequently given without any attempt being made to show the student the bearing of the subject in the interpretation of disease, or to train him so that in his later years he may be able to adapt the methods of investigation which he learned in the laboratory, to the study of morbid conditions. It is self-evident that (without any knowledge of disease) the extent to which the student in the earlier years of the course could be expected to appreciate the clinical significance of what he learns in the laboratory is limited, but this should not deter the in- structor from indicating whenever he can, the general application of scientific knowledge in the interpretation of diseased conditions. But Vlll PREFACE the chief remedy of the evil undoubtedly lies partly in the continuance of certain of the laboratory courses into the clinical years, and partly in the study of medical literature in which the application of physiology and biochemistry in the practice of medicine is emphasized. Notwithstanding the sufficient number of excellent textbooks in phys- iology available to the medical student, there is none in which partic- ular emphasis is laid upon the application of the subject in the routine practice of medicine. In the present volume the attempt is made to meet such a want, by reviewing those portions of physiology and bio- chemistry which experience has shown to be of especial value to the clinical investigator. The work is not intended to be a substitute, either for the regular textbooks in physiology, or for those in functional pathology. It is supplementary to such volumes. It does not start like the modern test in functional pathology, with a consideration of the diseased condition, and then proceed to analyze the possible causes and consequences of this disturbances of function which this exhibits; but it deals with the present-day knowledge of human physiology in so far as this can be used in a general way to advance the understanding of disease. In a sense it is therefore an advanced text in physiology for those about to enter upon their clinical instruction, and at the same time, a review for those of a maturer clinical experience who may- desire to seek the physiological interpretation of diseased conditions. In attempting to fulfil these requirements, it has been deemed essen- tial to go back to the fundamentals of the subject, and to explain as simply as possible the physical and physicochemical principles upon which so large a part of physiological knowledge depends. Physiology may be considered as an application of the known laws and facts of physics and chemistry to explain the functions of living matter, and it is only after the extent to which this application can be made has been appreciated, that the knowledge may be used to serve as the foundation upon which a superstructure of clinical knowledge can be built. In order that the volume might be maintained of reasonable size, it has been necessary to select certain parts of the subject for particular emphasis, the basis of selection being the degree to which our knowledge clearly shows the value of the application of physiological methods both of observation and of thought in the study of diseased conditions. This has not been done to the extent of omitting the apparently less essential parts, for these have been treated in sufficient detail to link the others together so as to preserve a logical continuity, and show the bearing of one field of knowledge on another. There are however certain parts of the science, particularly the physiology of nerve and muscle, of the special senses, and of reproduction, for which application in the general fields of medicine and surgery is limited, and these parts have been PREFACE IX omitted entirely. It has been judged that this perhaps somewhat arbi- trary selection is justified on the ground that the ordinary text in physiology covers these subjects sufficiently, except for the specialist, for whom on the other hand, no adequate review would have been pos- sible within the limits of such a volume as this. With reference to bio- chemistry, no attempt is made to review the properties or describe the characteristic tests of the various chemical ingredients of the body tis- sues and fluids. This is already sufficiently done in the textbooks on biochemistry, and in the numerous manuals on clinical methods. Bio- chemical knowledge is treated rather from the physiologist's stand- point, as an integral part of his subject, particular attention, neverthe- less, being paid to the far-reaching applications of this latest depart- ment of medical science, in the elucidation of many obscure problems of clinical medicine, such as those of diabetes, nephritis, acidosis, goiter and myxedema. To make the volume of value to those who may not have had time or opportunity to familiarize themselves with the techni- cal methods of the physiologist and biochemist as used in the modern clinic, a certain amount of space is devoted to a brief description of the methods that appear at present to be receiving most attention, and to be of greatest value. Finally, it should be mentioned that the principles of serum diagnosis and therapy are omitted, since these belong to a highly specialized science requiring an intensive training of its own. In the hope that the volume may be instrumental in arousing sufficient interest to stimulate a more intensive study of the various subjects which it introduces, a brief bibliography is given at the end of each section. The references selected are to papers that are more partic- ularly known to the author; they are not necessarily the most impor- tant publications on the subject, but are often chosen because of the useful reviews of previous work contained in them, rather than because of their own originality. Some of the papers, however, are referred to as authority for statements of fact which may arouse in the reader a desire to ponder for himself the evidence upon which these are based. The references are usually divided into two groups, "monographs" and "original papers," and it is only occasionally that specific reference is made to the former in the context. The original papers, on the other hand, are referred to by numbers. "With the general field of the subject so well covered by such excellent textbooks as Bayliss' "Principles of General Physiology," Stewart's, Howell's, Starling's, and Halliburton 's "Human Physiologies," and Leonard Hill's "Recent and Further Ad- vances in Physiology," the author has felt free to pick and choose from the monographs and original papers, topics that are ordinarily passed over cursorily in the textbook, and when this has been done, the refer- X PREFACE ences are somewhat more extensive. Such is the case for example in the chapters relating to the chemistry of respiration, to the metabolism of carbohydrates and fats, to the problems of dietetics and growth, to the physicochemical basis of neutrality regulation in the animal body, and to the action of enzymes. Acknowledgment is gratefully made for the assistance and advice in the preparation of the book, particularly to Doctor R. G. Pearce, for the contribution of several chapters, to which his name is attached, and for which he is entirely responsible; and to Doctor E. P. Carter, whose criticisms, after patient perusal of the unfinished manuscript, were of inestimable value in its final revision. Acknowledgment is also made to Doctor R. W. Scott and Professor F. E. Lloyd, for valuable criticism and advice, and to the former for a chapter on the " Clinical Applica- tion of Electrocardiographs. " To Miss Achsa Parker, M.A., the author owes a great debt of gratitude for the thorough and painstaking way in which she prepared the manuscript for the press, and for her never- tiring endeavors to have the spelling and punctuation in conformity with Webster's Dictionary. For assistance in the preparation of the index thanks are due to Miss Marion Armour and Mrs. MacFarlane, and for permission to use certain of the figures and illustrations, to the various authors and publishers who granted it. For the excellent man- agement and careful execution of the presswork, the author wishes to thank the publishers, whose courteous and friendly dealings have always made the work easier. J. J. R. MACLEOD. University of Toronto, Toronto, Canada. CONTENTS PART I THE PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES CHAPTER I PAGE GENERAL CONSIDERATIONS 1 The Laws of Solution, 3; Gas Laws, 3; Osmotic Pressure, 4; Biological Meth- ods for Measuring Osmotic Pressure, 6; Hemolysis, 7; Plasmolysis, 8. CHAPTEE II OSMOTIC PRESSURE (CONT'D.) 10 Measurement by Depression of Freezing Point, 10; The Eole of Osmosis, Dif- fusion, and Allied Processes in Physiological Mechanisms, 11, Physical Fac- tors Involved in Absorption, Excretion and Lymph Formation, 13. CHAPTEE III ELECTRICAL CONDUCTIVITY, DISSOCIATION, AND IONIZATION . . 16 Determination of Conductivity, 17; Biological Applications, 19. CHAPTEE IV THE PRINCIPLES INVOLVED IN THE DETERMINATION OF THE HYDROGEN-ION CONCEN- TRATION 22 Titrable Acidity and Alkalinity, 22 ; Actual Degree of Acidity or Alkalinity, 23; Mass Action, 23; Application to the Measurement of H-ion Concentration, 26; Application in Determining the Eeal Strength of Acids or Alkalies, 28. CHAPTEE V THE PRINCIPLES INVOLVED IN THE MEASUREMENT OF HYDROGEN-ION CONCENTRA- TION (CONT'D) 29 The Electrical Method, 29 ; The Indicator Method, 32. [ CHAPTEE VI KEGULATION OF NEUTRALITY IN THE ANIMAL BODY AND ACIDOSIS 36 Buffer Substances, 36; Theory of Acidosis, 38; Measurement of the Eeserve Alkalinity, 41; Titration Methods, 41; CO 2 -Combining Power, 42; Indirect Methods. 46; Eelationship Between CO 2 -content of Blood and Hydrogen-ion Concentration, 49. CHAPTEE VII COLLOIDS 51 Characteristic Properties, 51; Characteristics of True Colloidal Solutions, 52; Tyndall Phenomenon, 52; Eelative Indiffusibility, 52; Electrical Properties, 56; Brownian Movement, 58; Osmotic Pressure, 58. xii CONTENTS CHAPTER VIII PAGE COLLOIDS (CONT'D) 61 Suspensoids and Emulsoids, 61; Gelatiuization, 62; Imbibition, 63; Action of Electrolytes on Colloids, 63; Proteins as Colloids, 64; Surface Tension, 65; Adsorption, 66; Reactions which Depend on Adsorption, 67; Conditions that Influence or are Influenced by Adsorption, 68; Biological Processes Depend- ing on Adsorption, 70. CHAPTER IX FERMENTS, OB ENZYMES 71 The Nature of Enzyme Action, 72; Properties of Enzymes, 73; Reversibility of Enzyme Action, 77; Specificity of Enzyme Action, 79; Peculiarities of Enzymes, 80; Types of Enzyme, 81; Enzyme Preparations, 82; Conditions for Enzymic Activity, 82. PART II THE BLOOD AND THE LYMPH CHAPTER X BLOOD: ITS GENERAL PROPERTIES (BY R. G. PEARCE) 85 Quantity of Blood in the Body, 85; Water Content, 87; Proteins, 88; Fer- ments and Antiferments, 90. CHAPTER XI THE BLOOD CELLS (BY R. G. PEARCE) 92 Red Blood Corpuscles, or Erythrocytes, 92 ; Origin, 93 ; Rates of Regeneration, 94; Hemolysis, 96; Leucocytes, 97; Blood Platelets, 98. CHAPTER XII BLOOD CLOTTING 99 Visible Changes in the Blood During Clotting, 99; Methods of Retarding Clotting of Drawn Blood, 100; Nature of the Clotting Process, 102; Influence of Calcium Salts, 104; Influence of Tissues, 105. CHAPTER XIII BLOOD CLOTTING (CONT'D) 107 Theories of Blood Clotting, 107 ; Intravascular Clotting, 108 ; Measurement of the Clotting Time, 109; Blood Clotting in Various Physiological Conditions, 111; Blood Clotting in Disease, 111; Hemorrhagic Diseases, 113; Thrombus Formation, 113. CHAPTER XIV LYMPH FORMATION AND CIRCULATION CEREBROSPINAL FLUID 115 General Considerations, 115; Experimental Investigations, 118; Edema, 120; Cerebrcspinal Fluid, 121. CONTENTS XI 11 PART III CIRCULATION OF THE BLOOD CHAPTER XV PAGE BLOOD PRESSURE 124 The Mean Arterial Blood Pressure, 125; Mercury Manometer Tracings, 125; Spring Manometer Tracings, 128; Clinical Measurements, 129. CHAPTEE XVI THE FACTORS CONCERNED IN MAINTAINING THE BLOOD PRESSURE 135 Pumping Action of the Heart, 135; Peripheral Resistance, 135; Amount of Blood in the Body, 138; Effects of Hemorrhage and Transfusion, 140; Vis- cosity of the Blood, 141; Elasticity of Vessel Walls, 143. CHAPTER XVII THE ACTION OP THE HEART 145 The Pumping Action of the Heart, 145; Intracardiac Pressure Curves, 146; Comparison of the Curves, 148. CHAPTER XVIII THE PUMPING ACTION OF THE HEART (CONT'D) 151 Contour of the Intracardiac Curves, 151; Ventricular Curve, 151; Auricular Curve, 153; The Mechanism of Opening and Closing of the Valves, 154; The Heart Sounds, 156; Causes of Sounds. 157; Record of Heart Sounds (Elec- trophonograms), 158. CHAPTER XIX THE NUTRITION OF THE HEART 161 Blood Supply, 161; Perfusion of the Heart Outside the Body, 161; Heart- Lung Preparation, 163; Resuscitation of the Heart in Situ, 165; Relationship of the Chemical Composition of the perfusion Fluid in Cold-blooded and Warm-blooded Hearts, 166. CHAPTER XX PHYSIOLOGY OF THE HEARTBEAT 170 Origin and Propagation of the Beat, 170; Physiological Characteristics of Cardiac Muscle, 170; Myogenic Hypothesis, 171; Neurogenie Hypothesis, 172; The Pacemaker of the Heart and Heart-block, 174; Physiological Char- acteristics of Cardiac Muscle, 176. CHAPTER XXI PHYSIOLOGY OF THE HEARTBEAT (CONT'D) 182 Origin and Propagation of the Beat in the Mammalian Heart, 182; Conduct- ing Tissue in Mammalian Heart, 182; Site of Origin of Beat, 187. CHAPTER XXII PHYSIOLOGY OF THE HEARTBEAT (CONT'D) 191 Mode of Propagation in the Auricles and from the Auricles to the Ventricles, 191; Spread of Beat in the Ventricle, 193; Fibrillation of the Ventricles and Auricles, 195. XIV CONTENTS CHAPTER XXIII PAGE THE BLOODFLOW IN THE ARTERIES 198 The Pulses, 198; General Characteristics, 198; Eate of Transmission of Pulse Waves, 198; Contour of the Pulse Curves, 200; Velocity Pulse, 200; Palpable Pulse^202; Analysis of the Curve, 202; The Dicrotic Wave, 203; Causes of Disappearnce of the Pulse in the Veins, 205. CHAPTER XXIV RATE OF MOVEMENT OF THE B-LOOD IN THE BLOOD VESSELS 206 Velocity of Flow in a Vessel, 206; Mass Movement of the Blood in a Vas- cular Area, 208; The Visceral Bloodflow in Man, 212; Circulation, 212; Work of the Heart, 213; Circulation Time, 214; Movement of Blood in the Veins, 214. CHAPTER XXV THE OUTPUT OF THE HEART IN RELATION TO THE VENOUS INFLOW, CHANGE OF RATE, 'ETC 216 Output of the Heart per Beat, 216; Effect of Alteration in Rate of Heart Beat on Output of Blood, 218; Dilatation and Tonus, 219; Dynamics of Cir- culation, 219; Reserve Power of the Heart, 220; Testing Cardiac Efficiency, 220. CHAPTER XXVI THE CONTROL OF THE CIRCULATION 221 Nerve Control, 222; Vagus Control in the Cold-blooded and the Mammalian Heart, 222; Tonic Vagus Action, 226; Afferent Vagus Impulses, 227; Mech- anism of Action of Vagus on the Heart, 229; Termination of the Vagus Fi- bers in the Heart, 230 ; Sympathetic Control,, 232. CHAPTER XXVII THE CONTROL OF THE CIRCULATION (OONT'D) 234 Nerve Control of the Peripheral Resistance, 234; Detection of Constriction or Dilatation, 234; Detection of Vasomotor Fibers in Nerves, 236; Origin of Vasomotor Nerve Fibers, 237; Vasomotor Nerve Centers, 240; Independent Tonicity of Blood Vessels, 241. CHAPTER XXVIII THE CONTROL OF THE CIRCULATION (CONT'D) 242 Control of the Vasomotor Center, 242; Hormone Control, 242; Nerve Control, 243; Pressor and Depressor Impulses, 243; Reciprocal Innervation of Vas- cular Areas, 247; Influence of Gravity on the Circulation, 248; Capillary Circulation, 251. CHAPTER XXIX PECULIARITIES OF BLOOD SUPPLY IN CERTAIN VISCERA 254 Circulation in the Brain, 254; Anatomical Peculiarities, 254; Physical Condi- tions of the Intracranial Circulation, 256; Physiological Conditions of the In- tracranial Circulation, 258; Vasomotcr Nerves, 262; Intracranial Pressure, 263; Circulation through the Lungs, 264; Circulation Through the Liver, 265; The Coronary Circulation, 267. CHAPTER XXX CLINICAL APPLICATIONS OF CERTAIN PHYSIOLOGICAL METHODS 270 Electrocardiograms, 270; Interpretation of Electrocardiograms by the Triangle Method, 271; The Ventricular Complex, 274. CONTENTS XV CHAPTER XXXI PAGE CLINICAL APPLICATIONS OF CERTAIN PHYSIOLOGICAL METHODS (CONT'D) . . . 278 Electrocardiograms in the More Usual Forms of Cardiac Irregularities, 278; Sinus Arrhythmia, 278; Sinus Bradycardia, 278; The Extrasystoles, 278; Paroxysmal Tachycardia, 281; Auricular Fibrillation, 281; Auricular Flutter, 281; Heart-Block, 282; Ventricular Hypertrophies and Defects of the Divi- sions of the A-V Bundle, 284. CHAPTER XXXII CLINICAL APPLICATIONS OF CERTAIN PHYSIOLOGICAL METHODS (CONT'D) . . . 285 Polysphygmograms, 285; Venous Pulse Tracings, 285; Abnormal Pulses, 291. CHAPTER XXXIII CLINICAL APPLICATIONS OF CERTAIN PHYSIOLOGICAL METHODS (CONT'D) . . . 296 Measurement of the Mass Movement of the Blood, 296; The Normal Flow, 297; Clinical Conditions Which Affect the Bloodflow, 298. CHAPTER XXXIV SHOCK 301 Varieties of Shock, 301;' Experimental Investigations of Shock, 304; Hista- mine Shock, 307; Traumatic Toxemia Factor in Shock, 309; Cause of Second- ary Symptoms, 310; Treatment and Prognosis, 311. PART IV RESPIRATION CHAPTER XXXV RESPIRATION 316 Mechanics of Respiration, 316; Pressure of the Air in the Lungs, 316; Respir- atory Tracings, 320; The Intrapleural Pressure, 321; Influence on Blood Pressure, 323. CHAPTER XXXVI THE MECHANICS OF RESPIRATION (CONT'D) (BY R. G. PEARCE) 327 Variations in Dead Space, Residual Air and Mid and Vital Capacities in Various Physiological and Pathological Conditions, 327. CHAPTER XXXVII THE MECHA.NICS OF RESPIRATION (CONT'D) 332 Mechanism of the Changes in Capacity of the Thorax and Lungs, 332; The Movements of the Ribs, 332; The Action of the Musculature of the Ribs, 336; The Action of the Diaphragm, 337; The Effects of the Respiratory Movements on the Lungs, 342. CHAPTER XXXVIII THE CONTROL OF THE RESPIRATION 344 The Respiratory Centers, 344; Reflex Control of the Respiratory Center, 348. XVI CONTENTS CHAPTER XXXIX PAGE THE CONTROL OF KESPIRATION (CONT'D) 352 Hormone Control of the Eespiratory Center, 352; Tension of CO 2 and O 2 in Arterial Bloorl, 354; Tension of CO 2 and O 2 in Alveolar Air, 356; Tension of CO 2 and O 2 in Venous Blood, 359 ; Fallacies in Estimation of the Alveolar Gases, 361. CHAPTER XL THE CONTROL OF RESPIRATION (CONT'D) 363 Variations in the Alveolar CO 2 and the Acid Base Balance in Health and Disease, 363. CHAPTER XLI THE CONTROL OF RESPIRATION (CONT'D) 366 The Nature of the Respiratory Hormone, 366; Relationship between CO., of Inspired Air and Pulmonary Ventilation, 367 ; Possibility that CO 2 Specifically Stimulates the Center, 368; Relationship between Alveolar CO 2 and Respira- tory Activity, 371. CHAPTER XLII THE CONTROL OF RESPIRATION (CONT'D) 373 Alveolar CO, Tension in Conditions of Anoxemia, 373; Constancy of the Al- veolar CO 2 Tension under Normal Conditions, 373; The Nature of Changes Produced in the Body in Anoxemia, 378. CHAPTER XLIII THE CONTROL OF RESPIRATION (CONT'D) 382 Apnea, 382; Periodic Breathing, 385; Types of Periodic Breathing, 385; Causes of Periodic Breathing, 386. CHAPTER XLIV RESPIRATION BEYOND THE LUNGS 391 Transportation of Gases by tlje Blood, 392; Transportation of Oxygen, 392; Dissociation Curve of CO 2 , 396; Difference between Curves of Blood and Hemoglobin Solutions, 396; Rate of Dissociation, 399; Dissociation Constant, 401. CHAPTER XLV RESPIRATION BEYOND THE LUNGS (CONT'D) 403 Means by Which the Blood Carries the Gases, 403; Oxygen Requirement of the Tissues, 408; Mechanism by which the Demands of the Tissues for Oxy- gen are met, 412. CHAPTER XLVI THE PHYSIOLOGY OF BREATHING IN RAREFIED AND COMPRESSED AIR 415 Mountain Sickness, 415; Compressed Air Sickness (Caisson Disease), 420; Application of Foregoing Laws in Practice, 424. CHAPTER XLVII ADAPTATIONS OF THE CIRCULATORY AND RESPIRATORY SYSTEMS DURING MUSCULAR EXERCISE 427 Circulatory Changes Accompanying Muscular Exercise, 427; Mechanical Fac- tors, 428; Nervous Factor, 430; Hormone Factor, 431. CONTENTS XV11 CHAPTEE XLVIII PAGE ADAPTATIONS OF THE CIRCULATORY AND RESPIRATORY MECHANISMS DURING MUS- CULAR EXERCISE (CONT'D) 435 The Effect of Muscular Exercise on the Composition of the Alveolar Air, 435 ; Second-Wind, 438; Influence of Oxygen, 439; After Effects, 440; Effort Syn- drome, 441. CHAPTER XLIX OXYGEN UNSATURATION OF THE BLOOD CYANOSIS. THE THERAPEUTIC VALUE OF OXYGEN 443 Oxygen Unsaturation of the Blood, 443; Therapeutic Value of Oxygen, 445. PART V DIGESTION CHAPTER L GENERAL PHYSIOLOGY OF THE DIGESTIVE GLANDS 453 Microscopic Changes During Activity, 453; Mechanism of Secretion, 455; Other Changes During Activity, 456; Control of Glandular Activity, 457; Nervous Control, 458. CHAPTER LI PHYSIOLOGY OF THE DIGESTIVE GLANDS (CONT'D) 460 Hormone Control, 460; Nervous Control of Pancreas, 462. CHAPTER LII PHYSIOLOGY OF THE DIGESTIVE GLANDS (CONT'D) 465 Normal Conditions of Secretion, 465 ; Normal Secretion of Saliva, 466 ; Se- cretion of Gastric Juice, 467; The Intestinal Secretions, 476. CHAPTER LIII THE MECHANISMS OF DIGESTION 478 Mastication, 478; Deglutition or Swallowing, 479; The Cardiac Sphincter, 482; Vomiting, 483. CHAPTER LIV THE MECHANISMS OF DIGESTION (CONT'D) 485 Movements of the Stomach, 485; Character of the Movements, 485; Effect of the Stomach Movement on the Food, 488; Emptying of the Stomach, 491; Control of the Pyloric Sphincter, 491 ; Influence of Pathological Conditions on the Emptying, 494; Gastroenterostomy, 494. CHAPTER LV THE MECHANISMS OF DIGESTION (CONT'D) 497 Movements of the Intestines, 497; Movements of the Small Intestine, 497; Movements of the Large Intestine, 503; Effect of Clinical Conditions on the Movements, 504. XV111 CONTENTS CHAPTER LVI PAGE HUNGER, APPETITE AND THIRST 506 Hunger, 506; Bemote Effects of Hunger Contractions, 509; Hunger During Starvation, 510; Control of Hunger Mechanism, 5-11; Thirst, 514; Sensation of Thirst, 514. CHAPTER LVII BIOCHEMICAL PROCESSES OP DIGESTION 515 Digestion in the Stomach, 515; .Functions of Hydrochloric Acid, 516; Amount of Acid, 516; Source of Acid, 517; Action of Pepsin, 519; Clotting of Milk in the Stomach, 521. CHAPTER LVIII BIOCHEMICAL PROCESSES OF DIGESTION (CONT'D) 523 Digestion in the Intestines, 523; Pancreatic Digestion, 523; The Bile, 526; Chemistry of Bile, 528. CHAPTER LIX BACTERIAL DIGESTION IN THE INTESTINE 533 Bacterial Digestion of Protein, 535; Botulism, 537. PART VI THE EXCRETION OF URINE CHAPTER LX THE EXCRETION OF URINE (By R. G. P'EARCE) 541 Structure of Kidney, 541; Mechanism of the Excretion of Urine, 544; Theories of Renal Function, 545; Diuretics, 552; Albuminuria, 552; Influence of the Nervous System on the Excretion of Urine, 553. CHAPTER LXI THE AMOUNT AND COMPOSITION OF THE URINE IN HEALTH AND DISEASE (By R. G. PEARCE) 555 Amount, 556; Specific Gravity, 556; Depression of Freezing Point, 557; Reaction, 558; Solid Constituents, 560; Quantitative Changes in the Blood and Urine in Disease, 567. PART VII METABOLISM CHAPTER LXII METABOLISM 570 Energy Balance, 571; Methods for Measuring Energy, 572; Normal Values, 573; Standard for Comparison, 575; Influence of Age and Sex, 577; Influence of Diseases, 578; Material Balance of the Body, 579; Methods for Measur- ing Outputs, 579. CONTENTS XIX CHAPTER LXIII PAGE THE CARBON BALANCE 582 Respiratory Quotient, 582; Influence of Diet, 582; Influence of Metabolism, 584; Magnitude of the Respiratory Exchange, 585; Influence of Body Tem- perature, 586. CHAPTER LXIV A CLINICAL METHOD FOR DETERMINING THE RESPIRATORY EXCHANGE IN MAN (BY R. G. PEARCE) 589 The Mouthpiece, 589; The Valves, 590; Tissot Spirometers, 591; Douglas Bag, 592; Haldane Gas Apparatus, 593; Calculations, 596. CHAPTER LXV STARVATION 600 Excretion of Nitrogen, 600; Energy Output, 602; Nitrogenous Metabolites, 602; Excretion of P'urines, 603; Excretion of Sulphur, 603; Normal Metab- olism, 604; Nitrogenous Equilibrium, 605; Protein Sparers, 605. CHAPTER LXVI NUTRITION AND GROWTH 608 The Food Factor of Growth, 608; Relationship of Proteins to Growth and Maintenance of Life, 609. CHAPTER LXVII NUTRITION AND GROWTH (CONT'D) 617 Relationship of Carbohydrates and Fats to Growth, 617; Accessory Food Fac- tors, or Vitamines, 618. CHAPTER LXVIII DIETETICS 625 Calorie Requirement, 625; The Protein Requirement, 627; Accessory Food Factors, 630; Digestibility and Palatability, 630. CHAPTER LXIX THE METABOLISM OF PROTEIN 632 Introductory, 632; Chemistry of Protein and of the Amino Acids, 633. CHAPTER LXX THE METABOLISM OF PROTEIN (CONT'D) 641 Amino Acids in the Blood and Tissues, 641 ; Fate of the Amino Acids, 645. CHAPTER LXXI THE METABOLISM OF PROTEIN (CONT'D) 647 End Products of Protein Metabolism, 647 ; Urea and Ammonia, 649 ; Urea Ratio, 650 ; Influence of Liver on Ammonia-Urea Ratio, 651 ; Perfusion of Or- gans, 652; Clinical Observations, 654. CHAPTER LXXII THE METABOLISM OF PROTEIN (CONT'D) 656 Creatine and Creatinine, 656; Essential Chemical Facts, 656; Metabolism, 657 ; Influence of Food, Age, and Sex, 657 ; Origin of Creatine and Creatinine, 659. XX CONTENTS CHAPTER LXXIII PAGE THE METABOLISM OF PROTEIN (CONT'D) 662 Undetermined Nitrogen and Detoxication Compounds, 662; Ethereal Sul- phates and Glycuronates, 665. CHAPTER LXXIV URIC ACID AND THE PURINE BODIES 667 Chemical Nature of the Purines, 667; Chemical Nature of the Substances Con- taining Purine and Pyrimidine Bases, 669; History of Nucleic Acid in the Animal Body, 671 ; Balance Between Intake and Output of Purine Substances under Various Physiological and Pathological Conditions, 674. CHAPTER LXXV URIC ACID AND THE PURINE BODIES (CONT'D) 676 Source of Endogenous Purines, 676; Influence of Various Physiological Con- ditions, of Drugs, and of Disease on the Endogenous Uric-acid Excretion, 680; Uric Acid of Blood, 681. CHAPTER LXXVI THE METABOLISM OF THE CARBOHYDRATES 685 Capacity of the Body to Assimilate Carbohydrates, 685; Assimilation Limits, 685; Tolerance of the Body for Glucose, 688; Digestion and Absorption, 689; Sugar Level in the Blood, 690; Value of Blood Examination in Diagnosis of Diabetes, 691; Relationship Between Sugar Concentration of the Blood and the Occurrence of Glycosuria, 692. CHAPTER LXXVII THE METABOLISM OF THE CARBOHYDRATES (CONT'D) 694 Fate of Absorbed Glucose. Gluconeogenesis, 694; Storage of Sugar, 694; Sources of Glycogen, 694; Gluconeogenesis in Normal Animals, 699. CHAPTER LXXVIII THE METABOLISM OF THE CARBOHYDRATES (CONT'D) 701 Fate of Glycogen, 701; Regulation of the Blood Sugar Level, 703; Nerve Con- trol and Nervous Experimental Diabetes, 704; Nervous Diabetes in Man, 706; Hormone Control and Permanent Diabetes, 707; Utilization of Glucose in Tis- sues, 708; Diabetes and the Ductless Glands, 710; Relationship of Pancreas to Sugar Metabolism, 710; Pathogenesis of Pancreatic Diabetes, 712; Dia- betic Acidosis or Ketosis, 715; Starvation Treatment, 716. CHAPTER LXXIX FAT METABOLISM 718 Chemistry of Fatty Substances, 718; Digestion of Fats, 721; Absorption of Fats, 722. CHAPTER LXXX FAT METABOLISM (CONT'D) 726 Fat of Blood, 726; Methods of Determination, 726; Variations in Blood Fat, 727; Depot Fat, 730; Fat in the Liver, 731. CONTENTS XXI CHAPTER LXXXI PAGE FAT METABOLISM (CONT'D) 736 Production of Fatty Acid out of Carbohydrate, 736; Method by which the Fatty Acid is Broken Down, 737. CHAPTER LXXXII CONTROL OF BODY TEMPERATURE AND FEVER 742 Variations in Body Temperature, 742 ; Factors in Maintaining the Body Tem- perature, 743; Control of Temperature, 747; Fever, 748; Mechanism of Fever, 748; Changes in the Body During Fever, 750; Essential Cause of Fever, 752 ; Heat-regulating Center, 752 ; Significance of Fever in the Organism, 753. CHAPTER LXXXIII THE PHYSIOLOGICAL PRINCIPLES OF VENTILATION 754 Relationship Between Chemical Composition of the Air and the Well-being of the Body, 754; Relationship Between the Physical Conditions of the Air and the Well-being of the Body, 757 ; Relationship between the Conditions of Ventilation and Susceptibility to Infections, 759; Methods for Determining the Healthfulness of Air, 763. PART VIII THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS CHAPTER LXXXIV GENERAL CONSIDERATIONS, THE ADRENAL GLANDS 766 Methods of Investigation, 767; Adrenal Gland, 768; Cortex, 768; Medulla, 770; Adrenalectomy, 771; Adrenal Disease in Man, 772; Suprarenal Extracts, 773; Physiological Action, 774. CHAPTER LXXXV THE ADRENAL GLANDS, (CONT'D) 779 Variations in Physiological Activity, 779; Assaying the Epinephrine Content of the Gland, 779; Epinephrine Content of the Blood, 780; Autoinjection Method, 784; Association of the Adrenal with Other Endocrine Organs, 788. CHAPTER LXXXVI THE THYROID AND PARATHYROID GLANDS 791 Structural Relationships, 791; Thyroid Gland, 792; Condition of Gland, 792; Experimental Thyroidectomy, 794; Disease of the Thyroid, 795; Relationship with Other Endocrine Organs, 800; Parathyroids, 800; Experimental Para- thyroidectomy, 800; Injury or Disease of the Parathyroids in Man, 801. CHAPTER LXXXVII THE PITUITARY BODY 806 Structural Relationships, 806; Functions, 807; Clinical Manifestations of Deranged Pituitary Function, 816; Relationship with Other Endocrine Or- gans, 818. XX11 CONTENTS CHAPTER LXXXVIII PAGE THE PINEAL GLAND, THE GONADS, AND THE THYMUS 820 Pineal Gland, 820; Gonads or the Generative Organs, 821; Generative Glands of the Male, 821; Generative Organs of the Female, 822; Thymus, 824. PART IX THE CENTRAL NERVOUS SYSTEM AND THE CONTROL OF MUSCULAR ACTIVITY (Rewritten by A. C. Redfield) CHAPTER LXXXIX THE EVOLUTION OF THE NEUROMUSCULAR MECHANISM 827 Primitive Neuromuscular Mechanisms, 827; The Nerve Net, 830; The Cen- tral Nervous System, 830. CHAPTER XC THE CONDUCTION OF THE NERVOUS IMPULSE 836 Conduction in the Nerve Fiber, 837; The All or None Law, 837; Refractory Period, 839; Conduction between Neurons, 841; Resistance Due to Synapse, 842; Summation, 842; Inhibition, 843; Canalization, 844; Myoneural Junc- tion, 845. CHAPTER XCI THE NUTRITION OF NERVOUS TISSUE 846 Function of the Nerve Cell Body, 846; Degeneration and Regeneration of Nerve Fibers, 846; Metabolism of the Nerve Fiber, 850; Metabolism of the Central Nervous System, 851. CHAPTER XCII THE RECEPTORS 854 The Evolution of Specialized Receptors, 854; Quality of Sensation and Its Local Sign, 855; Referred Pain, 858; Cutaneous and Deep Sensibility, 859; Touch, 860; Heat and Cold, 861; Pain, 862; Distribution of Sensitivity in the Body, 863. CHAPTER XCIII THE AFFERENT PATHS OF SENSORY IMPULSES 866 Segmental Distribution of Afferent Nerves, 866; Ascending Pathways in the Spinal Cord, 868; Afferent Paths in the Brain Stem, 871; Afferent Impulses Which Fail to Produce Sensation, 872. CHAPTER XCIV THE SENSORY CENTERS OF THE BRAIN 876 The Sensory Center of the Optic Thalamus, 876; The Sensory Centers of the Cerebral Cortex, 878; The Visual Areas, 880; Sensory Hallucinations, 883. CONTENTS xxiii CHAPTEE XCV PAGE THE MOTOR AREAS OF THE CEREBRUM AND THE EFFERENT PATHWAY TO SKELETAL MUSCLE 884 The Motor Area of the Cerebral Cortex, 885; The Visuo-Motor Areas, 886; The Efferent Pathway in the Brain and Cord, 888; Distribution of Efferent Nerves, 890; Spinal Reflexes, 891. CHAPTER XCVI THE AUTONOMIC NERVOUS SYSTEM, OR THE EFFERENT P'ATHWAY TO SMOOTH MUS- CLES AND GLANDS 893 The Organization of Efferent Nerves to the Viscera, 893; The Double Inner- vation of the Visceral Organs, 896; The Function of the Autonomic Nervous System, 897; The Axon Reflex, 898; Function of the Bulbo-sacral Divisions, 899 ; The Mechanism for Emptying the Bladder, 900 ; Function of the Thorac- ico-Lumbar Division, 901 ; Effects of Impulses from the Viscera Upon Central Nervous Activity, 902. CHAPTER XCVII MUSCULAR CONTRACTION 904 The Tonic Contraction of Skeletal Muscle, 905; Tetanic Contraction of Skeletal Muscle, 906; The All or None Law, 908; Chemistry of Tetanic Con- traction, 910; Smooth Muscle, 912. CHAPTER XCVIII POSTURAL COORDINATION 914 Reflex Adjustment of Tone, 914; The Posture of the Body as a Whole, 917; Compensatory Movements of the Eyes, 918; Clinical Tests of Labyrinthine Mechanism, 920; The Tendon Jerks, 921. CHAPTER XCIX THE CENTRAL CONTROL OF POSTURAL REACTIONS; THE CEREBELLUM 924 The Influence of the Brain on the Local Tonic Reflex, 924; Function of the Cerebellum, 926; Localization of Function in the Cerebellum, 929; 'Compensa- tion for Cerebellar Injuries, 931. CHAPTER THE INTEGRATION OF ACTION WITHIN THE REFLEX ARC 933 The Receptors, 933; Summation, 934; Refractory Period, 934; Reciprocal Inhibition, 937; Action of Strychnine and Tetanus Toxin on Reciprocal In- hibition, 941; The Reflex Figure, 941; Rules for the Spread of Spinal Re- flexes, 944. CHAPTER CI THE INTEGRATION OF SIMULTANEOUS AND SUCCESSIVE REFLEXES 945 Principle of the Final Common Path, 945; Integration of Allied Reflexes, 946; Integration of Antagonistic Reflexes, 947. CHAPTER CII THE INTEGRATIVE ACTION OF THE CEREBRUM 951 Relation of the Cerebrum to the Distance Receptors, 952; Conditioned Re- flexes, 954. XXIV CONTENTS CHAPTER CIII PAGE THE HIGHER FUNCTIONS OF THE CEREBRUM IN MAN ; APHASIA 958 Psychopathological Applications, 960. CHAPTER CIV SUMMARY OF THE ORGANIZATION OF THE MAMMALIAN NERVOUS SYSTEM; SPINAL SHOCK 963 Spinal Shock and the Recovery of Reflexes in Animals, 965 ; Spinal Shock and the Recovery of Reflexes in Man, 967; The Cause of Spinal Shock, 969. ILLUSTRATIONS FIG. PAGE 1. Diagram of osmometer 5 2. Hematocrite 7 3. Plasmolysis in cells from Tradesoantia discolor 9 4. Apparatus for measurement of the depression of freezing point of solution . 11 5. Diagram of conductivity cells 18 6. Wheatstone Bridge for the measurement of electric resistance 18 7. Diagram to show type of electrodes used in studying electromotive force . . 30 8. Diagram of apparatus for the measurement of the H-ion concentration . . 31 9. Chart of tints as used in colorimetric measurement of H-ion concentration (Color Plate) 34 10. Diagram of apparatus for saturating blood and plasma with expired air . . 43 11. Van Slyke's apparatus for measuring the CCycombining power of blood in blood plasma ' 44 12. TJltramicroscope (slit type) for the examination of colloidal solutions ... 53 13. To show diffusion into gelatin of a crystalloid stain, and the nondiffusion of a colloid stain 54 14. Diagram from W. Ostwald showing the relative size of various particles and colloidal dispersoids compared with a red blood corpuscle and an anthrax bacillus 55 15. Capillary analysis of colloids 57 16. Diagram to show structure of gels 62 17. Diagram to illustrate surface tension 65 18. Traube's stalagmometer 66 19. Diagram of the graphic coagulometer 110 20. Coagulometer 110 21. Mercury manometer and signal magnet, arranged for recording the mean ar- terial blood pressure in a laboratory experiment 126 22. The arterial blood pressure recorded with a mercury manometer (lower trac- ing) along with a tracing of the respiratory movement of the thorax . 127 23. Hiirthle's spring manometer 128 24. Normal curve of arterial blood pressure obtained with spring manometer . . 128 25. Diagram based on experiments on dogs to show the systolic, diastolic and mean blood pressures at different parts of the circulatory system . . . 129 26. Apparatus for measuring the arterial blood pressure in man 131 27. Effect of cutting the vagus nerve on the arterial blood pressure 136 28. Effect of stimulating the peripheral end of the right vagus on the arterial blood pressure 136 29. Effect of stimulation of the left splanchnic nerve on the arterial blood pressure 137 30. Composite curves to show effects of hemorrhage and transfusion of various solutions on blood pressure 140 31. Diagram of experiment to show that the diastolic pressure depends on the elasticity of the vessel wall 144 32. Diagram of Wiggers' optical manometer ,147 XXV XXVI ILLUSTRATIONS FIG. PAGE 33. Optical records of intraventricular pressure 148 34. Superimposed pressure curves after being graduated 150 35. Diagram to illustrate optical method for recording pressure curves from auricles 152 36. Diagram to show the positions of the cardiac valves 156 37. Electrophonograms along with intraventricular pressure curves from three different experiments 159 38. Arrangement of apparatus for heart-lung preparation 164 39. Volume curve of ventricles of cat (lower curve) in a heart-lung perfusion preparation 169 40. Heart and cardiac nerves of Limulus polyphemus 173 41. Heart-block produced by applying clamp 175 42. Tracing of contraction of ventricle, showing the effect of the local appli- cation of heat to the auricle 175 43. Frog heart showing the position of the first and second ligatures of Stannius 176 44. Effects of stimuli of increasing strength on skeletal and cardiac muscle to illustrate the "all or nothing" principle in the latter ....... 177 45. The effects of successive stimuli on skeletal and cardiac muscle to show the prominence of the staircase phenomenon, or treppe, in the latter . . . 178 46. The effects of successive stimuli and of tetanizing stimuli on skeletal muscle and cardiac muscle 179 47. Myograms of frog's ventricle, showing effect of excitation by break induc- tion shocks at various moments of the cardiac cycle 180 48. Heart of tortoise as suspended 183 49. Dissection of heart to show auriculoventricular bundle 184 50. Photograph of model of the auriculoventricular bundle and its ramifications, constructed from dissections of the heart 184 51. Diagram of an auricle showing the arrangement of the muscle bands; the concentration point; and the outline of the node 186 52. Diagram to show the general ramifications of the conducting tissue in the heart of the mammal 186 53. Diagram to illustrate the development and spread of the wave of negativity in a strip of muscle (curarized sartorius) when stimulated at the end . 188 54. Simultaneous electrocardiograms to show the cause for extrinsic deflections 190 55. Diagram of experiment by Lewis showing the times at which the excitation wave appeared on the front of the heart 194 56. Diagram of Chauveau's dromograph 200 57. Diagram to show principle of Pitot's tubes for measuring velocity pulse . . 201 58. Cybulski's photohematotachometer 201 59. Dudgeon's sphygmograph 201 60. Pulse tracing (sphygmogram) taken by sphygmograph 202 61. Forms of apparatus for measurement of blood velocities 207 62. Plethysmograph for recording volume changes in the hand and forearm . . 210 63. Effect of venous supply on volume of heart 217 64. Simultaneous tracings from auricle and ventricle of turtle's heart .... 223 65. Effect of vagus stimulation on 'heart of turtle 223 66. Tracing to show that vagus stimulation may diminish transmission from auricles to ventricles 224 ILLUSTRATIONS XXV11 FIG. PAGE 67. Tracing to show that vagus stimulation may facilitate transmission from auricles to ventricles 225 68. Diagram to show the innervation of the heart in the frog or turtle. (Color Plate.) 228 69. Frog heart tracing showing the action of nicotine 231 70. Schematic representation of the innervation of the heart of the mammal. (Color Plate.) 232 71. Tracings showing the effects on the heartbeat of the frog resulting from stimulation of the sympathetic nerves prior to their union with the vagus nerve 233 72. Boy's kidney oncometer . . . 235 73. Fall of blood pressure from excitation of the depressor nerve ...... 244 74. The effect of strong stimulation (heat) of the skin of the foot on the ar- terial blood pressure and respiratory movements 245 75. Diagram showing the probable arrangements of the vasomotor reflexes . . 246 76. Aortic blood pressure, showing the effect of posture 249 77. Tracing to show the effect of gravity on the arterial blood pressure . . . 250 78. The effect of gravity on the aortic pressure after division of the spinal cord in the upper dorsal region 250 79. Capillaries from abdominal wall of guinea pigs after injection of india ink . 252 80. Tracing showing simultaneous records of the arterial blood pressure, the venous pressure, the intracranial pressure, the pressure in the venous sinuses 260 81. Electrocardiographic apparatus as made by the Cambridge Scientific Ma- terials Co 271 82. Normal electrocardiogram 273 83. Electrocardiogram (dog) taken simultaneously with curves from auricle and ventricle 274 84. Diagrams to show spread of electrical current in heart and the use of Ein- thoven's triangle 276 85. Sinus bradycardia 279 86. Auricular extrasystole 279 87. Ventricular extrasystoles arising in the right ventricle 279 88. Ventricular extrasystole arising in the left ventricle 279 89. Paroxysmal tachycardia 280 90. Auricular fibrillation . 280 91. Auricular flutter 282 92. Delayed conduction 282 93. Partial dissociation 283 94A. Electrocardiogram showing a lesion of the branch of the conducting bundle 284 94B. Illustrating the effect upon the electrocardiogram of a defect of the left di- vision of the bundle 284 95. Tracings of the jugular pulse, apex beat, carotid and radial pulses .... 286 96. Polysphygmograph 288 97. Normal jugular tracing 288 97. Superimposed pressure curves from aorta, ventricle and auricle, along with electrocardiogram and phonocardiogram 289 98. Polysphygmograms including jugular, apex and rndial tracings 290 99. Delayed conduction 291 100. Dropped beats . t , 292 XXV111 ILLUSTRATIONS FIG. PAGE 101. Premature beats (extrasy stoles) ventricular in origin 292 102. Paroxysmal tachycardia 293 103. Auricular flutter 294 104. Auricular flutter 294 105. Auricular fibrillation 295 106. Showing the appearance of the blood vessels in the ears of a rabbit in a state of deep shock. (Color Plate.) 304 107. Diagram showing amounts of air contained by the lungs in various phases of ordinary and of forced respiration 318 108. Diagram of structure of air sacs, atria, alveolar ducts, etc 318 109. Pneumograph 321 110. Effect of abdominal and chest breathing on the pulse and blood pressure of man 325 111. First dorsal vertebra, sixth dorsal vertebra and rib. Axis of rotation shown in each case 333 112. Lower half of the thorax from the 6th dorsal to the 4th vertebra, seen from the front 335 113. Intercostal muscles of 5th and 6th spaces 336 114. Hamberger's schema to demonstrate the functional antagonism of internal and external intercostals 336 115. Schema to demonstrate that the function of the internal intercartilaginous intercostals is identical with that of the external interosseous intercostals 337 116. Diagram to show the effect of high and low positions of the diaphragm on the costal angle 339 117. Diagram to show the effect of clinical displacements of the diaphragm on the costal angle 340 118. Diagram to show cuts required for isolation of the phrenic center .... 345 119. Diagram to show certain positions in the medulla and upper cervical cord, where sections may be made without seriously disturbing the respirations 346 120. Diagram to show where cuts are made to isolate the chief respiratory center from afferent impulses 347 121. Diagram showing principle for measurement of the tension of CO 2 in blood 355 122. The gas analysis pipette for the microtonometer shown in Fig. 123 . . . 356 123. Microtonometer, to be inserted into a blood vessel 356 124. Apparatus for collection of a sample of alveolar air by Haldane's method . 357 125. Fridericia's apparatus for measuring the CO 2 in alveolar air 358 126. Curves to show the relationship between the O 2 and CO 2 tensions in alveolar air and arterial blood 358 127. Same as Fig. 126, except that in this case the tension of CO 2 in the alveolar air was experimentally altered 359 128. Curves showing relationship between total CO 2 in solution and P H at varying CO 2 pressure' 364 129. Curve showing the respiratory response to CO 2 in the decerebrate cat . . 368 130. The behavior of the respiratory volume, the blood pressure and the pulse during progressive anoxemia 376 131. Curves showing variations in alveolar gas tensions after forced breathing for two minutes 383 132. Various types of periodic breathing 385 ILLUSTRATIONS XXIX FIG. PAGE 133. Quantitative record of breathing air through a tube 260 cm. long and 2 cm. in diameter 383 134. Barcroft's tonometer for determining the curve of absorption of oxygen by hemoglobin or blood 394 135. Barcroft's differential blood gas manometer 394 136. Barcroft blood gas manometer 395 137. Typical dissociation curve. (Color Plate.) 396 138. Average dissociation curves 397 139. Degree of variation in dissociation curve of man 398 140. Dissociation curves of human blood 399 141. Curves showing relative rates of oxidation and reduction of blood as in- fluenced by temperature and tension of CO 2 400 142. Curve of CO 2 tension in blood 405 142. CO 2 tension at various altitudes 417 143. Cells of parotid gland showing zymogen granules 454 144. Parotid gland of rabbit in varying states of activity examined in fresh state 454 145. Diagrammatic representation of the innervation of the salivary glands in the dog. (Color Plate.) 458 146. Pancreatic acini stained with hematoxylin 462 147. Three preparations of pancreatic acini stained by eosin orange toluidin blue 463 148. Diagram of stomach showing miniature stomach separated from the main stomach by a double layer of mucous membrane 468 149. Typical curve of secretion of gastric juice collected in 5-minute intervals on mastication of palatable food for 20 minutes 471 150. Cubic centimeters of gastric juice secreted after diets of moat, bread, and milk 475 151. Digestive power of the juice, as measured by the length of the protein column digested in Mett's tubes, with diets of flesh, bread, and milk .... 475 152. Loop of intestine after tying off the portions, cutting the nerves running to the middle portion and returning the loop to the abdomen for some time 477 153. The changes which take place in the position of the root of the tongue, the soft palate, the epiglottis and the larynx during the second stage of swal- lowing 480 154. Schematic outline of the stomach 486 155. Outlines of the shadows cast by the stomach at intervals of an hour each after feeding a cat with food impregnated with bismuth subnitrate . . 487 156. Skiagrams of human stomach at intervals after food, illustrating a gastric cycle 488 157. Section of the frozen stomach (rat) some time after feeding with food given in three differently colored portions 489 158. Tracings showing the relationship between contractions of the antrum (lower tracing) and the pyloric sphincter (upper tracing) 492 159. Diagram of the relationships of the contractions of antrum, sphincter, and t duodenum, and the passage of chyme from antrum to duodenum . . . 493 160. Apparatus for recording contractions of the intestine 498 161. Diagrammatic representation of the process of segmentation in the intestine 499 162. Intestinal contractions after excision of the abdominal ganglia and section of both vagi t 500 XXX ILLUSTRATIONS FIG. PAGE 163. The effect of excitation of both splanchnic nerves on the intestinal contrac- tions . . 502 164. The effect of stimulation of light vagus nerve on the intestinal contractions 502 165. Diagram of time it takes for a capsule containing bismuth to reach the various parts of the large intestine 504 166. Diagram of method for recording stomach movements 507 167. Tracing of the tonus rhythm of the stomach three hours after a meal . . 508 168. Tracings from the stomach during the culmination of a period of vigorous gastric hunger contractions 508 169. Showing augmentation of the knee-jerk during the marked hunger contrac- tions 509 170. Diagram of the uriniferous tubules, the arteries, and the veins of the kidney 542 171. Cross section of convoluted tubules from kidney of rat 543 172. Diagram of blood supply of Malpighian corpuscle and of convoluted tubules in amphibian kidney 549 173. Nerve supply of the kidney 553 174. Respiration calorimeter of the Russell Sage Institute of Pathology, Bellevue Hospital, New York 572 175. Chart of determining surface area of man in square meters from weight in kilograms and height in centimeters according to formula 576 176. Diagram of Atwater-Benedict respiration calorimeter 580 177. Nose clip, face mask, and mouth piece 590 178. Diagram of respiratory valves 590 179. The Tissot spirometer 591 180. The Douglas bag method for determining the respiratory exchange . . . 592 181. Haldane gas apparatus and Pearce sampling tube 593 182. Curve constructed from data obtained from a man who fasted for thirty- one days . 601 183. Curves of growth of rats on basal rations plus the various proteins indicated 610 184. Curves of growth of rats on basal rations plus the proteins indicated . . . 611 185. Photographs of rats of same brood on various diets 613 186. Curves of growth of rats as influenced by the accessory food factors . . . 620 187. Vividiffusion apparatus of J. J. Abel 642 188. Curves showing the amount of amino nitrogen taken up by different tissues after the cutaneous injection of amino acids 643 189. Curves showing the concentrations of amino-acid nitrogen in the blood dur- ing fasting and protein digestion 644 190. Curves showing the percentage of glucose in blood after a constant injection of an 18 per cent solution into a inesenteric vein 691 191. Child aged 4^ years suffering from hypernephroma 769 192. Arrangement of apparatus for recording contractions of a uterine strip, intes- tinal strip, or ring, etc .- 781 193. Tracing showing the effect of epinephrine on the intestinal contractions and on the arterial blood pressure 782 194. Arrangement of apparatus for perfusion of the vessels of a brainless frog . 783 195. Microphotographs of thyroid gland of a dog 793 196. Cretin, nineteen years old 796 197. Case of myxedma before and after treatment 797 198. Drawing from a photograph of mesial sagittal section through the pituitary gland of a human fetus . . 807 ILLUSTRATIONS XXXI FIG. PAGE 199. Tracing showing the action of pituitrin 011 the uterine contractions and blood pressure in a dog 812 200. Tracing showing the constructing action of pituitrin on the bronchioles and its effect on blood pressure in a spinal dog . . . . 813 201. Showing the appearance before and after the onset of acromegalic symptoms 815 202. Hand of a person affected with acromegaly 817 203. The evolution of the nervous system 829 204. Normal cell from the anterior horn, stained to show Nissl's granules . . . 831 205. Arborization of collaterals from the posterior root fibers around the cells of the posterior horn 832 20(3. Part of a ventral cornual cell from the calf's spinal cord stained to show neurofibrils 833 207. Schema of simple reflex arc 833 208. Diagram of nervous system of segmented invertebrate 834 209. Diagram illustrating the effect of areas of narcosis on the strength of the nerve impulse 839 210. The recovery of excitability in the nerve fiber after the passage of a nerve impulse . . . 840 211. Degeneration and regeneration of a sectioned nerve fiber 847 212. Evolution of the sense organs 855 213. Cold spots and heat spots of an area of skin of the right hand 860 214. Diagram showing the segmented arrangement of the sensory nerves . . . 867 215. Diagram of the afferent paths followed by sensory impulses within the spinal cord and brain 869 216. Afferent paths connecting the retina with the visual area of the cerebral cortex 881 217. Outer aspect of the brain of the chimpanzee showing the position of the motor centers . . 885 218. Diagram to illustrate the different arrangements of the internuncial neurons of the voluntary and autonomic nervous systems 894 219. Diagram of the autonomic nervous system. (Color Plate.) 894 220. Diagram showing the main parts of the autonomic nervous system. (Color Plate.) 896 221. Schematic representation of the autonomic nervous system. (Color Plate.) 898 222. Diagram of an axon reflex in a sensory nerve fiber of the skin 899 223. Electromyogram of the voluntary contraction of the flexor muscles . . . 907 224. The contraction of a single fiber of the sartorius muscle of the frog . . . 907 225. The all or none nature of the contraction of a single fiber of skeletal muscle 909 226. Eeciprocal inhibition 916 227. Records of the contraction of the isolated extensor muscle of the knee of the cat 917 228. Compensatory movements of the eyes and fins of the dogfish 919 229. The semicircular canals of the ear 919 230. A tracing of the knee-jerks of a normal man and of a man with a cerebellar injury 922 231. Schema of the parts of the mammalian cerebellum 929 232. Diagrams to represent a ventral view of the left half and a dorsal view of the right half of the cerebellum 930 XXX11 ILLUSTRATIONS FIG. . PAGE 233 and 234. The inferolateral and the posterior aspect of the human cerebellum indicating certain cerebellar localizations according to Barany . . . 931 235. Footprints after destruction of the cerebellum in a dog 932 236. Tracing from the hind limb of a spinal dog during the scratching movements 935 237. Diagram showing the reflex arcs involved in the scratch reflex 936 238. The region of body of dog from which the scratch reflex can be elicited . . 936 239. Record from myograph connected with the extensor muscle of the knee . . 939 240. Sherrington's diagram illustrating the mechanism of reciprocal inhibition 940 241. Reflex figures 942 242. Successive induction illustrated by the crossed-extension reflex 949 243. Postures assumed by the robber fly when the eyes are unequally illuminated 953 PHYSIOLOGY AND BIOCHEMISTRY IN MODERN MEDICINE PART I THE], PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES CHAPTER I GENERAL CONSIDERATIONS The work of the physiologist consists, in large part, in ascertaining to what extent the known laws of physics and chemistry find application in explaining the phenomena of life. He gathers from the vast store- house of physical and chemical knowledge whatever is of value in the interpretation of the various mechanisms that work together to com- pose the living machine, and having added to this knowledge he passes it on for use by those who are concerned in the study and treatment of disease. Many of the most important steps in the advance of physiological knowledge in recent years have depended upon the discovery of some hitherto unknown physical or chemical law, or upon the elaboration of some accurate method for the measurement of the phenomena upon which these or previously known laws depend. The discoveries of van't Hoff, Arrhenius, and Ostwald of the so-called laws of solution were soon followed by important observations on their relationship to the movement of fluids and dissolved substances through cell mem- branes; the discoveries of Hardy, Willard Gibbs, etc., of the behavior of colloids and of the phenomena of surface tension found application in explaining many hitherto inexplicable peculiarities in the activities of ferments; the discovery by Nernst, etc., of methods for the measurement of the electro-motive force of dissolved substances was applied to de- termine the actual reaction or hydrogen-ion concentration of animal 1 2 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES fluids, and to explain the generation of the electric currents which ac- company muscular, nervous, and glandular activity. It would be out of place here to devote much space to a detailed ac- count of such matters. They belong more properly in the domain of general than in that of human physiology. General physiology is con- cerned with the study of the essential nature of the vital processes; whereas human physiology is merely a branch of the subject in which special attention is devoted to the application of the truths of general physiology to the working of the human machine. For the physician and surgeon a knowledge of human physiology is as essential as is a knowledge of the construction of a piece of machinery for the engineer who attempts its repair, but obviously to acquire this knowledge the fundamental principles of general physiology must first of all be under- stood. For these reasons the introductory chapters are devoted to a brief review of the most important of the physicochemical principles upon which the working of the cell depends. From the viewpoint of the physical chemist the cell consists of an envelope of more or less permeable material inclosing a solution of various crystalloids and colloids, which are in a state of equi- librium with one another. This equilibrium is readily altered by various influences that may act on the cell, and the resulting changes manifest themselves outwardly by alterations in the shape and volume of the cell growth and motion; by the extrusion of some of its contents secretion; or by the propagation to other parts of the cell, or its processes, of the state of disturbed equilibrium nervous impulse. Besides the activities that are dependent upon physicochem- ical changes, purely chemical processes go on in the cell. Many of these consist in the breakdown and oxidation of complex unstable organic molecules, a process identical with that occurring in combustion outside the cell. Others involve the building up, stage by stage, of complex substances out of the elements or out of simpler molecules. Chemical transformations occur in the cell which, in the chemical laboratory, re- quire the most powerful reagents and physicochemical forces, either the strongest of acids, alkalies, oxidizing agents, etc., or extreme degrees of heat, electrical energy, etc. But this is not all, for in the cell these chemical transformations are capable of being guided to a very remark- able degree of nicety so as to produce intermediate products that are used for some special purpose either by the cell that produced them or, after transportation by the blood, etc., by cells in other parts of the organism. It is customary to speak of the cell as a chemical laboratory, but it LAWS OF SOLUTION O is more than this; it is a laboratory furnished not only with the equip- ment of the chemist but directed in the harmonious operation of its many activities by a guiding hand which far surpasses anything else known to man. Chemical transformations that require for their accomplishment the greatest skill proceed without apparent difficulty in the cell. To what are these changes due? What is the nature of the chemical rea- gents and forces, and what is the directive influence that guides them in their varied activities? To these, which are among the great ques- tions of general physiology, the reply may be given that the reagents are the ferments or enzymes, and that the directive influence operates through the susceptibility of enzymic activities to changes in the envi- ronment in which the enzymes are acting. In many cases these changes can be explained on a physicochemical basis as dependent upon the known laws of mass action or surface tension; in other cases they de- pend on purely chemical changes in the cell contents, such as changes in reaction or the accumulation of chemical substances that act like poisons on the enzyme. But there are still others that appear to depend on influences which as yet are quite unknown to the physical chemist, such as the changes in cell activity that can be brought about by the nerve impulse. These preliminary remarks will serve to indicate the problems with which we must first occupy our attention. They concern the physico- chemical nature of saline solutions and of colloids, and the general na- ture of enzyme action. The knowledge which we acquire will be found to be of value, not only because it will help us to understand the nature of the workings of the normal healthy cell, but because, here and there, it will indicate possible causes for derangement in cellular function and suggest rational means by which we may attempt to rectify the fault. THE PHYSICOCHEMICAL LAWS OF SOLUTION The Gas Laws Three fundamental principles of general chemistry serve as the basis for an understanding of the nature of solutions. The first is that if we take a quantity of any gas equal to its molecular weight in grams (called a gram-molecule or for sake of brevity a mol), it will occupy ex- actly 22.4 liters at a temperature of C. and a pressure of 760 mm. Hg. ; the second is that, as we compress a gas, its pressure will increase in exactly the same proportion as the volume diminishes (the volume of a gas is in- versely proportional to its pressure) ; the third is that all gases expand by 4 PHYSICOCHSMICAL BASIS OF PHYSIOLOGICAL PROCESSES 1/273 part of their volume at C. for every degree C. that their tempera- ture is raised.* The pressure of a gas is measured by connecting a pressure gauge or manometer with the vessel which contains the gas. Now, it is plain that if the 22.4 liters, which is the volume occupied by a gram-molecular quantity, were compressed so as to occupy a volume of 1 liter, its pressure would be 22.4 times that of 1 atmosphere, or 22.4 x 760 mm. Hg the temperature remaining constant. Under these conditions we must im- agine that the molecules of gas are crowded together by the compression, and if we further conceive of these molecules as being in constant mo- tion, then we can understand why the pressure should increase just in proportion as we confine the space in which they can move. One other property of gases must be borne in mind namely, their tendency to diffuse from places where the pressure is high to places where it is low until the pressure is the same throughout. OSMOTIC PRESSURE These fundamental facts regarding the behavior of gases suggested to van't Hoff the hypothesis that molecules of dissolved substances must behave in a similar manner to those of gases. To put this hypothesis to the test, it is necessary that we have some method for measuring the pressure of dissolved molecules. We can not, as in the case of a gas, use an ordinary manometer, for this would measure only the pressure of the solvent on the walls of its container and would tell us nothing of the pressure of the dissolved molecules. We must use some filter or membrane that will allow the molecules of the solvent but not those of the dissolved substance to pass through it. It is evident that if such a filter is placed, for example, between a solution of sugar in water and water alone, the molecules of the latter will diffuse into the solution until this has become so diluted that the pressure of the dissolved mol- ecules is equal on both sides of the membrane. Such a membrane is called semipermeable ; the diffusion of molecules through it is called osmosis, and the pressure which is generated, the osmotic pressure. If we prevent the water molecules from actually diffusing by opposing a pressure which is equal to that with which they tend to diffuse through the membrane, we can tell the magnitude of the osmotic pressure (Fig. 1). In applying these facts to test the hypothesis that molecules in solution *This implies that at -273 C. the gas would occupy no volume. Before this temperature is reached, however, the liquefaction of the gas sets in. The temperature -273 C. is known as absolute zero. An observed temperature pins 273 is called the absolute temperature. Another way of stat- ing the above law is therefore that the volume is directly proportional to the absolute temperature. At 273 C. the volume of a gas at C. would be doubled, or if expansion were prevented the pressure would be doubled. LAWS OF SOLUTION obey the same laws as those in gaseous form, we must employ a semi- permeable membrane which is rigid enough to withstand the pressure and which forms part of the walls of a closed vessel connected with a manometer. If we place in such an osmometer a solution containing the molecular weight in grams of some substance dissolved in one liter of solvent, a so-called gram-molecular solution, it is obvious that, if the gas laws are to apply, the osmotic pressure should equal that of 22.4 liters of a gas compressed to the volume of one liter; in other words, it should equal 22.4 x 760 = 17,024 mm. Hg. Although there are very considerable technical difficulties in making a semipermeable membrane that is strong enough to withstand such a pressure, yet this has been accom- w Fig. 1. Diagram of osmometer. The cylindrical vessel (0), with a bottom of unglazed clay, the pores of which are filled with a precipitate of copper ferrocyanide to form a semi- permeable membrane, is suspended in an outer vessel, and is closed above by a tightly fitting stopper pierced by a tube leading to a manometer (M). O contains a strong solution of cane sugar, and W contains water. The water molecules tend to pass through the semipermeable membrane into the cane sugar solution, and since the cane sugar molecules can not pass in the opposite direction, the pressure in O rises and is recorded in M. This equals the osmotic pressure. plished, and the fundamental principle has therefore been firmly estab- lished that substances in solution obey the same laws as gases. Further proof that the gas laws apply to solutions has been secured by showing that the osmotic pressure (of a dilute solution) is directly pro- portional to the concentration of the dissolved substance (the solute) and to the absolute temperature. It also obeys the law of partial pres- sures, which states that the total pressure exerted by a mixture (of gases or dissolved molecules) is the sum of the pressures which each constit- uent of the mixture would exert were it alone present in the space occupied by the mixture. 6 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES Since the osmotic pressure is analogous to the pressure of a gas and is therefore proportional to the molecular concentration (i. e., number of molecules in unit space), it follows that a semipermeable membrane can be used to determine the relative concentration of two solutions of the same substance. When a watery solution of some substance is placed in an osmometer that is surrounded by a similar but more dilute solution, water molecules will diffuse into the osmometer until the pres- sure is equal on the two sides of the semipermeable membrane; that is, the water will pass from the solution having a lower osmotic pressure into the solution having the higher pressure. When two solutions have the same osmotic pressure, they are said to be isotonic; when that of one is greater than that of the other, it is hypertonic; and when less, hypotonic. Biological Methods for Measuring Osmotic Pressure A practical biological application of these principles can very readily be made if, instead of a rigid semipermeable membrane such as that figured in the diagram, we employ one that is extensible and takes the form of a closed sac ; then as diffusion of water occurs the sac will either distend when it contains a stronger solution than that outside, or shrivel or crenate when the reverse conditions obtain. Many animal and veg- etable protoplasmic membranes are semipermeable, including the en- velope of red blood corpuscles. Thus, if we examine blood corpuscles under the microscope and add to them a saline solution of higher os- motic pressure than blood serum, they will visibly diminish in size and become irregular in shape; whereas if the solution is of lower osmotic pressure, they will distend. If no change occurs, the osmotic pressure of the cell contents must equal that of the saline solution in which the cells are immersed, from which it is clear that we can readily determine the magnitude of the osmotic pressure if we know the strength of the saline solution. Instead of measuring the individual cells under the microscope, we can measure the space they occupy in the fluid in which they are suspended. For this purpose a portion of the suspension is placed in a graduated tube of narrow bore, which is rotated in a horizontal position by a cen- trifuge after being closed at one end. The graduation at which the upper edge of the column of cells stands after centrifuging is a measure of the relative amounts of cells and of fluid in the suspension. Having found this value for cells suspended in an isotonic solution, as for exam- ple, blood corpuscles in blood serum, we may then proceed to ascertain it for the same cells suspended in an unknown solution; if we find that the cells now occupy a greater volume, the saline solution must have an os- LAWS OF SOLUTION 7 motic pressure that is lower than that of serum in approximate proportion to the readings on the tube in the two cases, and vice versa. The above apparatus, called a hematocrite (Fig. 2) has been very ex- tensively used in the collection of data concerning the relative osmotic pressures of different physiological fluids. Hemolysis Another way for determining the relative osmotic pressure of dif- ferent solutions consists in placing equal amounts (a few drops) of blood in a series of test tubes containing solutions of different strengths, and after allowing the tubes to stand for some time, noting in which of them laking of the blood corpuscles occurs. In solutions which are isotonic or hypertonic with the contents of the corpuscles, the latter will settle to the bottom of the tube and the supernatant fluid will be untinted with hemoglobin, but in solutions which are distinctly hypotonic, the sediment will be less distinct and the supernatant fluid red. Fig. 2. Hematocrite. The graduated glass tubes are filled with the two specimens of blood, or corpuscular suspension, and then rotated rapidly by a centrifuge. The relative heights at which the corpuscular sediment stands in the two tubes is proportional to the osmotic pressures of the fluid in which the corpuscles are suspended. By noting (1) the lowest concentration (percentage composition) of the solutions in which the corpuscles sink to the bottom and leave the supernatant fluid colorless, and (2) the highest concentration in which the corpuscles when they settle leave the supernatant fluid tinted red, we can determine the limiting concentrations for solutions of different .sub- stances. Thus, with bullock's blood the following results were obtained (Hamburger) : SUBSTANCE PERCENTAGE STRENGTH OF SOLUTION IN WHICH I II SUPERNATANT FLUID SUPERNATANT FLUID WAS COLORLESS WAS RED KN0 3 1.04 0.96 NaCl 0.60 0.56 K 2 S0 4 1.16 1.06 C^H^O,, (Cane sugar) 6.29 5.63 CH 3 COOH (Pot. acetate) 1.07 1.00 MgSO 4 .7H2O 3.52 3.26 CaCL, 0.85 0.79 8 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES The mean of these limiting concentrations is the critical concentration and indicates the strength of each solution that can be added to blood without causing any damage to the corpuscles. This critical concen- tration is not, as might at first sight be imagined, the same as that which is isotonic with the contents of the corpuscles, but distinctly below it. The reason for this becomes apparent if we observe the be- havior of corpuscles suspended in an isotonic solution which is then gradually diluted. As dilution proceeds, the corpuscles distend, until at last their envelopes burst and the hemoglobin is discharged. The lim- iting concentrations of a given salt vary for different corpuscles; thus, the concentration of sodium chloride solution that just causes laking of frog's blood corpuscles is 0.21 per cent, that of human blood 0.47 per cent, and that of horse blood 0.68 per cent. It is the strength of the corpuscular envelope rather than variations in the osmotic pressure of the contents that is responsible for these differences. The above described method of hemolysis, as it is called, can not be used for comparisons of osmotic pressure in cases in which the solution contains substances which alter the permeability of the corpuscular envelop; for example, it can not be used when urea, or ammonium salts, or certain toxic bodies are present. We may therefore ascertain whether a given substance has a damaging influence on the corpuscular envelope by finding whether hemolysis occurs when we suspend the cor- puscles in a solution that is known by physical methods to be isotonic with the corpuscular contents. We can further determine the approximate degree of this toxic influence by estimating by color comparisons (colorimetry) the amount of hemoglobin that has diffused out of the corpuscles. Plasmolysis An analogous method for determining osmotic pressure is that of plasmolysis, in which the behavior of certain plant cells is observed microscopically while they are in contact with solutions of different strengths. When the surrounding solution is isotonic with the cell contents, the latter fill the cell and extend up to the more or less rigid cell wall '(A in Fig. 3); but when the solution is hypertonic, the cell contents become detached from the cell wall at one or more places plasmolysis (B and C). The semipermeable membrane in this case is therefore not the cell wall but the layer of protoplasm on the surface of the cell contents. The method can be used only for detecting solu- tions that are hypertonic, for with those that are hypotonic the cells merely become turgid and exert more pressure on the more or less rigid cell wall. Many of the conclusions that have been drawn from LAWS OF SOLUTION results obtained by the plasmolytic method have recently been called in question, because no regard has been taken of the power of the colloids of the cell to absorb (imbibe) water (see page 63). The methods of hemolysis and plasmolysis have been used for the inves- tigation of many problems in medicine besides those pertaining strictly to osmotic pressure. In the case of certain toxic fluids, such as snake venom, tetanus toxin, etc., determination of the hemolytic power has proved of value in roughly assaying the damaging influence on other cells than blood corpuscles. Studies in hemolysis have also been especially valuable in working out the mechanism by which cellular toxins in general develop their action, and the conditions under which this action may be counter - Fig. 3. To show plasmolysis in cells from Tradescantia discolor. A. normal cell; B, plasmolysis in 0.22 M. cane sugar; C, pronounced plasmolysis in 1.0 M. KNO3; h, the cell wall; p, the protoplasm. (After De Vries.) acted, as by the development of antibodies. Furthermore, any solution that is to be injected into the animal body, either intravenously or subcu- taneously, should first of all be tested by the above methods in order to find out whether it is isotonic with the body fluids. If a hypertonic so- lution is injected, it will result in the abstraction of water from the tissue cells, whereas a hypotonic solution will cause the water content of these to increase. Advantage has recently been taken of this water-abstracting effect of hypertonic solutions in the treatment of wounds. By constantly bathing them with strong saline solutions, an outflow of water is set up from the tissue cells that border on the wound, and this tends to bring to the focus of infection the defensive substances that are present in animal fluids. CHAPTER II OSMOTIC PRESSURE (Cont'd) MEASUREMENT BY DEPRESSION OF FREEZING POINT The limitations in the use of the plasmolytic and hemolytic methods in the precise measurement of the osmotic pressure of the body fluids have rendered it necessary to find some physical method that will be generally applicable. Because of technical difficulties, it is impracticable to measure the pressure directly by employing an osmometer, so that some indirect method, depending on a readily measurable physical prop- erty which varies in proportion to the osmotic pressure of the dissolved substances, must be used. Fortunately, one such exists in the property which dissolved substances have in lowering the temperature at which the pure solvent solidifies; the freezing point of pure water, for example, is lowered when substances are dissolved in it, and the extent of this lowering, with certain reservations which will be explained later (page 16), is proportional to the molecular concentration of the solution and independent of the chemical nature of the substance dissolved. This lowering of temperature is designated by the Greek letter A, and to measure it a thermometer is used which is not only extremely sensitive but in which the level of the mercury column can be adjusted so that it stands at a convenient level on the scale corresponding to the freezing point of whatever solvent was used in making the solution under investi- gation (Beckman's thermometer) (Fig. 4). Having ascertained the exact position on the scale of this thermometer at which the pure solvent freezes, the observation is repeated with the solution, the osmotic pressure of which is to be determined. A gram-molecular solution in water (having therefore an osmotic pres- sure of 17.024 mm. Hg) has a freezing point that is 1.86 C. lower than that of pure water. This is known as the " freezing point constant," and it varies for different solvents, being 3.9 for acetic acid and 4.9 for benzene. If an unknown watery solution is found to have a freez- ing point that is A C. lower than that of wa'ter, its osmotic pressure , Ax 17.024 will equal r-^ mm. Hg. l.oo The depression of the freezing points produced by the various body 10 OSMOTIC PRESSURE 11 fluids has been compared, the objects in view being to see whether osmotic pressure is a property which changes under different physiological and pathological conditions, and to find out by comparison of the osmotic pressures of the fluids in contact with a membrane, whether physical forces alone can be held responsible for the transference of substances through it from one fluid to the other. THE ROLE OF OSMOSIS, DIFFUSION, AND ALLIED PROCESSES IN PHYSIOLOGICAL MECHANISMS The Transference of Substances Through Cell Membranes. An ac- investigations in which the foregoing meth- count of some of the Fig. 4. Apparatus for measurement of the depression of freezing point of solutions. The solution is placed in the large test tube with the side arm, and in it is suspended the bulb of a Beckmann thermometer with a platinum loop to serve for stirring. The upper end^of the mercury column of the thermometer is shown magnified at the upper left corner. ihe amount of mercury in the thermometer tube can be regulated by tapping the upper end with the thermometer in various positions. The test lube is protected by an outer tube, which is then placed in a vessel containing a freezing mixture. ods have been used will illustrate their value in revealing the mech- anism involved in the transference of water and dissolved substances through cell membranes, as occurs in absorption of food in the intestine, in the formation of lymph and urine, and so forth. In em- ploying physical methods in the elucidation of such problems, it is always most necessary to proceed with great care, since the physical 12 PHYSICOCHEJMICAL BASIS OF PHYSIOLOGICAL PROCESSES chemist works with pure solutions, while the physiologist has to use fluids that are always complicated and frequently very variable in com- position. We must simplify the problem as far as possible by having clearly before us the exact nature of the biological problem which a com- parison of physicochemical values, such as osmotic pressure, may ena- ble us to elucidate, and we must consider the other physical forces which may assist or modify the particular one we are investigating. In the physical experiments described above, the semipermeable mem- brane may be conceived of as composed of pores of such a size that they permit only the smallest of molecules those of water to pass through them. Semipermeable membranes with larger pores may, how- ever, exist that is, membranes which permit water molecules and mole- cules of simple chemical substances to pass, but hold back those com- posed of large complex molecules. Such a semipermeable membrane would allow the saline constituents but not the proteins of blood serum to pass. It is, however, no longer semipermeable towards all of the dis- solved substances, and the process of diffusion through it is more gener- ally designated as one of dialysis than of osmosis. Since the passage of dissolved molecules through membranes de- pends upon the principle of diffusion, its rate will be proportional to the osmotic pressures of the solutions on the two surfaces of the mem- brane and to the size of the molecules, small molecules diffusing more quickly than large ones. Suppose a membrane permeable to sodium chloride and water is placed between two fluids containing sodium chloride in solution, but in greater concentration in one of them than in the other: although the sodium chloride will diffuse from the stronger to the weaker solution, the water will tend to diffuse still more quickly (be- cause its molecules are smaller) in the opposite direction, until the number of sodium-chloride molecules in a given volume of solution is equal on both sides of the membrane. For a time, therefore, the volume of the stronger solution will increase. The differences which exist in the dif- fusibility of dissolved molecules are analogous to those which have long been known to exist in the diffusibility of gases, but the relation between rate of diffusibility and molecular weight is not so simple as the ratio between these two quantities in gases. These relationships, however, indicate several further possibilities in the explanation of the mechanism of exchange of substances through membranes, and must not be overlooked, as they often are, in the interpretation of physiological phenomena. An excellent review of the possible conditions is given by Starling in his " Human Physiology." 4 For example, let us suppose the substances dissolved in the fluid on the two sides of a semipermeable membrane, such as the peritoneum, to be different in diffusibility, as cane OSMOTIC PRESSURE 13 sugar, which does not readily diffuse, and sodium chloride, which diffuses quickly; the osmotic flow will take place from the sodium-chloride solu- tion to that of cane sugar even though the sodium-chloride solution is stronger than the sugar. Furthermore, the simple laws of osmosis may be upset by an attrac- tive influence of the membrane toward certain substances [due to their becoming dissolved or adsorbed in it (see page 66)] but not toward others. Many membranes of this nature are known to the chemist (e. g., rubber membranes in contact with gases, pyridine solutions, etc.), and it is probable that such a property of selective solubility may play a not unimportant role in the transference of substances across animal membranes (Kahlenberg 5 ). These few conditions which may modify the direction of the osmotic flow, are indicated here to show how involved such problems are, and how careful we must be not to assume that, because a substance is trans- ferred through a living membrane contrary to the simpler laws of os- mosis and diffusion, it must involve the expenditure of forces different from those operating in dead membranes. Another force comes into operation in causing transference of sub- stances through membranes namely, that of filtration. This is a purely mechanical process, in which molecules are forced through the pores of a filter (i. e., membrane) by differences in pressure on its two sides. We are now in a position to consider in how far the above physical forces explain certain physiological problems. The Physical Factors Involved in Absorption, Excretion and Lymph Formation. 1. Is the absorption, into the Hood and lymph circulating in the intestinal walls, of substances in solution in the intestinal contents, entirely dependent upon the processes of filtration, diffusion and osmosis f The absorption of w r eak solutions of highly diffusible substances is probably very largely a matter of osmosis and diffusion, and water passes quickly into the blood because of osmotic attraction, but that other forces ordi- narily come into play is very clearly established by the following ob- servations. If a piece of intestine is isolated from the rest by placing two ligatures on it, and the isolated loop filled either with a solution con- taining the same saline constituents in similar proportions as in blood serum, or better still, with some of the same animal's blood serum, it will be found after some time that all of the solution becomes absorbed into the blood; the contents of the loop are therefore absorbed into the blood, even though the osmotic pressures of the dissolved substances are the same on both sides of the membrane (Weymouth Eeid 6 ). The intestinal membrane seems to possess towards readily diffusible 14 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES substances a permeability which varies, not at all with the physical diffusibility of the substance, but with its value from a physiological standpoint. Thus, sodium sulphate and sodium chloride diffuse through ordinary membranes with about equal facility, and yet if a solution con- taining these two salts is placed in the intestine, the chloride will be absorbed into the blood much more quickly than the sulphate. Sodium sulphate in watery solution diffuses through a membrane fifteen times more quickly than cane sugar, but from the intestinal lumen, cane sugar is absorbed ten times more quickly than sodium sulphate. If. however, the vitality of the epithelium is destroyed, as by first of all bathing it with a solution of sodium fluoride, then the sulphate and chloride will be absorbed at an equal rate. Although diffusion and osmosis can not therefore play any significant role in the normal process of absorption from the intestine, we must not entirely discount them; under certain circumstances, these physical forces may assert their influence as, for example, when concentrated saline solutions are present. Such solutions will attract water from the blood, and, other things being equal, more will be attracted the less permeable the epithelium happens to be towards the saline employed. Sulphates and phosphates will attract more water than chlorides or acetates. This property of the saline solutions to attract water coun- teracts the natural tendency for the water to be absorbed, and the large volume of fluid stimulates peristalsis. 2. Do the physical processes of filtration, diffusion and osmosis suf- fice to account for the production of urine by the kidneys? Under normal conditions the molecular concentration of the urine, as determined by the depression of freezing point, is considerably greater than that of the blood. This indicates that excretion must have occurred contrary to the laws of diffusion and osmosis; in other words, that the renal cells must have compelled dissolved molecules to be transferred from the blood to the urine, although the difference in concentration would cause them to pass in the opposite direction. This force, sometimes called for want of a better name " vital activity," must depend on the operation of processes that are quite distinct from those of diffusion, etc.; but that they are necessarily of a nonphysical nature (e. g., vital) is less probable than that they depend on some physical process the nature of which our present knowledge does not permit us to understand. By comparing the osmotic pressures of urine and blood, attempts have been made to measure the work done by the kidney in the produc- tion of urine. Thus, it has been found that A for normal urine (human) is about 1.8, and for blood about 0.6, from which it may be calculated that in the production of 1 kilogram of urine 150 kilogrammeters of OSMOTIC PRESSURE 15 work are expended.* But that such comparisons of the osmotic pres- sure of blood and urine are fallacious as an indication of the work of the kidney is evidenced, not alone by the results of the above calcula- tions, but also by the fact that under certain circumstances (as after copious diuresis) the osmotic pressure of the urine may be considerably lower than that of the blood. For some time after the application of osmotic pressure measurements to the study of biological problems, it was thought that determination of A in urine might be of clinical value as a criterion of renal efficiency, especially in one kidney as compared with the other. For this purpose A was determined in samples of urine removed from each ureter by catheterization. The tests of renal efficiency based on the rate of excre- tion and on the specific gravity of the urine, following ingestion of a fixed amount of water, have been found of much greater value. 3. Is the formation of lymph purely a physical process? The osmotic pressure of normal lymph is nearly always somewhat below that of blood serum, although occasionally it has been found to be a trifle higher. Physical processes, such as filtration, might therefore suffice to account for its formation under most conditions. But when we con- sider the excessive production of lymph that occurs as a result of cel- lular activity or following the injection of certain substances, called "lymphagogues," it is not so easy to explain the production in such terms, although some interesting attempts have been made to do so by those that are wedded to the mechanistic view. For example, the very marked increase in lymph flow which occurs as a result of muscular exercise or glandular activity has been attributed to the fact that dur- ing such processes large molecules become broken down into small ones in the cell protoplasm, so that the osmotic pressure is raised and water is attracted into the the cell until the latter becomes distended and a process of filtration into the neighboring lymph spaces occurs (see page 119). There are several other physiological processes of secretion and excre- tion which might be considered in the present relationship, but the above instances will suffice to illustrate the general principle upon which all of them have to be considered. *Osmotic pressure corresponding to A = -0.6 C. ermals 5,662 mm. Hg (75 m. of H 2 O), and that corresponding to A "1-8 C. equals 16,986 mm. Hg (225 m. HoO). The difference is there- fore equal to a column of water 150 m. high. According to these calculations it would appear that the kidney in producing the average daily output of 1500 c.c. urine performs 225 kilogrammeters of work in comparison with the 14,000 kilogrammeters which the heart is computed to perform in the same time (page 213). CHAPTER III ELECTRICAL CONDUCTIVITY, DISSOCIATION, AND IONIZATION The osmotic pressure is not infrequently found to be considerably greater than that expected from the strength of the solution. Although A of a gram-molecular watery solution of cane sugar (342 gm. to the liter) is 1.86 (see page 10), that of sodium chloride (58.5 gm. to the liter) is considerably greater. If the hypothesis regarding the relationship of molecular concentration to osmotic pressure is to hold good, it becomes necessary to explain this apparent inconsistency; one must account for a greater number of dissolved units than is represented by the actual number of dissolved molecules (i.e., weight of dissolved substances). It was observed that the power to conduct the electric current electrical conductivity in the case of solutions (e. g., of sugar) which have an osmotic pressure that corresponds to the weight of dissolved substances is practically nil, whereas the conductivity of those solutions which give higher osmotic pressure is quite pronounced. Arrhenius made the hy- pothesis that the conductivity depends on the splitting of molecules into two or more portions or ions, each of which carries either a positive or a negative electric charge, and that it is only when such dissociation occurs that the electric current can be conducted through the solution, the ions serving as it were as floats carrying the electric current. When sodium chloride is dissolved in water, it splits into Na carrying a positive charge and Cl carrying a negative charge, or Na + Cl~, as it is written; on the other hand, when sugar is dissolved, the molecules remain unbroken and no electric charges are set free. Substances which thus dissociate are called electrolytes, and those which do not, nonelectrolytes. When the electric current is passed through a solution of electrolytes, the ions which carry a positive charge move to the electrode or pole by which the current leaves the solution that is, in the same directions as the current; and since this electrode is called the cathode, these are called cations. Hydrogen and the metals belong to this group. The ions carrying a negative charge go in the opposite direc tion, against the current that is, towards the electrode by which the cur- rent enters, or the anode; they are therefore called anions. They include oxygen, the halogensrand the acid groups, such as S0 4 , C0 3 , etc. It must be understood that this dissociation into ions is already present 16 ELECTRICAL CONDUCTIVITY, DISSOCIATION, IONIZATION 17 in the solution before any electric current passes through it, the ions being however uniformly distributed throughout that is, arranged so that the negative charges of the anions precisely neutralize the positive charges of the cations. The electric current causes the electrodes to be- come charged, the one positively, the other negatively, so that an attrac- tive force is exerted on the ions of opposite sign. This causes the nega- tively charged ions to migrate towards the positive electrode, and the positively charged, towards the negative electrode. It is this migration of the ions that endows the solution with conducting qualities. In water, or in a solution of a nonelectrolyte, molecules of H 2 or non- electrolyte exist thus: H H 2 H 2 H 2 H 2 H 2 H 2 H 2 H 2 In a solution of an electrolyte, many of the molecules split into ions thus : Na* Cl- Na+ Cl- Na* 01- Na> Cl- Na+ Cl- Na> 01- Na+ Cl- Na+ Cl- Na+ 01- When an electric current passes through a solution of an electrolyte, the ions tend to arrange themselves thus: Cathode- Anode* Na+ Na> Na* Cl- Cl- 01- Na+ Na+ Na+ Cl- Cl- 01- Na+ Na+ Na* Cl- Cl- 01- It follows from the above considerations that the conductivity of a sub- stance in solution will depend on the degree to which it undergoes dissocia- tion. Furthermore, if we assume that in so far as osmotic pressure phenomena are concerned, each ion behaves in the same way as a mole- cule, then it follows that the electrical conductivity must be proportional to the extent to which the osmotic pressure is greater than we should ex- pect it to be from the amount of substance actually dissolved. In the Determination of the Conductivity it is obviously necessary to use standard conditions of depth and width of the fluid through which the current is passed, and to have some standard of comparison. The value is then known as the specific conductivity, the standard for comparison being the conductivity of a hypothetical liquid which, if enclosed in a centimeter cube, would offer a resistance of 1 ohm between two opposite sides of the cube acting as electrodes. The actual determination is usu- 18 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES ally made in a cylindrical vessel of hard glass (from soft glass enough alkali might be dissolved to affect the results), the electrodes being circu- lar plates of platinum firmly cemented at a known distance from each other (Fig. 5).* This conductivity cell, as it is called, is connected with a suitable apparatus for measuring the resistance offered by the Fig. 5. Diagram of conductivity cells. The platinum discs are represented by the thick black lines. They are held in position by thick-walled glass tubes, through which they are connected with the terminals by platinum wires. (From Spencer.) solution to the passage of an electric current (Wheatstone Bridge) (see Fig. 6). The resistance is of course inversely proportional to the con- ductivity. As a saline solution is progressively diluted, its specific conductivity naturally decreases (since there are now fewer molecules between the Fig. 6. Wheatstone Bridge for the measurement of electric resistance: a-b, bridge wire; c, the movable contact. two opposite faces of the centimeter cube, and the space between ions or molecules is increased). This result will not, however, tell us whether the salt itself is undergoing any alteration in conducting power as a con- sequence, for example, of greater dissociation. To ascertain this we must *This distance is determined not by direct measurement but by calculation from results obtained by testing the actual resistance of a solution whose specific resistance is accurately known. ELECTRICAL CONDUCTIVITY, DISSOCIATION, IONIZATION 19 obtain figures relating to the same quantity of salt at each dilution. If we multiply the specific conductivity by the volume of solution in c.c. which contains 1 gram-equivalent (see page 22), a value will be secured which represents the conducting power of a gram-equivalent. This is known as the equivalent or molecular conductivity* and is represented by the sign A. When it is determined for progressively diluted solutions, A gradually increases, indicating that the efficiency of the electrolyte itself as a conductor increases with dilution, because it dissociates more. The extent of this increase is found to become less and less as dilution proceeds. By plotting the values of the molecular conductivity of suc- cessive dilutions as a curve, the value at infinite dilution can be ascertained by extrapolation. This value is represented by A a. Now, let us see how these facts bear out the theory of electrolytic dissocia- tion. According to this hypothesis the conductivity depends on the num- ber of ions (see page 17), and since it is at a maximum at infinite dilu- tion, the value A must represent the total number of ions that can be pro- duced by the dissociation of 1 gram-equivalent, and A that at some other dilution. If, therefore, we divide A by Ace we obtain a value (called a) which must represent the degree to which the electrolyte is ionized at the various dilutions at which A is measured. From what has been said re- garding the osmotic pressure of similar solutions, it is evident that the value a could also be calculated by finding the extent to which the de- pression of freezing point (A) is greater than would be expected from the number of dissolved molecules. As a matter of fact, it has been found that the two methods yield practically identical values for many substances, thus furnishing almost incontrovertible proof in support of the dissociation hypothesis. In the cases of weak acids and bases, it is possible to secure a value, called the dissociation constant (K), which represents the rela- tive values of a at all dilutions. Since the activity of acids and bases is dependent upon the number of H- and OH-ions, respectively, set free by dissociation, it follows that it must be proportional to K. It will be necessary, however, to postpone a further consideration of the application of this constant until we have studied mass action (page 23). Biological Applications. The practical value of a knowledge of the laws of electrical conductivity rests, not so much on any direct application that can be made of it in explaining physiological processes, as on the es- sentially important bearing which it has in enabling us to understand the nature and operation of other physicochemical laws. Without a clear com- prehension of the elemental laws of dissociation, it is impossible to con- sider such problems as those which concern the activities of enzymes (mass *In other words, the molecular conductivity is the specific conductivity divided by the number of gram-equivalents contained in 1 c.c. 20 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES action, etc.), the occurrence of electrical currents during the physiological activity of muscles, glands, and nerves, and the all-important question of the reaction or H-ion concentration of the body fluids. There are, however, several instances in which measurements of electrical conductivity and of dissociation have direct physiological value. The circu- lation time of the bloodflow through an organ can be determined by first finding the electrical resistance of a short piece of the vein of the organ, and then observing the change in resistance which is produced when the conductivity of the blood in the vein is altered by the arrival in it of saline injected into the artery. The interval elapsing between the injec- tion into the artery and the changes in resistance in the vein equals the circulation time (G. N. Stewart). The same investigator has used measurements by electrical conductiv- ity to study the passage of electrolytes out of the red blood corpuscles into the serum. Under normal conditions the blood serum has a certain elec- trical conductivity equal to that of a 0.9 per cent sodium-chloride solution. The conductivity of the defibrinated blood is only about one-half that of serum, because it contains corpuscles which are nonconductors and there- fore obstruct the free passage of the ions, just as a suspension of quartz powder in a sodium-chloride solution lowers the conductivity of the lat- ter. If anything occurs therefore to occasion a passage of the saline con- tents of the corpuscles through their walls into the serum, an increase in the electrical conductivity will be produced. The value of this method in the investigation of changes in permeability of the red corpuscles is de- pendent on the fact that such migration of electrolytes out of the cor- puscles may occur before any of the less diffusible hemoglobin itself has escaped. The rise in conductivity precedes the hemolysis (see page 7). Although determinations of the specific conductivity of blood and urine under various pathological conditions have also been made, the results have not been found to possess any diagnostic value or clinical signifi- cance. Measurements of the electrical conductivity of blood have, how- ever, been used by Wilson 7 and by Priestley and Haldane 8 to detect the degree of dilution when large quantities of water are ingested. Another application of conductivity measurements in biochemistry has been made in studying the digestive action of proteolytic enzymes (Ba} r - liss). The general action of the enzymes is to break the large undisso- ciated molecules of the higher proteins (albumin, casein, etc.), into smaller molecules (amino acids, etc.), which are partly ionized. As diges- tion proceeds, therefore, the conductivity of the digestion mixture pro- gressively increases, and is a measure of the rate of digestion. Applications of the dissociation hypothesis in physiology concern the explanation of such phenomena as the production of electric currents ELECTRICAL CONDUCTIVITY, DISSOCIATION, IONIZATION 21 during muscular, glandular, and nervous activity. The exact details of the application are not as yet sufficiently understood to warrant our at- tempting to do more than indicate the general lines along which the problems are being investigated. To do so we must delve a little further into physicochemical research, when we shall find that there are two further facts concerning ionized molecules that must be of importance in connec- tion with our problem. The first is that the contribution which each ion makes to the equivalent (or molecular) conductivity of a solution is inde- pendent of the other ion with which it is associated; and the second, that ions differ considerably in their conducting power. Since the univalent ions, K., Na., CL', N0 3 ', carry charges of equal magnitude,* and yet all do not conduct to the same degree, they must move at different velocities through the solution. We are driven, therefore, to the conclusion that, exposed to the same electrical force, different ions have different mobili- ties ; that is to say, when an electric current passes through a solution of an electrolyte, the positively charged ions move towards the cathode at a different rate from that at which the negatively charged ions move towards the anode. Confirmation of this conclusion is obtained by exam- ination of the concentration changes around the two electrodes of an electrolytic cell. The actual velocity of each ion can be determined by experimental means. The inequality in concentration of ions in different regions of a tissue is no doubt the fundamental cause for the electrical currents that are set up by injury and activity. *Thus Faraday showed that the amounts of the various ions liberated by electrolysis are in the same ratio as their chemical equivalents. I I/ e^x^U * CHAPTER IV THE PRINCIPLES INVOLVED IN THE DETERMINATION OF THE HYDROGEN-ION CONCENTRATION TITRABLE ACIDITY AND ALKALINITY All acids have one property in common namely, that they contain hydrogen and when the acid becomes neutralized, it is this element which becomes replaced by some other cation. Evidently, then, the strength of an acid is proportional to the number of displaceable hydro- gen atoms which it contains. It may contain other hydrogen atoms which are so bound up in the molecule that they do not become displaced when an alkali is mixed with the acid. For example, in organic acids like acetic, CH 3 COOH, it is only the H atom attached to the COOH group, but not those attached to the CH 3 group, that is replaceable. It must therefore be possible to prepare for every acid a solution having exactly the same neutralizing power as that of any other acid; that is, the same volume of solution must be required in each case to neutralize a given quantity of alkali, the point of neutralization being judged by the change in color of indicators. As a standard a gram-molecular solu- tion of an acid with one displaceable H ion, such as hydrochloric, is chosen. This we call a "normal acid" (N). To prepare a normal solu- tion of acids having two displaceable H atoms, such as H 2 S0 4 , we can not however use a gram-molecular quantity, but must take one-half of it; and similarly in the case of those with three H atoms, such as H 3 P0 4 , a one-third gram-molecular solution will be a normal acid solution. For practical purposes, use is very generally made of solutions that are some fraction of the normal, e. g., tenth or decinormal (written N/10), or hun- dredth or centinormal (N/100). In a similar way, alkaline solutions can be prepared, a normal alkali being one which exactly corresponds in strength with a normal acid (i.e., can exactly neutralize it). Now, the characteristic of alkalies is that they produce in solution "OH" or hydroxyl ions; so that the process of neutralization must consist in the union of the H ions of the acid with the OH ions of the alkali to form water: KOH + HC1 = KC1 +H 2 0. We can, therefore, prepare normal solutions of alkalies by dissolving in 1 liter of water such quantities of alkali (in grams) as will yield the OH required to react with the available hydrogen in normal acid solutions. 22 HYDROGEN-ION CONCENTRATION 23 Actual Degree of Acidity or Alkalinity. According to the foregoing method of titration a normal solution of a powerful mineral acid, such as hydrochloric, is no stronger than a normal solution of a weak acid, such as acetic or lactic. It requires no fewer c.c. of N alkali to neutralize it. But the normal solution of the powerful acid is more acid to the taste, is more toxic, dissolves metals more readily, and in all its other chemical and physiological properties acts much more quickly than the weak acid, so that the titrable acidity or alkalinity can not express the real strength of the acid or alkali, or the actual degree of acidity or alkalinity. It is in this connection that the dissociation hypothesis aids us, for it suggests that the degree to which the acid becomes dissociated into H- and the remainder of the molecule will determine its real strength (see page 16). The question is, how are we to measure the latter? One action of H ions which we may measure is that known as catalytic that is, the power to accelerate reactions, such as the splitting of cane sugar (C^H^On) into glucose and levulose, which otherwise would proceed very slowly (see page 75). If then the real strength of an acid depends on the degree of dissociation which it undergoes, figures representing the catalytic power should correspond with those representing the relative conductivi- ties of the acids in equivalent concentration (see page 19). That this is actually the case is shown in the following table, in which the above values of various acids are given compared with HC1, which is taken as 100. ACID CATALYTIC POWER RELATIVE CONDUCTIVITY HC1 100 100 Dichloracetic 27 25 Monochloracetic 4.8 4.9 Formic 1.5 1.7 Acetic 0.40 0.42 It will be evident that, if we could measure the concentration of free H ions in a solution that is, of H ions that are not matched by OH ions we should have a faithful index of its real acidity. This measurement has been rendered possible by the application of two other physico- chemical principles namely, those of mass action and electromotive force. Since the object of this volume is to present the scientific basis for the various methods that are used in modern medicine, it will be nec- essary for us to review the main principles of these two actions. We shall see that they apply, not only in the measurement of H-ion concentration, but in many other physiological processes. Mass Action When materials take part in a reaction, some molecules are decom- posing while others are being formed. After some time, however, a 24 PHYSICOCHKMICAL BASIS OF PHYSIOLOGICAL PROCESSES condition is reached in which the changes in one direction are exactly offset by those in the other. An equilibrium is said to have become estab- lished between the reacting substances. Bearing in mind that the ions and molecules entering into these reactions are constantly moving about and coming in contact with one another, it is easy to see that if we were to add an additional quantity of one kind of molecule or ion, there would be a change all along the line until a new equilibrium was established. If, on the other hand, we were to remove one kind of molecule or ion as fast as it was formed, the equilibrium could never be established, and the reaction would proceed until all of this material had disappeared. The natural rate at which any chemical reaction proceeds is dependent upon a number of conditions, such as chemical affinity, temperature, catalysis, and concentration. Of these conditions that of concentration is most readily measured. If we maintain all of the conditions other than that of concentration unchanged, and designate this combined in- fluence as K (constant), we shall find that the speed of the reaction is proportional to the molecular concentration of the reacting substances (i. e., the number of gram-molecular weights per liter). In other words, the speed with which two substances, a and b, unite to form other sub- stances, c and d, will be expressed by the equation, k (a)x (b) ? k' (c) x(d)j* which means that, when the reaction is complete, the composition of the mixture will be dependent upon the ratio between k and k'. Since however these are both constants, their quotient is also constant (K), and (a) x (b) we have the equation, -~^ ~~ = K, indicating that no matter how (c) x (d) the concentrations a, b, c, and d are varied, reaction will take place in one direction or the other until the concentrations have become adjusted so that K remains unchanged. As an example of the application of these laws, let us take the reaction which occurs between alcohols and organic acids to form the substances called esters a reaction which is analogous to that between mineral alkalies and acids to form neutral salts, and which is of special interest to us because it is the reaction involved in the splitting of animal fats. The equation for the reaction is: C 2 H 5 OH + CHsCOOH <=* C 2 H B OOCCH 3 + H 2 O. (ethyl (acetic (ethyl acetate, alcohol) acid) an ester) Or expressed according to the law of mass action: [C 2 H 5 OH] x [CH 3 COOH] [C a H 6 OOCCH 3 ] x [H.O] : *The brackets indicate that gram molecular quantities are used. HYDROGEN-ION CONCENTRATION 25 Now it is clear that if we increase, say, H 2 in the above equation, then in order that K may remain unchanged C 2 H 5 OOCCH 3 must diminish or the substances which form the numerator of the equation must increase, or both these changes must occur. As a matter of fact, in such a case as the above, both of these adjustments take place, for, as the ester breaks down, it must thereby increase the concentration of acid and alcohol. Since in aqueous solutions the reaction occurs in the presence of an excess of water, it is evident that the tendency for an ester is to break down into alcohol and acid, and this must occur in all reactions in the body fluids in which water enters into the equation. Physiological Applications. The application of the law of mass action in the explanation of biochemical processes is very extensive. Most of the reactions which enzymes or ferments are capable of influencing are of the same general nature as that represented above, and the products of their activities are usually the substances on the side of the equation in which no water molecules appear i. e., they are hydrolytic reactions. Enzymes merely accelerate the reaction (page 72), so that if we start with a mixture of the substances on either side of the equation, all they do is to accelerate the production of a sufficient concentration of those on the other side, until the equilibrium point is reached. For example, an enzyme present in pancreatic juice, called lipase, accelerates the breakdown of such esters as neutral fat, which consists of the triatomic alcohol, glycerol, combined with the fatty acids palmitic (C 15 H 31 COOH), stearic (C 17 H 35 COOH) and oleic (C 7 H 33 COOH): C S H 5 (O 00 C 1T H M ), + 3H,0?3C 1T H M COOH + q,H II (OH),. (the neutral fat, (the fatty acid, (glycerol) tristearin) stearic) Under ordinary conditions the reaction proceeds until nearly all the neutral fat has become decomposed because of the preponderance of water, but if we start with a mixture of fatty acid and glycerol with just enough water to permit the enzyme to act, the reaction will pro- ceed in the opposite direction i. e., so that some neutral fat will be synthesized. This is called the reversible action of enzymes (page 77). Because of the universal presence of water, it is plain that such re- versible reactions could not alone be held responsible for the synthe- sis of neutral fat or of similar substances in the animal body. The only way by which synthesis could occur under these conditions would be if the substance produced along with the water were removed from the site of the reaction as soon as it was formed. This might occur by the precipitation of the substance or by its becoming surrounded by an en- velope of some inert material. In the synthesis of neutral fat which 26 PHYSICOCHKMICAL BASIS OF PHYSIOLOGICAL PROCESSES occurs in the epithelium of the intestine out of the fatty acid and glycerol absorbed from the intestinal contents, it is possible that the last men- tioned process occurs. In other cases the substance may be carried away by the blood or lymph or urine as fast as it is formed. The Law of Mass Action as Applied to the Measurement of H-ion Concentration. Let us now return to the reaction or H-ion concentration of substances in solution. As the standard of neutrality, pure water is chosen. Let us consider, then, how the laws of mass action can be applied in order to enable us to determine the H-ion concentration of pure water. It has been stated above that chemically pure water is in- capable of conducting the electric current. This however is not strictly the case, for it conducts to a very slight degree. According to the dis- sociation hypothesis, it must therefore be represented as containing molecules of H 2 and ions of H and OH', and according to that of mass action there must be a balanced reaction between the molecules and ions represented thus: . Since the concentration of H- and OH' is extremely small, there must always be such an overwhelming preponderance of H 2 molecules that no changes in the concentration of H and OH' will be capable of appre- ciably affecting the concentration of H 2 ; in other words, one may omit the denominator of the equation and write it [H ] x [OH'] =K. If then we know the value of K, it will only be necessary to measure the concentration of either H or OH' in order to express in numerical terms the reaction of the solution. It has been found that the value of K is about 1 x 10' 14 ,* and since the concentrations of H and OH' are nec- essarily equal in pure water, it follows that [H] = [OH] = Ix 10~ 14 , i. e., each ion has a concentration of 1 x 10' 7 . This means that water con- tains approximately 1 gram mol. each of H- and OH' ions, or 1 gram H- and 17 grams OH' ions, in 10+ 7 or 10,000,000 liters. A consequence of the above law is that no matter how much the concentration of one ion is increased by adding another substance, the solution must still contain some of the other ion. Thus, in acid solutions the concentration of H must increase and the concentration of OH' must decrease in such pro- portion that the two multiplied together equals about 1 x 10~ 14 . The H-ion concentration can be used therefore to express the reaction of neutral, acid and alkaline solutions. In place of water, let us substitute decinormal hydrochloric acid *The sign 10- 14 is simply a convenient way of expressing the degree of dilution. It gives the number of times the value standing in front of it must be divided by 10 in order to find the concentration. HYDROGEN-ION CONCENTRATION 27 (0.1 N Hd) that is, a hydrochloric acid solution containing one tenth of the molecular weight of hydrochloric acid in grams dissolved in a liter of water. At this dilution HC1 is 91 per cent dissociated ; therefore the H-ion concentration (or C H as it is written for short) is 0.091 N, or, in mathematical notation, 9.1 x 10 2 . Method of Expressing C H . To avoid the necessity of having to use several figures to express C H , as has been done above, Sorenson has intro- duced a scheme by which only one figure is required. This figure, des- ignated by P H , is found by subtracting from the power of ten (i. e., the figure standing behind 10) the common logarithm of the figure ex- pressing the normality of the acid. In a decinormal HC1 solution, therefore, we must subtract from the power 2, the common log. of 9.1, which is .96 (ascertained from logarithm tables), leaving 1.04. . Take another example: decinormal acetic acid is dissociated only to the ex- tent of 1.3 per cent ; C H is therefore 0.0013 normal, or 1.3 x 10' 3 . Since the logarithm of 1.3 is .11, P H equals 3-.11, or -2.89.* The only objection to the use of the exponent P H as an expression of the H-ion concentration is that it increases in magnitude as the acidity becomes less; this is because the negative sign of the power is disre- garded. As stated above, it is usual to express the strength of alkalies as well as acids in terms of C H , or P H , because it is easier to measure the concentration of H ions than of OH ions. A 0.1 NaOH solution is 84 per cent dissociated; therefore the "OH" ion is 0.084 N (i. e., 0.084 gram equivalents OH per liter), and since the product of the H- and OH' concentrations must always equal 10' 14 - 14 (at 20 C.), it is clear that as the H ion increases in concentration, the OH ion must reciprocally de- crease. Expressed according to the above scheme, the 0.084 N NaOH solution gives P H 13.06; thus, 0.084 = 8.4 x 10' 2 ; the log. of 8.4 is .924, and this subtracted from the power -2 = 1.08 as POH, or 14.14 - 1.08 = 13.06 as P H .** Similarly, P H of 0.1 N NH 4 HO solution is 11.286. Its dissociation is 1.4 per cent; therefore the solution contains only 0.0014 gram equivalents HO i.e., 1.4 xlO- 3 P OH = 3 - 0.146 = 2.854 .-. P H 14.14-2.854 = 11.286.f *If we wish to express the value of PH in ordinary notation, we must find the antilogarithm of the difference between the value of PH and the next higher whole number; e. g., if PH = 7.45, the antilogarithm of 0.55 being 3.55, the CH is 3.55 x 10" 8 , or 0.000,000,0355 N, or 3.55 gm. mol. H ion in 100,000,000 liters. **It must be remembered that the power of a number indicates the number of times by which that number must be multiplied by ten; thus, Pn- 6 does not mean that the H ion is six times less than PH, but 1 x 10 x 10 x 10 x 10 x 10 x 10, or 1,000,000 times less. Similarly, Pn' 3 is 1000 times as great as Pa- 6 , not twice as great. A solution containing almost exactly 1 gram molecule of dissociated hydrogen in 10,000,000 fiters constitutes a neutral solution (Pn = 7). tThe expressions PH and CH may be used indiscriminately, but when the numerical value is given, it is most convenient to use the former. 28 PHYSICOCHEM1CAL BASIS OF PHYSIOLOGICAL PROCESSES Application of the Law of Mass Action in Determining the Real Strength of Acids or Alkalies. We have seen that it is the degree of dissociation upon which the real strength of an acid depends and that this varies with dilution (page 19). The equilibrium between the un- dissociated and dissociated molecules may therefore be shifted in either direction by changing the concentration; in other words, the process of dissociation is a reversible reaction, and may be represented as AB + A' + B . The law of mass action must apply in such a case, and as a matter of fact it has been found that a constant can be calculated, which is known as the dissociation constant.* It is an expression of the inherent ability of the acid to dissociate into ions, and is therefore the best measure of the strength of the acid. This is strictly the case for all of the weaker acids, but strong mineral acids (and bases) do not give a satisfactory constant, so that the comparison must not be made between them and weaker ones. That the dissociation constant expresses the rela- tive strength of organic acids can be shown by comparing its value with that of the rate at which cane sugar is inverted (see page 23), this being proportional to the concentration of the H ions present. K for some or- ganic acids is : Acetic, 0.000018 ; Formic, 0.000214 ; Benzoic, 0.00006 ; Sal- icylic, 0.00102. *The equation is yr yy- = K, where it is supposed that in volume V of the solution there is 1 gram-equivalent of electrolyte, and that the degree of dissociation is a; the quantity of undis- sociated electrolyte stated in a fraction of a gram-equivalent will be 1-a, and the quantity of each ion a. To illustrate, let us take acetic acid in various dilutions: V a K x 10 5 0.994 0.004 1.62 2.02 0.00614 1.88 15.9 0.0166 1.76 18.1 0.0178 1.78 CHAPTER V THE PRINCIPLES INVOLVED IN THE MEASUREMENT OF THE HYDROGEN-ION CONCENTRATION (Cont'd) THE METHODS OF MEASUREMENT The Electrical Method In order to understand the principle of the standard method used for measuring the H-ion concentration, it is necessary that a few words be said concerning the factors governing the development of electric cur- rents in chemical batteries. There may be a further application of this knowledge in connection with the generation of the electric currents which occurs during physiological activity, as in active glands and muscles. When a metal is immersed in a solution of one of its salts, it has a tendency to give off ions into the solution. Similar ions are, however, already present in this solution, and these, by their osmotic pressure, tend to oppose the passage of the ions coming from the metal. The force with which the metal sends out its ions into the solution is called the electrolytic solution pressure. If this is equal to the osmotic pres- sure of the metallic ions in the solution, there will be no electric current generated, but if it is greater or less than the osmotic pressure of the metallic ion, an electric current will be set up. When the solution pres- sure is the greater, the metal will become negatively charged, because its ions carry positive charges into the solution (cations) ; on the contrary, when the osmotic pressure is greater than the solution pressure, the metal will have a positive charge, owing to the receipt of the positive cations from the solution. Because of a force called electrostatic attraction, the ions given off from the metal can not travel any measurable distance from the oppositely charged mass of metal, so that from one of the electrodes alone it is impossible for us to lead off any electric current. For this purpose we must form a circuit. This is done in the manner shown in Fig. 7 by connecting side tubes coming from the electrode vessels with an inter- mediate vessel containing a solution of high conductivity and by con- necting the metals by wires. If the circuit is formed between the same metals in solutions of the same concentration, no electric cur- rent will be generated, because the two electrode potentials will be 29 30 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES equal and in opposite directions to each other. On the other hand, should the concentration of the metallic ion in the solutions be unequal, the electromotive force will flow from the one electrode to the other, and the pressure with which it flows will be equal to the difference in con- centration of the two solutions. This is the principle of a concentration cell, and if we know the concentration of one of the solutions composing it, and then proceed to measure the electromotive force, we can obtain the concentrations of the other solution by difference. To do this we must employ a formula which takes into consideration the relation be- tween the potential and the concentration of the solution. The potential of an unknown electrode composed of a metal in con- tact with a solution of one of its salts may also be determined by making it one pole of a battery of which the other pole is composed of a stand- ard electrode of unchanging known potential. An electrode of the latter Fig. 7. Diagram to show type of electrodes used in studying electromotive force. The metal in each electrode is connected (through a glass tube) with a platinum wire, to which the appai-atus for measurement of the voltage is connected. The metal dips into a solution contained in the electrode vessel and filling the side tube. The latter dips into an inter- mediate vessel containing saturated KC1 solution. The currents flow through the circuit under the following conditions: (1) dissimilar metals dipping into the same fluid; (2) similar metals dipping into different fluids; (3) dissimilar metals dipping into different fluids. type can most readily be made by bringing pure mercury in contact with a saturated solution of calomel (Hg 2 Cl 2 ) in normal potassium chlo- ride solution (Fig. 8). Under suitable conditions (i. e., when the circuit is completed), a potential of 0.560 v. is developed in this so-called calomel electrode* that is, positive ions of mercury are deposited on the mercury from the calomel solution at this pressure. Suppose that we connect a calomel electrode, through the intermediation of some solution which *The calomel electrode consists of a suitably shaped glass vessel containing pure mercury, con- nected by means of a platinum wire with a conductor, and filled with a saturated solution of pure mercurous chloride in normal KC1 solution up to such a level that it also fills a side tube connected with a vessel containing a saturated solution of potassium chloride. Into this vessel also runs a similar side tube from the unknown electrode. By having an excess of utidissolved calomel in the solution in the calomel electrode its saturated condition is maintained during the chemical changes which accompany the production of the electric current. HYDROGEN-ION CONCENTRATION 31 will serve as a good conductor, with another electrode, the two elec- trodes being also connected by wires with electrical apparatus for measuring the total potential of the battery; then by adding +0.560 v. to or subtracting this value from the total potential (depending on the sign of the unknown electrode) we can tell the potential of the unknown electrode. We have discussed these principles of electrochemistry because they form the basis upon which depends the standard method for the deter- mination of the H-ion concentration of fluids. Suppose, for example, that in place of using a metal in the construction of one electrode, we use an electrode consisting of a layer of pure hydrogen gas in contact with a solution in which are free H ions; then the rate at which H ions Fig. 8. Diagram of apparatus for the measurement of the H-ion concentration. The cur- rent generated in the battery (composed of calomel electrode, connecting vessel with KC1 solu- tion, and the H electrode) or that from the normal element is transmitted through a reversing key to the bridge wire, where the voltage is compared with a steady current flowing through the bridge wire from an accumulator. The capillary electrometer is used to detect the flow of current at various positions of the movable contact on the bridge wire. (Modified from Sorensen.) become added to the solution from the H layer, or taken from it, will de- pend on the concentration of H ions in solution. In order to secure a hydrogen electrode fulfilling the above requirements, it is necessary to employ some means by which a layer of hydrogen may be furnished, and fortunately this can be done by taking advantage of the property which spongy platinum possesses of absorbing large quantities of this gas. It is also necessary to keep an atmosphere of pure H in contact with the fluid. As is the case of the simpler cells described above, there are two types which we might use for measuring the electromotive force gen- erated in the unknown electrode: a concentration cell composed of two 32 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES hydrogen electrodes, of which one contains a solution of known H-ion concentration, and the other the solution in which this is unknown; and a cell of which one electrode is a standard calomel electrode and the other, a hydrogen electrode containing the unknown solution. The exact arrangement of the apparatus in which the calomel elec- trode is used will be seen in the accompanying sketch. The hydrogen electrode, it will be noticed, is a very small V-shaped tube, in which is suspended a platinum wire coated with spongy platinum and dipping into a solution which nearly fills the tube. The space above the solution is filled with pure hydrogen. This and the calomel electrode are con- nected with suitable electric measuring instruments, and the circuit is completed by connecting the two electrodes by means of an intermediate vessel containing a saturated solution of potassium chloride. This con- necting solution is used because it has been found that the electric cur- rents set up at the contact between different solutions are so small that they can be disregarded.* As outlined above, the hydrogen electrode is that which is used to determine the H-ion concentration of blood, the particular point about it, in comparison with the apparatus used for simpler solutions, being that the hydrogen is not changed in the course of the experiment. This precaution is to prevent the carbon dioxide of the blood from being " washed out'* of it by a frequently changing atmosphere of hydrogen. Many inaccuracies in the earlier results obtained by this method were due to the removal of carbon dioxide, which, as we shall see later, is one of the chief acids contributing to the H-ion concentration of blood. The Indicator Method As pointed out in a previous chapter (page 22), the method of titra- tion for acidity or alkalinity in which a standard solution of alkali or acid is added until a certain change in the color of a suitable indicator is detected, does not afford any information regarding the H-ion con- centration actually present in the solution. It tells us the total con- centration of available acid or base, both dissociated and undissociated. By modification of the method of procedure, however, we may also use indicators for determining the H-ion concentration. The principle of this method depends on the fact that there are certain dyes which change quite distinctly in tint with very slight changes in the H-ion concentration, so that if we use dyes which possess this property at a point near that of neutrality (i. e., between P H 6.5 and P H 8), we can es- *A description of the technic for measuring the electric potential developed by the cell would be out of place here. Suffice to say that the strength of the current is compared with that of a current of known strength furnished by a normal cell, the comparison being made by a bridge wire F, a capillary electrometer H being employed to detect the direction and degree of current. HYDROGEN-ION CONCENTRATION 33 timate the H-ion concentration of the body fluids with very remarkable accuracy, provided certain precautions are taken to circumvent the disturbing influence which the protein and salts in these fluids may have on the color change. To understand this use of indicators, it is important to bear in mind that one solution reacting neutral to one indicator may have a H-ion concentration which differs very markedly from that of another solu- tion reacting neutral to another indicator. This is because indicators react to different H-ion concentrations. A solution that is neutral to phenolphthalein has a PH of about 9, whereas one neutral to methyl or- ange has a P H of about 4. This can be very clearly shown by titrating a solution of phosphoric acid with decinormal alkali. After a certain amount of alkali has been added it will be noticed that methyl orange changes from red to yellow, but after it has changed and is therefore alkaline as judged by this indicator, it still remains distinctly acid to- wards phenolphthalein (shows no red tint) even though considerably more alkali is added. The methyl orange is, therefore, itself unrespon- sive to weak acids such as remain after the greater part of the phos- phoric acid has been neutralized by the alkali. The series of indicators which has been employed for this purpose is given in the accompanying table, along with the P H limits through which they change in color. LIST OF INDICATORS CHEMICAL NAME COMMON NAME CONCEN- TRATION COLOR CHANGE RANGE PH Thymol sulfon phthalein per cent (acid range) Thymol blue 0.04 Red-yellow 1.2-2.8 Tetra bromo phenol sul- fon phthalein Brom phenol blue 0.04 Yellow-blue 3.0-4.6 Ortho carboxy benzene azo di methyl aniline Methyl red 0.02 Red-yellow 4.4-6.0 Ortho carboxy benzene azo di propyl aniline Propyl red 0.02 Red-yellow 4.8-6.4 Di bromo ortho cresol sulfon phthalein Brom cresol purple 0.04 Yellow- purple 5.2-6.8 Di bromo thymol sulfon Brom thymol phthalein blue 0.04 Yellow-blue 6.0-7.6 Phenol sulfon phthalein Phenol red 0.02 Yellow-red 6.8-8.4 Ortho cresol sulfon phthalein Cresol red 0.02 Yellow-red 7.2-8.8 Thymol sulfon phthalein Thymol blue 0.04 Yellow-red 8.0-9.6 (see above) Ortho cresol phthalein Cresol phthalein 0.02 Colorless-red 8.2-0.8 These dyes may now be obtained in this country. (W. M. Clark and H. A. Lnbs.) 9 34 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES Briefly stated the method for measuring the H-ion concentration con- sists in preparing a series of solutions containing known concentrations of H-ion that is to say, of known P H and adding to each solution an equal amount of an indicator which exhibits easily distinguishable changes in tint at H-ion concentrations approximating those believed to be present in the unknown solution. The same indicator is added to the unknown solution, which is then placed side by side with the stand- ards to find with which of them it most closely matches. The series of solutions of known H-ion concentration is prepared by mixing fif- teenth normal solutions of Na 2 HP0 4 and KH 2 P0 4 in varying propor- tions as given in the following table: PREPARATION OF STANDARD SOLUTIONS The solutions are mixed in the proportions indicated below to obtain the desired PH:* PH 6.4 6.6 6.8 7.0 7.1 7.2 7.3 7.4 7.S 7.6 7.7 7.8 8.0 8.2 8.4 Primary Potas. 73 63 51 37 32 27 23 19 15.8 13.2 11 8.8 5.6 3.2 2.0 Phos., c.c. Secondary Sodium 27 37 49 63 68 73 77 81 84.2 86.8 89 91.2 94.4 96.8 98.0 Phos., c.c. (From Levy, Rowntree and Marriott.) *Standard phosphate mixtures are prepared according to Sorensen's directions as follows: 1/15 mol. acid or primary potassium phosphate. 9.078 grams of the pure recrystallized salt (KH2PO4) are dissolved in freshly distilled water and made up to 1 liter. 1/15 mol. alkaline or secondary sodium phosphate. The pure recrystallized salt (Na 2 HPC>4. 12H 2 O) is exposed to the air for from ten days to two weeks, protected from dust. Ten molecules of water of crystallization are given off and a salt of the formula! NaoHPO 4 .2H 2 O is obtained; 11.876 grams of this are dissolved in freshly distilled water and made up to 1 liter. The solution should give a deep rose red color with phenolphthalein. If only a faint pink color is obtained, the salt is not sufficiently pure. The indicator method is extremely accurate when used with pure solutions of acids, but, as mentioned above, it is apt to be inaccurate, at least with most indicators, when protein or inorganic salts are pres- ent in the solution, and of course it is quite unusable with colored fluids such as blood. In order to overcome these difficulties, the dialysis method has recently been evolved. It consists in placing the fl u id blood, for example in a dialyser sac composed of celloidin and about as large as a small test tube. The sac is placed in a wider test tube of hard glass containing an isotonic solution of sodium chloride that has been carefully tested to ascertain that it is strictly neutral. The amount of blood or serum required for this method is only 2 or 3 c.c., and the amount of salt solution placed outside the sac should be about the same. It takes only from five to ten minutes for dialysis to occur. The celloidin sac is then removed, a few drops of the indicator are thoroughly mixed with the dialysate, and the tube compared with the series of standards until the corresponding tint is matched. This indicates the H-ion concentration in the dialysate. The tints produced by using sulphonephenolphthalein are reproduced as nearly as possible P H 7-0 P H 7-/ P H 7-2 P H 7-3 P U Fig. 9. Chart showing approximately the tints produced by adding sulphophenolphthalein to a series of phosphate solutions of the H-ion concentrations indicated in each case by PH. HYDROGEN-ION CONCENTRATION 35 in the accompanying chart. The H-ion concentration of the unknown solution is that of the tint with which it matches in the series. It might be thought that this method would be inaccurate because of the loss of carbon dioxide from the blood. By actual experiment, how- ever, it has been found that, if the blood is collected with certain pre- cautions, the error is negligible. The method is, therefore, a most useful one clinically. To minimize the chances of loss of free C0 2 , S. H. Dale and Evans have elaborated the technic of the dialysis method by tying the sac onto an ebonite perforated stopper which exactly fits the mouth of the test tube containing the neutral saline solution. The blood is delivered directly from the blood vessel by a narrow glass tube which passes to the bottom of the sac through the hole in the stopper. After enough blood has been collected the tube is withdrawn and the hole in the stopper is tightly closed by a piece of cork. We have found this a useful method but we do not believe that the further modification described in their paper for determination of the P H of the dialysis is as reliable as that described above. 20 The following table gives the hydrogen-ion concentration or true reaction of the body fluids. FLUID PH FLUID PH Blood 7.4 Urine 6.0 Saliva 6.9 Gastric juice (adult) 0.9-1.6 Gastric juice (infant) 5.0 Pancreatic juice (dog) 8.3 Small intestinal contents 8.3 Small intestinal contents (infant) 3.1 Bile from liver 7.8 Bile from gall bladder 5.3-7.4 Perspiration 7.1 Perspiration 4.5 Tears 7.2 Muscle juice (fresh) 6.8 Muscle juice (autolyzed) Variable Pancreas extract 5.6 Peritoneal fluid 7.4 Pericardial fluid 7.4 Aqueous humor 7.1 Vitreous humor 7.0 Cerebrospinal fluid (fresh) 7.4 Cerebrospinal fluid (after standing) 8.3 Amniotic fluid 7.1 Amniotic fluid 8.1 Milk (human) 7.0-7.2 Milk (cow) ' 6.6-6.8 Milk (goat) 6.6 Milk (ass) 7.6 (W. M. Clark and H. A. Lubs.) a TER VI THE REGULATION OF NEUTRALITY IN THE ANIMAL BODY *~AND ACIDOSIS Nothing is more constant in the animal economy than the H-ion con- centration (C H ) of the fluids which bathe the tissues. This regulation is fundamentally of a physicochemical nature, depending on the inter- action of alkalies with acids, of which c#tobo*iic and phosphoric acids are the most important.* When different amounts of acids or al- kalies are added to water, the rajige of variation in H ion is very extensive, whereas in blood the'range is very limited indeed, not extending beyond P H 7 and P H 7.52 (i. e., C H ^)ever goes above that of a 0.000,000,1 N solution or below that of a 0.000,000,03 N solution). In other words blood can withstand considerable additions of acid or al- kali without much change. Buffer Substances. The chemical reactions upon which this remark- able constancy in reaction depends have been explained by Lawrence J. Henderson. 10 The fundamental equations are as follows: M.HPO, + HA MH 2 PO 4 + MA, and MHC0 3 + HA = H 2 C0 3 + MA, when M = a basic radicle, and A, an acid radicle. Now it has been discovered that weak acids, like carbonic and phosphoric, possess the remarkable property of maintaining the reaction tolerably constant when they are present in a solution which also contains an excess of their salts. Under these circumstances the concentration of ionized hydrogen is almost exactly equal to the product of the dissocia- tion constantf of the acid (see page 19) multiplied by the ratio be- tween free acid and salt; in other words, [HA] If carbonic acid is present in a solution of bicarbonates so that there "Under certain circumstances, proteins may also act either as acids or as alkalies. They are therefore calhd amphoteric. The neutralizing properties of proteins are, however, of little conse- quence in the neutrality regulation in the animal body (Bayliss 20 ). tThe dissociation constant tms already been referred to as a figure which expresses the tendency of a weak acid or base to dissociate in an aqueous solution. "It expresses the proportion in which the nondissociated part is capable of existing in the presence of its ions," and therefore is a gauge of the strength. The dissociation constant amounts to about 0.000,000,5 for carbonic acid; that is, the dissociation of HoCOs into H'-f-HCO^' at room temperature will be such that the concentra- tion of H-ion equals a 0.000,000,5 N solution. 36 ACIDOSIS 37 are equivalent quantities of free H 9 C0 3 and bicarbonate i. e., | the H-ion concentration will be exactly the same as the dissociation con- stant of carbonic acid (i.e., 5 x 1O 7 ) ; therefore 0.000,000,5 N (P H = 6.3)*, or about five times the value of neutrality, 0.000,000,1 N (P H = 7). If ten times as much free carbonic acid as bicarbonate is present, then the H-ion concentration will be fifty times that of neutrality, i. e., x 0.000,000,5 = 0.000,005 (P H = 5.31) ; if there is ten times less carbonic acid than bicarbonate, the H-ion concentration will be one-half that of neutrality, i. e., 4wTT = T7i x 0.000,000,5 = 0.000,000,05) (P H = 7.31) ; or if twenty times less, one fourth (P H = 7.6). Since a large amount of bicarbonate is actually present in blood (enough to yield from 50 to 65 c.c. C0 2 per 100 c.c. of blood), and the free carbonic acid undergoes fluctua- tions which are only trivial when compared with those which have been chosen in the above examples, it is clear that there must be very little change in the H-ion concentration of the blood in comparison with the variations which would occur were no bicarbonate present. Another weak acid which acts like carbonic in maintaining neutral- ity is acid phosphate (MH 2 P0 4 ), and for the same reason namely, that its dissociation constant is of similar magnitude to the H-ion concen- tration. Although the blood plasma itself contains much less phosphate than bicarbonate, the tissues contain a considerable amount, which en- ables them to maintain their neutrality. This action of bicarbonates and phosphates is styled the buffer action, meaning that it serves to damp down the effect on the H-ion concentration which additions of acids or alkalies would otherwise have. As pointed out by Bayliss, however, a better word to use would be "tampon action,'* since the substances actually soak up much of the added H- or OH' ions. It is not confined to the fluids of the higher animals, but is very widely distributed throughout nature ; for example, in the ocean and in the fluids of marine organisms and animalcules (see L. J. Henderson). 11 Although the actual reaction by which neutrality is maintained is purely of a physicochemical nature, some provision must obviously be made so that the acid and basic substances that take part in it may be supplied and those produced by the reactions removed as occasion re- quires. The source of supply is partly exogenous and partly endogenous. The exogenous source is the basic and acid substances present in the food; and although we do not ordinarily attempt to control the amounts of these substances ingested, we may do so, as, for example, by the persistent administration of soda in cases of pathological acidosis. The endogenous source depends on the constant production in metabolism *For calculating this value see p. 27 38 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES of acids such as carbonic, phosphoric, lactic, and sulphuric, and of alkalies such as ammonia and fixed alkali, a considerable reserve of which is undoubtedly available in the animal organism. The removal is affected by three pathways: (1) through the lungs gaseous carbonic acid is eliminated; (2) through the kidneys, the fixed acids; and (3) through the intestines, some of the phosphoric acid. Carbonic acid is produced in large amounts in the normal process of metabolism, and is excreted in a gaseous condition by the lungs. Varia- tion in its excretion is the most important mechanism for controlling fl 'temporary changes in C H . In order to make this clear, it may be well to revert for a moment to the physicochemical equation by which carbonic acid is enabled to maintain neutrality. This may be written: C H = TT r*o molecular ratio ^-^r^ Tne rat i mav De increased either by adding JMaxlbUg free carbonic acid to the blood (as by causing an animal to respire some of the gas), or by the addition of some other acid (e. g., oxybutyric, as in diabetes) which will decompose some of the NaHC0 3 and produce H 2 C0 3 . The increase which these changes would cause in C H of the blood is prevented by the remarkable sensitivity of the respiratory cen- ter to changes in C H . An increase which is much less than can be measured by physicochemical means stimulates the center, causing in- creased pulmonary ventilation, so that the carbonic acid is immediately eliminated through the lungs. This elimination does not stop when the old level of carbonic-acid concentration is reached, but proceeds until TT p an( i C H consequently become greater, the condition is called uncompensated acidosis, but if the center does respond so that C H is held constant (although NaHC0 3 is decreased), the condition is one of compensated acidosis. For practical reasons, therefore, the study of pathological acidosis de- pends on an estimation of the bicarbonate content of the blood or, since it is simpler to carry out and is of equal value, of the plasma. When plasma is obtained by removing blood from a vein of the arm and cen- trifuged immediately out of contact with air (so that C0 2 may not be lost from it) it contains approximately 60 vols. per cent of C0 2 . Since we know that the partial pressure of C0 2 in blood is equal to 42 mm. Hg (ascertained from determinations of the alveolar C0 2 ) (see page 361), we can calculate how much of the 60 vols. per cent must be in simple solution by application of the law of solution of gas in a liquid (page 353'). One cubic centimeter of plasma at body temperature and at 760 mm. Hg (atmospheric pressure) dissolves 0.54 c.c. C0 2 , so that at 42 42 mm. it will dissolve 7^7- x 100 x 0.54 = 3 vols. per cent. Transcribing ToU [H 2 C0 3 ] 3 1 the figures to our equation we get [NaHC0 3 ] 60 20 This definition of acidosis leaves out of regard all conditions that may TT pr\ raise the ratio -^ rr f by the addition of H 2 C0 3 without decomposing JNaJtLvjUs any of the NaHC0 3 , such, for example, as occurs when an excess of free carbonic acid is present in the blood plasma. Since increases in free C0 2 are not infrequent in both health and disease e. g., asphyxial con- ditions the above definition is not sufficiently comprehensive. When we come to study the control of the respiratory center, we shall see that FT PO an increase in the ratio -A-^rf ^ sufficient magnitude to cause an actual increase in C H can be brought about by causing an animal to respire air containing an excess of C0 2 a true acidosis, but one for which no place is found in the above definition. *This agrees sufficiently with the result as calculated from the known values of the equation 2 Thus, if we take CH as 0.35 x 1Q- 7 , X as 0.605 for blood conditions, and K. s 4.4X10- 49.) K as 4.4X10- (Michaelis and Rona), we get ' = 0.605 x 0.35 x 10-' = 1 (gee a]go p 4.4 X 10 ' 21 40 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES Nevertheless, Van Slyke's definition has a real value, because it em- phasizes the importance of a determination of the bicarbonate as a cri- terion of the degree of the forms of acidosis usually met with in disease. The bicarbonate under such conditions may become reduced either be- cause of the appearance of improperly oxidized fatty acids, like /?-oxy- butyric and acetoacetic, when carbohydrate metabolism is upset as in diabetes or starvation, or because the acids produced by a normal metabolism are inadequately eliminated by the kidneys, as in nephritis. Accordingly, if the respiratory mechanism and increased mass move- ment of the blood (for an increase in C H accelerates this also) should TT pA fail to eliminate C0 2 quickly enough so as to keep the JL ^ ratio at one twentieth, then C H will rise. This is not likely to happen until a large part of the NaHC0 3 has been used up, so that an estimation of that actually present must be a reliable index of the proximity to this condition. A sustained increase in C H is incompatible with life. The NaHC0 3 is the buffer, the factor of safety which prevents its occurrence. Although it is only in arterial blood (i. e., after elimination of excess of C0 2 by the lungs has been accomplished) that constancy in the ratio NaHCO, can be expected, it is fortunate, for practical reasons, that venous blood collected during muscular rest and without stasis should be only slightly different. When acids are added to the blood, they will first of all be neutralized by the "buffers" of the plasma namely, NaHC0 3 (and protein), as we have seen. But this is only the first line of defense against acidosis, for buffer substances present in the corpuscles may also be used. This intra- corpuscular reserve of base is rendered available by transference of HC1 from the plasma into the corpuscle so releasing base in the former to combine with the added acid (e.g., H 2 C0 3 ), according to the equation: H 2 C0 3 + NaCl 5 NaHC0 3 + HC1. The HC1 on entering the corpuscle combines with alkali which it receives from the hemoglobin and also from phosphates according to the equation: HC1 + Na 2 HP0 4 ^ NaH 2 P0 1 + NaCl. This is a particularly important detail of the buffer action of the blood, not only because it shows us how the hemoglobin and phosphates of the corpuscles are rendered available for neutralizing acids added to the plasma, but also because the transference of acid must go on with the other cells of the body so that the plasma, itself rather poor in buffer substances, has all those of the body at its disposal. ACIDOSIS 41 THE MEASUREMENT OF THE RESERVE ALKALINITY OF THE BODY FLUIDS Titration Methods There are several methods by which the reserve alkalinity of the blood may be measured. The simplest in theory consists in seeing how much standard acid must be added to a measured quantity of blood plasma in order to reach the neutral point as judged by change in tint of some indicator. The indicators employed (e. g., methyl orange) are such as change their tints at H-ion concentrations that are well to the acid side of neutrality (i. e., at a high C H or low P H ). To bring the plasma to this point of neutrality the added acid will need to neutralize, not only the bicarbonate of the plasma, but other acid-binding substances as well. This will give us a false impression of the acid-binding powers of the plasma, since, at the normal C H of the blood, proteins do not absorb acids to anything like the extent they do at higher degrees of C H . Another objection to the method is that the proteins interfere with the sensitive- ness of the indicators. The objections can be removed by determining the end point electro- metrically or by indicators that change tint at about P H 7. The most practical way is to determine the change in C H produced by adding a known volume of standard acid to blood plasma. The resulting change in CH will then be greater the less the alkaline reserve. In the electro- metric method irregularities that might be caused by variable amounts of carbonic acid in the blood to start with are best controlled by removing the C0 2 from the plasma after adding the standard acid. The procedure therefore consists in mixing 1 c.c. plasma with 2 c.c. N/50 HC1 in a small separating funnel, which is then evacuated so as to remove the C0 2 , after which the fluid is transferred to a hydrogen electrode and CH measured (see page 29). In normal blood this should be 10 5 - 6 (P H 5.6). In acidosis, where there is a depleted alkaline reserve, the 2 c.c. of acid will cause a much greater change in C H in diabetic blood to below 5 or lower. The technic involved in the above method is, however, too exacting for routine clinical work. For such purposes the colorimetric method of Levy and Eowntree may be employed. The Method of Levy and Eowntree.is A test tube made of hard ("nonsol") glass of about 20 c.c. capacity, containing about a gram of powdered neutral potassium oxa- late, is filled with newly drawn blood, immediately stoppered and placed on ice. Quan- tities of 2 c.c. each of the blood are then placed in a series of seven small (nonsol) test tubes and allowed to stand for five to six minutes in order to permit a narrow layer of plasma to separate on the surface (this prevents laking of the blood during the sub- 42 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES sequent addition of acid or alkali). The blood in the first tube is used for the de- termination of the normal H-ion. In each of the next three tubes are added respec- tively 0.1, 0.2 and 0.3 c.c. N/50 HC1, and to the last three, similar quantities of N/50 NaOH. After inverting the tubes so as to mix the contents, the blood in each is trans- ferred to celloidin sacs and the C R determined according to the method described else- where (page 32). The tubes are noted in which a change in tint from that of the normal blood is evident, and the results are expressed as the c.c. of N/50HC1 or NaOH which must be added to blood to change its R . Thus, the alkali buffer is the c.c. of N/50 NaOH which can be added to 2 c.c. of blood without change of C H of the dialysate, and the acid luffer the c.c. of N/50 HC1. The method suffers from the following drawbacks: 1. Very small quantities of acid and alkali are employed. 2. It is often difficult to tell just exactly when a slight difference in tint has been produced. 3. Even with the precautions described above, it is impossible to be sure that the amount of CO in the different samples of blood is the same, which means, of course, that some bloods will, on this account alone, be able to bind more alkali than others. The Method of Van Slyke. A method based on somewhat the same principle, but which is more accurate because it meets the above objections, is that suggested by Van Slyke, Stillman and Cullen.i* Plasma is freed of CO 2 by placing it in a vacuum, and is then mixed with an equal volume of N/50 HC1 (or NaOH) and the C H deter- mined by the electric method (see page 29). In the case of normal blood, after such an addition of acid, a practically normal C H will be found, whereas in the blood of cases of acidosis it will be very distinctly increased (i.e., P H lower). Total CO, -combining Power The above objections to the titration of blood plasma or dialysate with standard solutions of acids are removed if we measure the com- bining power of the blood alkali towards carbonic acid itself at normal blood reaction. This may be done either in blood immediately after its removal from the animal or in blood that has been first of all saturated outside the body with carbonic acid at a partial pressure equal to that existing in the body. Since for practical reasons venous blood must be used in the clinic at least the former of these methods suffers from the fault that varying amounts of carbonic acid will be added to the blood during its passage through the tissues, and the error thereby incurred will become greatly aggravated if venous stasis has been pro- duced in drawing the specimen for analysis. But the chief reason why this method has not been extensively employed, as pointed out by Van Slyke, is the technical difficulty of making the necessary analysis. It is most satisfactory to collect venous blood after a period (one hour at least) of muscular rest (so that there is no excess of CO o ) and without venous stasis, and to centrifuge without permitting any considerable loss of carbonic acid. The latter precaution is necessary because there is a migration of acid radicles, e. g., HC1, from plasma into corpuscles when the CO o of the former is increased, and in the reverse ACIDOSIS 43 direction when the CO 2 is decreased. If the CO 2 in the blood were not the same dur- ing centrifuging as it is in the body, the separate plasma would not contain the same amount of alkali i. e., its reserve alkalinity would be altered. Although theoretically, therefore, centrifuging should be performed in an atmosphere containing the same partial pressure of CO* as exists in the body (i. e., the alveolar air) (see page 361), this has been found impracticable for general use, and is unnecessary if loss of CO 2 from the specimen of blood is prevented by allowing it to flow into the syringe very slowly (without any suction). It is mixed in the syringe with powdered (neutral) potassium oxalate (enough to make a 1 per cent solution with the blood), and imme- diately delivered into a centrifuge tube under paraffin oil, which by floating on its surface serves to diminish free diffusion of CO 2 to. the outside air (even though such oils dissolve more CO 9 than water). To mix the blood with the oxalate, the syringe should be moved backward and forward several times, but it must not be shaken. After centrifuging, about 3 c.c. of plasma are removed and saturated with CO 2 at the same tension as in alveolar air (i. e., 5.5 per cent). This is done by placing the plasma in a separating funnel of 300 c.c. capacity, laying the funnel on its side and displacing the air in it by alveolar air secured by quickly making as deep an inspira- Fig. 10. Diagram of apparatus for saturating blood or plasma with expired air. _ The glass beads in the bottle condense excess of moisture. The separating funnel, as soon as it has been filled with expired air, should be closed by a stopper and the stopcock turned off. It is then rotated so that the blood forms a film on its walls. tion as possible through the tube and bottle containing glass beads (Fig. 10). The glass beads remove excess of water vapor from the air. The funnel must be restop- pered before the end of the expiration, so that no outside air enters. It is then ro- tated, for about two minutes, in such a way that the plasma forms a film on its walls. If it is necessary to postpone the saturating of the plasma, this should me pipetted off from the corpuscles and preserved in hard glass test tubes coated with paraffin. From ordinary glass enough alkali is soon dissolved out to vitiate the results. After satura- tion of the plasma with CO^, the funnel is placed in the upright position and the plasma allowed to collect in "the narrow portion, after which 1 c.c. is removed with an accurate pipette and analyzed for CO . The analysis may be done by using either the Van Slyke or the Haldane-Barcroft apparatus. The Van Slyke method is as follows: The apparatus is filled to the top of the graduated tube with mercury (Fig. 11) by raising the mercury reservoir F, care being taken that D and E are also filled. One c.c. of the CO o -saturated plasma is then delivered into A (which has been rinsed out with CO 2 -free ammonia water), and the stopcock I turned so that by cautiously lowering the level of the reservoir F, the plasma runs into B (but no trace of air). 44 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES The same procedure is repeated with 1 c.c. water, so as to wash in all of the plasma/ arid finally 0.5 c.c. of 5 per cent H SO is sucked in, after which stopcock I is turned off. The reservoir F is then lowered sufficiently to allow all of the mercury, but none of the blood, to run out of B and C. A vacuum is thus produced in B and C. As the level of the mercury falls in B and C, the plasma effervesces violently,* be- cause it is exposed to a vacuum. To be certain that all traces of CO have been dislodged from the solution, the apparatus is inverted several times. To ascertain how much CO 2 has been liberated, stopcock II is now turned so as to bring C and E into communication, and by cautiously lowering the reservoir the fluid in C is allowed to run into the bulb E. Stopcock II is thereafter turned so as to connect C and D, and the reservoir raised so that the mercury runs into C as far as the CO that has col- Fig. 11. Van Slyke's apparatus for measuring the CDs-combining power of blood in blood plasma. For description, see context. lected in the burette will permit it to go. After bringing the level of the mercury ir. F to correspond to that in the burette, the graduation at which this stands is read. It gives the c.c. of CO liberated from the plasma. Under the above conditions normal plasma binds about 75 per cent of its volume of CO 2 ; therefore, since the total capacity of the pipette is 50 c.c., the mercury should stand at 0.375 c.e. on the burette. For accurate measurement it is necessary to allow for the CO 2 that remains dissolved in the water, etc., as well as for barometric pressure and temperature. This is best done by the use of a table based on the known solubility of CO., under the various condi- tions obtaining, which is given in 'van Slyke's paper. 12 *This may be prevented by adding a small drop of caprylic alcohol. ACIDOSIS 45 The Haldane-Bar croft apparatus that is most suitable for the above analysis is shown in Fig. 136, page 395.* One c.c. of CO 2 -free ammonia water is placed in the bottle and the 1 c.c. of plasma delivered beneath it. The bottle is then connected with the manometer with the precautions described elsewhere in this volume. When temperature conditions have been allowed for, saturated tartaric acid is mixed with the plasma solution and the gas evolved measured by the displacement of the fluid in the manometer. The apparatus may also be used with blood in place of plasma. In this case, however, it is necessary that the oxygen be removed before adding the tartaric acid. This precaution is necessary, since acid can dislodge some of the H 2 from hemoglobin. The blood is therefore first of all laked with ammonia containing some saponin, then shaken with 0.25 c.c. saturated potassium ferricyanide solution, and finally with the saturated acid solution. If blood is used, the estimations must be made on strictly fresh blood, since on standing the CO 2 combining power greatly de- teriorates. From what has been said in the introductory part of this chapter it is clear that the plasma furnishes only the first line of defense of the body against excess of acid; the corpuscles form the second line of defense, so that a much truer estimate of the ' ' reserve alkalinity ' ' is afforded when the C0 2 -combining power of whole blood rather than that of separated plasma is used. The C0 2 -combining power of the whole blood has been studied by Christiansen, Haldane and Douglas, 22 by Morawitz and Walker, 23 and more recently by Haggard and Henderson, 21 Van Slyke, 28 and Joffe and Poulton. 29 Another question remains to be considered, namely, whether arterial or venous Hood should be employed. For various reasons arterial blood is preferable. In the first place the percentage of C0 2 actually present in it is proportionate to the alkaline reserve, because the respiratory center is so sensitive to the slightest excess of this acid (see page 353) that it stimu- lates respiration so as to remove the excess and thus maintain the C0 2 of the arterial blood at exactly the point which corresponds to its alkaline reserve. It is, therefore, unnecessary -to expose the blood to an atmosphere *This form of Haldane-Barcroft apparatus is not quite the same as the differential manometer that is used for measurement of the O 2 -combining power of hemoglobin (page 395). In the form used for the present purpose, a side tube at the bend of the U-tube is connected with a small rub- ber bag, which can be compressed by a screw. When the gas is evolved in the bottle, it presses down the fluid in the proximal limb of the manometer correspondingly and raises that in the distal limb. Since the calculation of the amount of gas evolved depends on finding the pressure produced without any change in volume, it is necessary after the gas has been evolved to compress the rubber bag until the meniscus of fluid in the proximal limb of the manometer is brought back to its original level. The height at which the fluid stands in the distal limb then obviously corresponds to the pressure created by the evolved gas. The equation for determining the amount of gas evolved depends on the gas Jaw, which states that the pressure of a gas is inversely proportional to its volume (page 353). Suppose that the volume of gas evolved was equal to the volume of the bottle, then, since the volume has been kept constant, the pressure would be doubled that is, the fluid in the distal limb would equal that of 1 atmosphere, or 10,400 mm. of water or 10,000 of clove oil, which is the fluid actually used to fill the manometer. Any other observed pressure would therefore correspond to the volume of evolved gas according to the equation, V = V01 ' f b ttle (and tubi " g t0 meni8CU8 > X mm. Pressure in Manometer. 10,000 (when clove oil is used) In using the apparatus in the above manner, only one of the bottles is employed, and the tartaric acid is added from a pocket in the stopper by a simple manipulation. 46 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES containing C0 2 before measuring the C0 2 content. In the second place, the arterial blood represents the mixed blood of the body, and not that of one locality only, as is the case with blood removed from a peripheral vein. If venous blood is collected with the precaution that the muscles in the corresponding area have been at rest for some time it appears that there is practically no difference between the alkaline reserve of arterial and ve- nous blood; but if there has been any muscular contraction, the venous blood will have a lower reserve than the arterial, because of the lactic acid thrown into it by the active muscles. But even when we take the precau- tion of avoiding muscular action it is probable that there is not a strict parallelism between the buffer action of arterial and venous blood, as in cases in which the demands on the alkaline reserves are such that those of the tissues are being called on as well as those of the blood itself. Even when the whole blood is used, however, we do not necessarily measure the total reserve of tlie ~body, a final reserve being afforded by the alkalies and possibly certain of the proteins of the tissue cells. Now it is clear that there can be. no test tube method by which measurement of the magnitude of all of these defensive agencies is possible; and we are there- fore compelled to supplement them by certain indirect methods. Indirect Methods The chief criticism against the use of the C0 2 carrying power of blood or blood plasma, is therefore, that it tells little if anything concerning the acid-absorbing powers of the tissues. Is there not, therefore, some test of the acid buffer which can be applied to the intact animal ? One such is the percentage of C0 2 in alveolar air (see page 361). 1. Determination of the Tension of C0 2 in Alveolar Air. Since this method is employed more particularly in investigating the hormone con- trol of the respiratory center, we shall defer a description of it until later (page 361). For the present, however, it should be remarked that the alve- olar C0 2 can be a precise gauge of the acid-base equilibrium only provided that the respiratory center is perfectly normal, and that there is no in- terference with the diffusion of C0 2 from the blood into the alveolar air. In order to place an estimate on the relative value of this method com- parisons have been made between the C0 2 tension of the alveolar air and the C0 2 absorbing power of the blood. This has been done both in nor- mal and pathological subjects. In normal subjects the comparisons have been made under conditions, such as the taking of food and during mus- cular exercise, in which slight alterations in the acid-base equilibrium are known to occur. Van Styke, Stillman and Cullen 9b found that the ratio plasma C 2 varies from 1.27 to 1.80 in different resting individu- mm. alveolar C0 2 als, there being apparently a characteristic ratio for each individual, and ACIDOSIS 47 that the taking of food invariably raises the alveolar OCX-combining power. This would seem to indicate that it must be the excitability of the 'respi- ratory center rather than the acid-base equilibrium that becomes altered so as to cause variations in alveolar C0 2 . Technical difficulties have also to be overcome in the collecting of the alveolar air, for it is now well established that the original method of Hal- dane and Priestley is approximately accurate only when it is carried out under strictly controlled conditions so strict that they can not be prac- tised in the clinic and even then, as R. G. Pearce, Carter, Krogh, Sie- beck and others have shown, we can not be certain of the results. At best, therefore, the alveolar C0 2 can serve as an accurate index of the acid-base equilibrium of the blood only under strictly controlled conditions. 2. The Measurement of the Acid Excretion by the Kidney, As might be expected, the acid-base equilibrium of the body may also be gauged by measurement of the acid excretion of the urine, in which the acids are contained partly in combination with ammonia or a fixed base, and partly in a free state. We shall first of all consider the methods of acid excretion and then examine the evidence showing that the total acid excretion is proportional to the alkaline reserve as measured by the above described methods. EXCRETION OF ACID IN COMBINATION WITH AMMONIA. The production of ammonia is essentially an endogenous process, and when excessive quantities of acid make their appearance in the organism, the fixed alkali may not be sufficient to neutralize it all, so that ammonia, derived from the breakdown of ammo acids (page 650), instead of being converted into urea is employed to neutralize the excess of acid. Most workers have in this way explained the very large ammonia excretion that has long been known to occur in such conditions as diabetic acidosis. Some recent workers are, however, inclined to question the significance of ammonia in this connection, believing that the increased ammonia ex- cretion is, like the acetone bodies themselves, a product of perverted metabolism. Be this as it may, it is no doubt true that ammonia is used for neutralizing acid in disease, although it may not be an important factor in the maintenance of neutrality under normal conditions. It is a factor of safety, in that it helps to care for an increase in acid when the normal mechanism of the body is overtaxed. EXCRETION OF PHOSPHATES. The more permanent control of neutrality depends on the excretion of phosphates by the kidney. The principle governing this process is exactly the same as that already discussed in connection with carbonic acid. In the one case it is the volatile acid C0 2 , and in the other, the fixed phosphoric acid that is concerned in the reaction. The ratio between the acid salts of phosphoric acid, MH 2 P0 4 , 48 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES and the alkaline salts, M 2 HP0 4 , in blood is approximately 1 to 5, but in the urine this ratio varies according to the amount of H ion that must be eliminated from the blood. In other words, a definite amount of phos- phoric acid is enabled to carry variable amounts of H ion out of the body by causing the amount of alkali excreted in combination with it to be- come altered. For example, in the form of MH 2 P0 4 a given amount of P0 4 obviously carries out more H ion than when it is excreted as M 2 HP0 4 . The adjustment between these two salts is a function of the kidney. We may accordingly measure the amount of alkali retained by the organism by finding how much standardized alkali must be added to a given quantity of urine until the reaction of the blood is obtained. Since the latter value is constant, the titration can be done simply by titrating the urine with an indicator such as sulphonephenolphthalein, which changes tint at about P H of blood. A more serviceable indicator to use, however, is phenolphthalein, be- cause its end point is such that when human urine just reacts neutral to it that is, when the titrable acid approaches zero the C0 2 -absorb- ing power of the plasma is at its maximum of 80 vols. per cent and the ammonia excretion by the urine is zero (Van Slyke). It is advantageous, therefore, to use this indicator, because it happens to have its turning point situated for a reaction which is well to the alkaline side of neu- trality, and which is reached in urine when the blood is at its maximal acid-combining power and no ammonia is being used for neutralization purposes. As the C0 2 -combining power of the blood decreases, there should, therefore, be a proportionate increase in ammonia and in the titrable acidity of the urine. 3. Determination of Alkali Retention. Another valuable criterion of the alkaline reserve is the amount of alkali required to change the re- action of the urine. In health the C H of the urine varies from 0.000,016 N (P H = 4.8) to about 0.000,000,035 N (P H = 7.46) with a mean of about 0.000,001 N (P H = 6). These extremes are rarely overstepped in disease, but frequently the average is considerably different. In car- dio-renal disease, for example, the mean acidity may be approximately 0.000,005 N (P H = 5.3), or five times the normal value, A certain de- gree of acidosis is therefore common enough in this condition a fact which has indicated the advisability of administering sodium bicarbon- ate. It has been found that 5 grams or less of soda, given by mouth to a normal person, causes a distinct diminution in tHe C H of the urine, whereas in pathologic cases it may be necessary to give more than 100 grams before a similar effect is observed (L. J. Henderson and Palmer 15 and Sellards 16 ). This test has been found of particular value in the diagnosis of acidosis ACIDOSIS 49 accompanying certain forms of renal disease (chronic interstitial nephri- tis), which raises the question as to whether the retention may not be due to faulty elimination of the bicarbonate rather than to its retention, in order that a deficient alkaline reserve maybe corrected. It has not been a very simple matter to disprove entirely this possible explanation, and ex- periments of a variety of types have had to be devised in connection with the problem. One of them consists in determining the effect of a second dose of bicarbonate administered to an acidosis patient to whom a suffi- cient amount had previously been given to render the urine just alkaline. It has been found that a few grams now suffice, indicating, apparently, that the alkaline reserve must have been restored to its normal level. Even to this experiment the objection can be raised, however, that the large doses were retained because the threshold of the kidney for the ex- cretion of bicarbonate was a Very high one, and that the second, smaller administration just sufficed to overstep this threshold. Sellards' careful work with this method seems quite clearly to establish its value, however, and for practical purposes it is probably the most prac- ticable test -of acidosis at present available in routine clinical work. It has the important* advantage, furthermore, of being simple and of requir- ing no elaborate apparatus. It may be advantageous in this place to classify the possible causes which might lead to a want of stability in th'e C H of the blood ; that is, to threatened acidosis or alkalosis, not of acidosis in the narrow sense im- pliecl in Van Slyke's definition, but in the broader sense of any disturb- ance in the acid-base equilibrium. Relationship Between OCX-content of Blood and Hydrogen-ion Concentration From the equation given on page 39, it is evident that P H can be cal- culated for each partial pressure of C0 2 at which blood is equilibrated, provided we know the total C0 2 -combining power and the H 2 C0 3 . We have seen how the former may be determined (page 42). The free H 2 CO :J is determined by multiplying the mm. of C0 2 pressure by the factor 0.0672 which is derived from the equation _-. x - in which 0.511 is the 7bU 1 coefficient of solubility of C0 2 in blood at 3'8 C. (cf. gas laws, page 354). If blood be exposed to various C0 2 tensions which are placed on the abscissa of a chart, as in Fig. 128, page 363, and the total C0 2 with which it combines at each tension be placed on the ordinates, a curve (C0 2 -dis- sociation curve) is obtained and a slanting line may also be drawn on the chart to show the C0 2 in simple solution (i.e., as H 2 C0 3 ). Now C H at each C0 2 tension will be proportional to the ratio between the dis- 50 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES tances of the curves and the slanting line from the abscissa; i.e., P H , since it is the reciprocal of C H , will be inversely proportional. Lines repre- senting P H may therefore be drawn across the chart, their exact position being most conveniently calculated from the equation P H = 6.10 + log (total C0 2 - 0.0672p) (a modification of . that already given on page 39, 0.0672p and in which p is the partial pressure of C0 2 and 0.0672 the factor by which it is necesary to multiply the tension in order to find the volume per cent of C0 2 in simple solution). As a practical detail in determining the curve it may be mentioned that the blood should be exposed to C(X tensions of 40, 72 and 18 m.m., in the order given (Haggard and Hender- son). If the lowest tension is used first there is danger of laking of the blood with consequent error because of the acid qualities of hemoglobin. CHAPTER VII COLLOIDS Substances which can be obtained in the crystalline state and which, when in solution, are capable of readily diffusing through membranes, are designated as crystalloids, and are to be distinguished from another, larger group of substances not having these characteristics or having them only in very minor degree the colloids. In every field of chem- istry the properties of colloids have been studied extensively during recent years, but in no field more than in that which covers the chem- istry of biological fluids and tissues, into whose composition colloids enter much more extensively than crystalloids. The subject of colloidal chemistry has indeed become so extensive that an attempt to do more than indicate some of the most important characteristics of colloids would take us far beyond the limitations of this book. The far-reaching applications of the subject in physiology and medicine are only begin- ning to be realized. The term "colloid," or "colloidal," does not refer to a class of chemical substances, but rather to a state of matter which is quite independent of the chemical composition of the substance. We are familiar with more colloids in the organic than in the inorganic world, yet they are plentiful in both, and the same substance may at one time be colloidal and at another noncolloidal. Indeed, under appropriate conditions prob- ably all substances may assume the colloidal state not solids and liq- uids alone, but gases as well. It is mainly with liquids, however, that we are concerned in biochemistry. CHARACTERISTIC PROPERTIES The distinction between molecular* and colloidal solutions is a rela- tive one. Suppose, for example, that we take a piece of gold in water and divide it up into smaller and smaller parts. At a certain stage, the particles will be so fine that they will remain in suspension and be in- visible by ordinary means. They are then said to be in the colloidal state. If we divide them further until they become molecules of gold, a molecular solution will be obtained. In the colloidal state, there are *Molecular solutions include those of nonelectrolytes, such as sugar, and electrolytes, such as inorganic salts. 51 52 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES two distinct phases in the solution, one solid and the other liquid, and between the two, because of the great subdivision of the original par- ticle, is an enormous surface of contact. The solution is heterogeneous, and at the interface between the two "phases" the physical forces which depend on surface e. g., surface tension (see page 65) are enormously developed, and are responsible for the peculiar properties of colloidal solutions as compared with those of molecular solutions, which may, therefore, be styled homogeneous. The solutions of crystalline substances which we have hitherto been concerned with, are homogeneous. Between these two groups of solutions is an intermediate one namely, suspensions (as suspensions of quartz or carbon, or oil emulsions). Be- sides being turbid in transmitted light, the solutions may be seen by means of the ultramicroscope to contain particles. These can be sepa- rated by filtration from the fluid they are suspended in, except in the case of many emulsions in which the particles can squeeze their way through the filter pores by changing their shape. On standing or being centrifuged suspensions may also separate into their constituents, al- though this can be greatly hindered by the addition of a suspending substance such as gelatin or certain bodies having a so-called protec- tive action (as peptone, proteose, etc.). True Colloidal Solutions 1. The Solution Is More or Less Turbid. Frequently this can be recog- nized by holding the solution in a thin-walled glass vessel against a dark background, but the turbidity may be so slight that it requires for its detection the use of the Tyndall phenomenon. This is familiar to all in the effect of a beam of sunlight let in through a small aperture into an otherwise darkened room. In the course of the beam suspended dust particles, which are invisible in an equally illuminated room, be- come visible, and thus render very distinct the pathway of the beam. If a colloidal solution contained in a glass vessel, preferably with paral- lel sides, is held in the course of such a beam, the Tyndall phenomenon will be seen in the liquid, which is not the case with molecular solutions. Focused artificial light may be employed for intensifying the effect. The light that is sent out at right angles to the beam is plane-polarized, which means that the particles reflecting the light must be smaller than the mean wave length of the light forming the beam. It should be under- stood that the individual particles themselves may not be rendered visible to the naked eye by the beam, although in such cases they can often be seen by using intense illumination and a dark-field (ultramicro- scope) combined with suitable magnification (Fig. 12). 2. Colloids Do Not Readily Diffuse. To demonstrate this, test tubes COLLOIDS 53 are half filled with, a 5 per cent solution of pure gelatin or a 1 per cent solution of pure agar, and, after the jelly is set, the solution under examination is poured on the surface; or, when it is of high spe- cific gravity, the tube of gelatin, etc., is placed mouth downwards in the solution. In the case of colloidal solutions very little if any diffu- sion into the gelatin or agar will occur, even after several days; whereas true molecular solutions will diffuse for a considerable distance. When colored solutions are used, the diffusion can readily be recognized by inspection (see Fig. 13), but when they are colorless, the presence or absence of diffusion must be determined by removing the column of gelatin or agar and dividing it into slices of equal size, which are then examined chemically for the substance in question. A further test is afforded by the failure of colloids to diffuse through membranes [dialysis). This was the method originally used by Thomas Graham to distinguish between molecular and colloidal solutions. The solution under examination is placed in a dialyzer, which is then im- mersed in a wide vessel containing the pure solvent. The older forms Fig. 12. Ultramicroscope (slit type) for the examination of colloidal solutions. The arrange- ment of diaphragms, etc., in this form removes the absorptive effects of the surfaces of the glass vessel or slide used to contain the colloidal solutions. of dialyzer consisted in general of a bell-shaped glass vessel closed be- low with parchment paper, but more recently so-called diffusion sacs have been adopted. These consist of pig or fish bladders or of col- lodion sacs. The latter are made by placing some collodion dissolved in ether in a test tube, which is then tilted so that the collodion runs out except for a thin layer which remains adherent to the walls. When the collodion has set, the sac can be removed after loosening it by allow- ing a little water to flow between the sac and the walls of the test tube. The sac containing the colloidal solution is then suspended in water or some of the solvent used in preparing the colloidal solution, care being taken that the menisci of the fluids inside and outside of the sac stand at the same level. Sometimes, especially when collodion sacs are used, some colloid may at first diffuse through, but if the outer fluid (the dialysate) is renewed and the dialysis allowed to proceed, this ceases. 54 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES When a fluid solution exhibits both of the above properties (i. e., the Tyndall phenomenon and indiffusibility), there can be no doubt as to its being in a true colloidal state, but there are substances, such as congo red, or protein solutions of certain strengths, which may exhibit a very slight diffusibility in a dialyzer but not show the Tyndall phenomenon. Substances of this group constitute transitional types between molecular and colloidal solutions, and to determine their true nature it is neces- Fig. 13. To show diffusion into gelatin of a crystalloid stain in b and the nondiffusion of a colloid stain in a. (From W. Ostwald.) sary to employ refined methods such as those of ultramicroscopy, ultra- filtration, etc., which can not be described here. 3. The Size of Colloidal Particles. It will be apparent that the essential property upon which the above-mentioned phenomena depend is the size of the particle. Particles which can still be seen under the microscope are called microns. They have been computed to have a dimension of 0.1 fj. (0.001 mm.) or more, and they form suspensions. Particles which are invisible microscopically under the ordinary conditions of illumina- COLLOIDS 55 tion, but are still visible when the ultrainicroscopic illumination is used, are called submicrons. They have a dimension between 0.1 p and 1 pp (0.000,001 mm.),* and they constitute the colloids. Particles smaller than 1 pp are called amicrons, this term being. used to include the mol- ecules and ions present in molecular solutions. (The amicron of hydro- gen is, for example, computed to be 0.067 to 0.159 pp, and that of water vapor, 0.113 pp.) This classification of dissolved substances according to the size of the particles and molecules shows the relationship of one Fig. 14. Diagram from W. Ostwald showing the relative size of various particles and colloidal dispersoids compared with a red blood corpuscle and an anthrax bacillus. class of substances to others. An idea of the relative sizes of colloidal particles and molecules in comparison with such familiar objects as a blood corpuscle and an anthrax bacillus is given in Fig. 14. The fluid in which the "particle" is suspended is called the dispersion medium, or external phase, and the particle itself the dispersoid, or internal phase. It is the enormous development of surface which determines the dif- *M 0.001 mm., and /*/* = 0.000,001 mm. 56 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES ference in the properties of a colloidal solution from those of a suspen- sion of the same substance. Thus, the difference between a colloidal solution of platinum (prepared by allowing an electric arc to form be- tween platinum electrodes in water) and pieces of platinum in water depends on the fact that the surface of the platinum in the former case has been increased many million times. When the subdivision becomes still greater and the particles gain the size of molecules, the phenomena due to surface development become suppressed and those due to con- centration in unit volume become accentuated. The properties depend- ent on osmotic pressure, diffusibility, etc., are exhibited by all dispersoids, whether ions, molecules or particles, but some of these properties are much more pronounced when the dispersoids are of large dimensions, and others when they are small. In other words, the phenomena due to surface, such as those of surface tension (see page 65), become apparent only when the dispersoids have the properties of matter in mass; when the dispersoids become molecular in size, they manifest the properties characteristic of true solutions. 4. Electrical Properties of Colloids. Most colloids carry a charge, which may be either positive or negative toward the dispersion medium. Both crystalloids and colloids therefore carry electric charges; in the former case, however, the charge does not reveal itself until the molecules in solution have become dissociated, when each ion carries a charge of opposite sign (see page 16), whereas 1 in the case of colloids, each col- loid particle usually carries a charge which is always of one sign, either positive or negative. Colloids may therefore be grouped into positive and negative, according to the charges which they carry, and there is a third group in which the charge may be either positive or negative ac- cording to the nature of the dispersion medium. A colloid not carrying a charge to begin with can be caused to assume one by the action of electrolytes, for the electrical properties of colloids, as well as those of inert powders suspended in water, are readily in- fluenced by the charges present in the ions of the dispersion medium. The H- and OH' ions are especially liable to exert this influence. The particles of inert powders in suspensions (kaolin, sulphur, etc.) carry a positive charge when the water in which they are suspended is acidi- fied, and a negative charge when it is made alkaline. In general, it may be said that suspensions of most powders and of insoluble organic acids in water (e. g., charcoal, cellulose, kaolin, caseinogen, mastic, free acid of congo red, etc.) are electro-negative. Of true colloids ferric hydrox- ide (ferrum dialysatum) and serum globulin are positive in acid solu- tions; arsenious sulphide and serum globulin are negative in alkaline solution, and serum globulin in neutral solutions has no charge. COLLOIDS 57 To ascertain the nature of the charge various methods may be em- ployed, of which the following are important: 1. The method of electrophoresis. The colloid solution is placed in a U-tube, each side of which carries a platinum electrode dipping into the solution. After a strong continuous electric current has been allowed to pass for some time through the solution, it will be found that the colloid collects at the anode (where the current enters) when it is a negative colloid (since unlike electric charges attract each other), and at the cathode when it is positive. In the case of colored solutions, the migration can be readily seen, but otherwise it may be necessary to ana- lyze the solution at the two poles. Fig. 15. Capillary analysis of colloids. Strips of filter paper, after being suspended with the lower ends dipping into colloidal solutions. Those on the right hand were positive colloids, which did not rise in the strips, but formed a sharp line of demarcation at the lower end on account of precipitation. Those on the left hand were negative colloids. (From W. Ostwald.) 2. The method of capillary analysis. For this purpose a long strip of filter paper is arranged vertically over the solution, with its lower end dipping into it. In the case of negative colloids the colloid, as well as the dispersion medium, rises uniformly on the strip of paper (it may be to a height of 20 cm.) ; whereas with positive colloids the dispersion medium alone rises, the colloid itself doing so only to a very slight ex- tent, but becoming so highly concentrated at the interface between the solution and the paper that it coagulates on the end of the strip of paper, where it forms a sharp line of demarcation (Fig. 15). 3. The method of mutual precipitation of colloids. When a positive 58 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES and a negative colloid are mixed in such proportions that the electric charges are neutralized, precipitation usually occurs. When it does so, we can tell the nature of the electric charge of an unknown colloid by its behavior when a colloid of known electric sign is added to it. For example, if ferric hydroxide (positive) causes a precipitate to form when it is added to an unknown colloidal solution, the electric charge of the latter must be negative; if it does not precipitate with ferric hydroxide, but does so with arsenious sulphide (negative), it must be positive. 5. Brownian Movement. Like the particles in fine mechanical suspen- sions, those of colloidal solutions, especially when examined ultra- microscopically, exhibit the so-called Brownian movements, which have been described as "dancing, hopping and skipping." These movements occur in straight lines, which are suddenly changed in direction and are quite independent of external sources of energy, such as change in temperature (although they become quicker as the temperature of the solution is raised), earth vibrations, chemical changes, or the electric charge of the colloid. The movements become more rapid the smaller the particles, and they become sluggish as the viscosity of the solution in- creases. Addition of electrolytes decreases the movement by causing the particles to clump together. The density and viscosity of the disper- sion medium, the electric charge of the dispersoid and the presence of Brownian movements, are the forces which operate together to prevent sedimentation of the particles in a colloidal solution. 6. Osmotic Pressure. As one of the distinguishing properties of col- loids we have seen that their diffusibility, as into gelatin or agar jel- lies, is extremely slow when compared with that of a molecular solution. This does not mean, however, that colloids are possessed of no power of diffusibility if left long enough. Indeed the existence of the Brownian movement indicates that such diffusion must occur, and therefore it should be possible, by the application of the same principles as those which govern molecular solutions (e. g., by using a semipermeable mem- brane), to measure the osmotic pressure. Many studies of the osmotic properties of colloidal solutions have been undertaken, especially by those who are interested in the possibility that the colloids of blood serum (serum albumin and globulin) may cre- ate an osmotic pressure. If this should prove to be the case, it would be necessary for the osmotic pressure to be overcome by mechanical pressure such as that supplied by the heart (i. e., the blood pressure) in the various physiological processes of filtration and diffusion taking place through cell membranes (as in the formation of urine in the kidney). For measuring the osmotic pressure of colloids, osmometers similar COLLOIDS 59 to those already described (page 4) can be employed. Most of the recent work has been done either with collodion sacs, or with unglazed clay cups impregnated with some gel, such as silica or gelatin. When such an osmometer, filled with some colloidal solution (like a solution of pure albumin) and provided with a vertical glass tube, is placed in an outer vessel containing water, the fluid will be seen to rise in the ver- tical tube, the height to which it rises being proportional to the osmotic pressure. But the observed pressure does not necessarily give us the osmotic pressure of the pure colloid, for to this, even when highly purified, there is almost certain to be attached a considerable amount of inorganic salt, which may be responsible for the osmosis. It has indeed been maintained by some observers that electrolytes form an integral part of certain colloids, being bound to them perhaps by adsorption (see page 66), and that they are essential to the maintenance of the colloidal state. In any case, since electrolytes are always present, the osmotic pressure of the pure colloid can be measured only when means are taken to discount their influence. Sev- eral devices have been used, of which the following may be mentioned: 1. Addition to the fluid outside the osmometer of a percentage of salt equal to that found by chemical analysis to be present in the colloid. (This method is untrust- worthy.) 2. The use of a limited quantity of fluid on the outside of the osmometer so that equality of saline content soon becomes established, by diffusion, in the fluids on the two sides of the membrane. 3. The use of a membrane which is permeable to electrolytes but not to colloids. Even when the greatest care is taken in its measurement, the osmotic pressure of a given colloid has been found to vary considerably not only according to the method used in its preparation, but also according to the amount of mechanical agitation (shaking, stirring, etc.) to which the colloid solution has been subjected. Eegarding the influence of the method of preparation, it was found in one series of experiments that albumin that had been repeatedly washed (but still contained considerable ash) gave no osmotic pressure, whereas another preparation that had been purified by crystal- lization twice (and contained much less ash) had a pressure of 3.38 mm. Hg. Ac- cording to these results the ash content of the colloid is not fundamentally responsible for its osmotic pressure. As to the influence of mechanical agitation, the osmotic pres- sure of a gelatin solution is increased by shaking, while that of a solution of egg albu- min is decreased. The property upon which the osmotic pressure depends is undoubtedly the state of dispersion of the colloid particles, and until we know all of the factors which may in- fluence this, measurements of osmotic pressures of colloids can scarcely be of very much value. Nevertheless, that this property has some physiologic bearing is clear from the effect which colloids have in restoring the blood pressure after hemorrhage (page 141). Further evidence that the osmotic pressure of colloids has not the significance that it has in the case of molecular solutions is furnished by the fact that the osmotic pres- sure is only approximately proportional to the concentration of the solution; it may either increase or decrease relatively to the strength of the solution. Temperature also has quite a different influence on the osmotic pressure of colloids from that which ^ has on the osmotic pressure of molecular solutions, and it frequently has an influence which persists after the solution is brought back to its original level. 60 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES The influence of added substances on the osmotic pressure of colloidal solutions is of considerable interest to the biologist, for, whereas in the case of molecular solu- tions this is purely additive, in the case of colloids the added substance may at one time cause the osmotic pressure to increase, at another, to decrease. It has been found that the osmotic pressure of gelatin solutions at first decreases, then rapidly increases as the H-ion concentration is raised. The addition of alkali increases the osmotic pressure until a maximum is reached, beyond which it begins to fall. Both acids and alkalies lessen the osmotic pressure of egg albumin. Electrolytes always decrease the osmotic pressure of gelatin and albumin solutions, and the degree to which they exert this influence depends on the nature of the cation and anion composing the electrolyte. In the order of their depressing influence the cations arrange themselves: Heavy metals > alkaline earths > alkalies ; and the anions: SO 4 > 01 > NO 2 > Br > I > CNS. The influence of a given electrolyte varies extraordinarily with the reaction of the eolloid, a fact which must be carefully regarded in all work in this field. CHAPTER VIII COLLOIDS (Cont'd) SUSPENSOIDS AND EMULSOIDS According to whether colloids form solutions that are more or less viscid than the suspension medium, they are divided into emulsoids and suspensoids. Examples of the former class are silicates and gelatin, and of the latter, dialyzed iron and arsenious sulphide. The following char- acteristics are used to distinguish between suspensoids and emulsoids: 1. Measurement of the time it takes, at a standard temperature, for a given volume of the fluid to flow out of a standard pipette (10 c.c.) shows the viscosity to be, roughly, inversely proportional to the time of outflow. In the case of suspensoids the viscosity is no different from that of the dispersion medium alone, and does not vary much when the solution is cooled. The viscosity of emulsoids even in very dilute solutions is, on the other hand, considerably greater than that of the dispersion medium itself, and it becomes greatly increased by cooling. 2. Suspensoids are much more readily coagulated by the addition of electrolytes than emulsoids. This is particularly true when water is the dispersion medium (so-called hydrosols), and when electrolytes hav- ing a polyvalent ion (such as Al or Mg.) are employed. Thus, practically all suspensoids are coagulated in the presence of 1 per cent of alum, which has no influence on emulsoids. We shall return to this phase of our subject later on. The division of colloids into emulsoids and suspensoids is more or less arbitrary, since one class may be changed into the other, the determining factor being the water content of the dispersoid. The water content of suspensoids is low (lyophobe), while that of emulsoids is high. By changing the relative amounts of water and solid of which a colloidal solution is composed, the nature of the dispersoid may be changed. If the water is diminished, the dispersoid behaves as a suspensoid and be- comes readily precipitated. The practical importance of this fact is that it explains the salting out of proteins a process extensively used in their separation. Ordinarily these behave as emulsoids, but the addi- tion of salt raises the osmotic pressure of the dispersion medium, and thus attracts water from the dispersoids, with the result that they come 61 62 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES to behave as suspensoids, and are accordingly precipitated by the elec- trolytes. Another property of emulsoids of biological importance is the pro- tection which they can afford against the precipitating influence of electrolytes on suspensoids. If a colloidal solution of gold is mixed with a trace of gelatin, the subsequent addition of salts will be found to produce no precipitation. The explanation of this is that the emulsoid becomes distributed as a film on the suspensoid particles, thus practically converting them into emulsoids. Gelatinization One of the best known properties of emulsoids is that of gelatiniza- tion, which has an interesting bearing on many problems of biology. After the gel has set, an enormous pressure is required to squeeze out any water from it, indicating that the water no longer forms the con- tinuous phase but must be enclosed in vesicles formed of more solid material. By observing solutions of pure soaps under the ultramicroscope it has been noted that as the solution cools, the gel at first forms a polarized cone of light, but the very fine particles which are responsible for this effect soon increase in number and size so that they obstruct one another in their Brownian movements and adhere, giving an appearance of fine felt-like threads throughout the solution. 25 A sort of impervious sponge work of the more solid phase is therefore formed, the more fluid phase being inclosed in the meshes. If, as in the accompanying diagram, the dispersion medium is repre- sented by white and the dispersoid in black, the relationship between the two in a suspensoid is as in B, and that in a gel as in A. To express COLLOIDS 63 any of the dispersion medium in A, it will require a pressure sufficient to cause the more fluid phase to penetrate the more solid. If the gel is treated with reagents like formaldehyde, the liquid can be readily pressed out. This occurs during fixation for histological purposes. Imbibition Closely related to gel formation is the process of imbibition the power of taking up large quantities of water without actually forming liquid solutions. Besides gelatin the dried tissues pf plants and animals exhibit the phenomenon, and it is undoubtedly of importance in many physiological processes such as growth and the passage of water into cells, etc. The materials present as vacuoles in plant cells attract water from the environment of the cell by imbibition, and thus exert on the cell wall a pressure which, acting along with the osmotic pressure, maintains the turgor of the cell. The initial growth of pollen is also dependent upon imbibition, and important observations on this process., under varying conditions, are likely to furnish us with useful informa- tion concerning the significance of imbibition in connection with growth of cells in general. By measuring the rate of increase in length of long, narrow strips of gelatin placed in Petri dishes containing solutions of varying composition, the factors that influence the imbibition process can be quantitatively investigated. Working in this way, F. H. Lloyd" has found that for all acids there is a certain concentration (about N/320 H 2 S0 4 ) which induces a maximum rate of swelling, and another, much weaker (N/2800 H 2 SO 4 ), in which the rate of swelling is even less than in pure water. In higher con- centrations of acid than N/320, the gelatin at first swells very quickly; but the rate slows off so that it soon comes to be less than that with intermediate concentrations. Regarding alkalies, at high concentrations the effect is similar to that of acids. Salts alone seem to repress the swelling below that of water. It should be pointed out that the concentrations of acid and alkali in the above observations are much greater than those that could occur in the animal body. The experiments recall the attempts made some years ago by Martin Fischer to explain edema as due to excessive imbibition of water by the proteins of the tissues because of increased acidity of the blood and tissue fluids. That imbibition might possibly play some role in such processes is not denied, but Fischer disregards entirely the now well-established facts that hydrogen- ion concentration is one of the most constant properties of the blood, that very low concentrations of acid may diminish rather than increase imbibition, and that it is manifested only in the absence of inorganic salts.* Moreover, the fluid in edema can often be drained off by hollow needles, and it passes by gravity from one part of the body to another, neither of which processes would be possible if imbibition were the essential factor concerned. If further evidence against this hypothesis should be demanded, it might be found in the utter failure of the therapeutic measures alkali administration that are recommended to combat the edema. Action of Electrolytes on Colloids (apart from their effect on osmotic pressure). It has been stated above that the charge which a colloidal *Determinations of the hydrogen-ion concentration of the blood recently published from Fischer's laboratory do not inspire confidence. 64 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES particle assumes may be neutralized by a charge of opposite sign car- ried by an ion present in the dispersion medium. The neutralization of the electric charge causes coagulation of the suspensoids but not of the emulsoids. Of the positive and negative ions into which the elec- trolytes dissociate, the one producing the coagulation is that which is opposite in sign to the electric charge of the colloidal particle. A quantity of electrolyte which is capable of producing complete pre- cipitation when added all at once to suspensoids will be ineffective when added in small quantities at a time. This phenomenon, which is also known to be exhibited when toxins and antitoxins are mixed together, is probably owing to the fact that precipitation depends on inequality and irregular distribution of electric charges, a condition which becomes established when the electrolyte is suddenly added, but not so when it is gradually added. The particles in the latter case become, as it were, acclimated to the electric charges introduced by the addition of the electrolyte. Proteins as Colloids. The most prominent colloids in the field of bio- chemistry are the proteins. On account of complexity of structure, however, certain factors intervene which render the investigation of their behavior very difficult. As we shall see later, proteins are made up of combinations of amino acids, each of which contains basic (NH 2 ) and acid groups (COOH). The various amino acids are linked together in protein by the COOH of one uniting with the NH 2 of another, with the elimination of water thus, CO ! OH + H i HN but some NH 2 and COOH groups are left uncombined. According to the relative number of these uncombined radicles, the protein (or polypeptid, see page 636) will exhibit faintly acid or basic or neutral properties. With acids, for example, a salt will be formed by union with the NH 2 groups, which will dissociate into the anion of the acid and a large organic cation; whereas with alkalies union will occur with the COOH group, and the salt on dissociating will form a small cation of the metal of the salt and a large complex anion. We may therefore obtain the protein with either a- positive or a negative electric charge by altering the chemical nature of the fluid in which it is dissolved, so that the reaction towards other colloids and towards electrolytes will vary. One feature of proteins of importance in this connection is that known as the isoelectric point, at which the protein exists with a maximum of electrically neutral molecules. This point is reached by adding acid to a protein solution. The acid represses the dissociation of the protein acting as an acid, and therefore diminishes the number of free hydrogen ions; and at the same time a salt is formed which then dissociates to COLLOIDS 65 produce cations. The sum of the cations and anions therefore becomes a minimum, the concentration of the two being equal, and this point the protein, being electrically neutral, has its solubility reduced to a minimum. SURFACE TENSION Before we consider a very important property of colloids known as adsorption, by means of which they are able to perform many reactions that do not conform with the laws of mass action, it will be well to say a few words concerning the physical phenomenon upon which this depends namely, surface tension. The creation of this force is due to the fact that, whereas the molecules within a liquid are subjected to equal forces of attraction on all sides, at the surface these forces act on one side of the molecules only, and therefore tend to pull them inwards. This causes the surface to pull itself together so as to occupy the least possible area, and it is this force which constitutes surface tension. The surface behaves as if stretched. There are various simple experi- A. Fig. 17. Diagram to illustrate surface tension. The rings A and B inclose soap films in which a very fine loop of silk is suspended. In A it is loose but in B, where the film inclosed in the loop has been broken, it is drawn into a circle by the tension of the soap film. (From Bayliss.) ments that reveal the presence of surface tension. If a film is made on a loop of wire by dipping it in soap solution, a fine silk thread can be floated in the film, so that it forms a loop that is quite loose. If the portion of the film inside the loop is destroyed by touching it with filter paper, the film will break in the loop, which will now be pulled into a circular shape by the tension of the film around it (Fig. 17). For the measurement of surface tension, various methods are used. The size of drops of liquid falling from an orifice is dependent on sur- face tension; the larger the drops, the greater the surface tension. If the number of drops obtained by allowing a liquid to drop from a stand- ard orifice in a given time is counted, we have a measure of the surface tension. Account must of course also be taken of the specific gravity of the liquid. The instrument used for this purpose is called a stalagmometer (Fig. 18). Another method depends on the fact that the height to which a i\uid rises in a capillary tube is dependent on 66 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES surface tension (and inversely on the diameter of the capillary). The difference in the heights to which two liquids rise in capillary tubes of known bore permits us to compare their surface tensions, and if this is known for one of the solutions, it can be determined for the other. Besides existing between liquid and air, surface tension also exists at Fib. 18. Traube's stalagmometer. The surface tension is proportional to the number of drops formed in a given time. The right-angled tubes are for thin liquids, and the straight one for blood and other viscous fluids. the interface between two immiscible liquids, and at that between sus- pended solid particles and liquid, as in colloidal solutions. Since, as we have seen, the surface area (interface) is enormously increased in these solutions, a very great surface energy is present, for this is equal to the surface tension multiplied by the surface area. ADSORPTION The surface tension between liquid and air is lowered when organic substances are dissolved in the liquid, but is slightly raised when inor- ganic salts are dissolved. The degree of lowering varies markedly ac- cording to the organic substance dissolved, being very pronounced with bile salts, upon which fact the well-known (Hay) test for the presence of bile in urine is based. Between liquid and liquid, as well as between solid and liquid, the surface tension is always lowered "by dissolving sub- stances in the liquid. Now, at the interfaces between solid particles and liquid there must be a local accumulation of free surface energy, which will be equal to the surface tension multiplied by the surface (inter- face) area. A constant tendency exists for such free energy to be de- creased and, since dissolved substances have this effect, they will become concentrated at the interface. This is known as the principle of Willard GMs, and it is of fundamental importance to. the biochemist, because COLLOIDS 67 on it depends the phenomenon known as adsorption, which in the case of colloidal solutions may therefore be defined as the local concentra- tion or condensation of dissolved substances at the interface between the two phases. The amount of substance concentrated at the interface can be calculated by a formula which takes into account the concentra- tion of the dissolved substance, the temperature, and the surface tension at the interface (the Gibbs formula). After adsorption has occurred, vari- ous reactions of a chemical, electrical or purely physical nature (e. g., dif- fusion) may follow at a rate which depends on the amount of the condensation. A. P. Matthews has recently challenged this view of adsorption. 30 Reactions Which Depend on Adsorption 1. Decolorization of liquids by charcoal. That no chemical reaction occurs in such a case is readily shown by the ease with which the pigment can be extracted from the charcoal. 2. Adsorption of gases by such solids as charcoal and spongy platinum. In these cases there must be great condensation, even a liquefaction of the gas, during which heat must be evolved. By adsorbing oxygen and hydrogen, spongy platinum causes these gases to combine and form water. The hemoglobin of blood may take up oxygen by a similar process. 3. Formation of solid surface films on solutions of protein, saponin, etc. The con- densation may lead to coagulation, which explains why, if the froth produced by beat- ing the white of an egg is allowed to stand, it can not be again beaten into a froth, the albumin having gone out of solution by surface coagulation. An interesting phenomenon depending on the surface tension occurs when the protoplasmic contents of a ciliated infusorian is pressed out in water. A new membrane forms on the protoplasm because of surface con- centration of all constituents which lower surface energy. By application of the principle of Willard Gibbs, A. B. Macallum 18 concludes that not only adsorption, as exhibited in a colloidal solution, but also the local accumula- tions of material often seen in cells, are associated with changes in sur- face energy. His conclusions are based largely on microscopic studies of various forms of cell exhibiting different degrees and types of activity, and ingeniously stained for potassium by cobalt hexanitrite. By such a means the potassium stains intense black. In vegetable cells, local accumulations of potassium occur either near the interface between the clear and the chlorophyl-containing parts of the cell (spirogyra) or under a portion of the cell wall from which later a protrusion grows out to form the first stage in conjugation. The outgrowth from the cell, as well as the accumulation of potassium, may be the result of a low surface tension. In unicellular animal organisms, such as Vorticella, much less potassium is present, being confined to the base of the cilia, which Macallum believes indicates that the structures are produced as an outcome of low surface tension. 68 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES In the cells of higher animals, deposits of potassium are also localized ; in striated muscle, for example, they occur in a zone at each end of the doubly refractive band and immediately adjacent to the singly refrac- tive band. Changes in surface tension, associated with changes in the distribution of potassium, are believed by many to be responsible for muscular contraction. In nerves and nerve cells, potassium is concen- trated at the axon and at the surfaces of the cells. Interesting sugges- tions are offered to explain the relationship among changes in surface tension at the terminations of axons (synapses, terminations in gland and muscle cells) brought about by the nerve impulse acting as a change in electric potential. Surface condensation of potassium has also been observed at the lumen border of gland cells (pancreas), and on the lu- men surface of the cells of the renal tubules. Such observations indicate in what way surface tension may be called into play to control cellular activities. The field is new and almost unexplored, but there is already much to indicate that surface energy plays a most important role in the performance of many cellular activities. Conditions That Influence or Are Influenced by Adsorption Electrical Changes. Besides mere concentration, other forces come into play to assist or retard adsorption. One of the most important of these is electrical. Most solids \vhen present as particles in a fluid carry a negative charge of electricity, some a posi- tive one. In conformity with the Willard Gibbs law, a constant tendency will exist for this free energy to be diminished by the neutralization of the electric charge. This can occur. by deposition on the interface of other particles carrying an electric charge of opposite sign or by the action of that present on ions. Charcoal in suspen- sion in water, for instance, has a negative charge. If colloidal iron, which has a pos- itive charge, is added to the solution, it will become deposited on the charcoal, as will also the cations of an inorganic salt. On account of electric adsorption, dyestuffs and bile salts are adsorbed much more freely than they would be if the process depended solely on surface condensation; that is, if the Gibbs formula is used to calculate the adsorption, it will give values that are much below those actually found. If the dissolved substance and the particles both have the same electric sign, ad- sorption will not occur. Filter paper, for example, has a negative charge and can not therefore adsorb a negative dye such as congo red (as shown by the depth to which it becomes stained) ; whereas it readily adsorbs night blue, which is positively charged. If the negative charge of the paper is lowered, it becomes capable of adsorbing some of the negative congo red. This can be effected either by placing the paper in al- cohol or by adding inorganic salts (NaCl) to the water with which it is in contact. The positive-charged ions of Na, produced by dissociation, neutralize some of the nega- tive charge on the paper, and allow a certain amount of adsorption of the negative- charged congo red to occur. As would be expected, acids and alkalies are capable of greatly altering the electric charges by the II and OH ions which they contribute. Chemical Forces. If the nature of the phase at the surface of which adsorption occurs is such that it can enter into chemical combination with the substance ad- sorbed, reactions will occur that do not obey the laws of mass action. By adsorption, COLLOIDS 69 reactions of a certain type may be encouraged over other reactions, even although, the necessary reacting substances may be present in the solution (specific adsorption). The adsorbing substance itself is not, however, usually susceptible of chemical change even when it exists as very minute particles, as in the case of colloidal solutions. Nevertheless, adsorption may accelerate chemical reactions by bringing together in concentrated form substances of high chemical reactivity. In such cases the adsorbing substance itself does not enter into the chemical reaction, and can be recovered at the end in an unchanged condition. It acts as a catalyst (page 72). As we shall see later, enzymes act in this way i. e., their rate of reaction is controlled by adsorp- tion.* The distinguishing feature of such adsorption phenomena is that a curve of . the reaction (drawn by plotting amount of chemical change against concentration of react- ing substances) is a parabola, indicating that the laws of mass action (page 23) are no longer followed. In order to be able to determine whether some particular process as, for example, a fermentation process, or the absorption of oxygen by blood is caused by adsorption, we must compare its curves, constructed according to the same principles, with the typical adsorption curve. A formula may be used in constructing the curves. In arriving at this formula, two facts have to be remembered: (1) As ad- sorption proceeds and less and less of the free energy on the adsorbing surface re- mains to be neutralized, the reaction slows off, until equilibrium is reached. The more dilute the solution, the greater is the proportion of its contents to be adsorbed, which means that if a is the amount of substance adsorbed from a certain solution, then, from a solution of twice that strength, somewhat less than 2 a will be adsorbed i. e., a multiplied by some root of 2. Although the formula is one belonging to the class known as parabolic, it must not be assumed that every reaction which happens to give such a parabolic curve (such as the combination of O with hemoglobin under certain conditions) (see page 396) must be one dependent on adsorption. It must be understood that although the substance that is removed from a solution by adsorption is no longer capable of contributing to the conductivity or the osmotic pressure of the solution, it is nevertheless not so firmly fixed that it can not be set free again by purely mechanical means, as by constant dilution of the fluid. If char- coal which has adsorbed sugar is placed in a dialyzer made of membrane, the pores of which allow sugar but not charcoal to pass through, the sugar will gradually be re- moved if the dialyzer is immersed in running water. A certain equilibrium exists be- tween the substance adsorbed and the same substance still remaining in solution. If the latter is constantly diminishing by dialysis, the adsorption compound must break down to maintain the equilibrium. It is clear, however, that the process of removal will be extremely slow. The ability of adsorbed substances to withstand removal by washing is taken advantage of by nature in holding back foodstuffs in the soil. *Another instance of the influence of surface energy on the course of chemical reactions is seen in the accelerative influence of charcoal on such reactions as the oxidation of formic acid, glycerol, etc. Surface tension may also cause retardation of chemical reactions, as is seen in the turbidity (due to the separation of chloroform) which gradually develops when a ^ NagCOs solution is mixed with a -^-chloral hydrate solution. The surface remains clear, because surface energy has prevented the reaction. An important effect of surface tension on chemical reactions is also seen in the relationship between it and the absorption coefficient of gases (volume of gas dissolved by unit volume of liquid). The lower the surface tension, the greater the solubility of the gas. Oxygen and nitrogen are, for example, much more soluble in alcohol, hydrocarbons or oil than in water. This shows the futility of attempting to prevent the loss of gases from fluids such as blood by covering them with oils or hydrocarbons. 70 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES Biological Processes Depending on Adsorption Instances in which adsorption undoubtedly plays a most important part in physiological processes are as follows: 1. The action of enzymes (see page 71). 2. The combination of toxin with antitoxin occurs according to the laws of adsorption rather than those of mass action. In this case it is im- portant to note that when the toxin of diphtheria is added in small suc- cessive quantities to diphtheria antitoxin, more toxin is neutralized than when the toxin is all added at once. A similar phenomenon can also be observed by adding filter paper to congo red, more of the pigment being adsorbed when the paper is added in small quantities than when added all at once. The explanation is that relatively more adsorption of a given substance occurs from a dilute than from a strong solution (cf. page 69). 3. The sensitizing of leucocytes by opsonins, as well as the subsequent ingestion of bacilli by the sensitized leucocytes, both of which follow the course of an adsorption reaction. 4. The formation of adsorption compounds, such as the inorganic salts and proteins and the complex lecithin compounds that can be extracted from egg yolk or brain tissue. In such compounds the laws of chemical proportion no longer hold, and properties may be exhibited that are quite different from those of either one of its components. When yolk of egg is extracted with ether, for example, a compound of lecithin with vitellin goes into solution, although vitellin itself is quite insoluble in ether.* There can be no doubt that adsorption compounds of this character are very abundant in living cells, and that they are constantly being formed and broken down. 5. It is possible that the distribution of a substance in protoplasm is largely dependent on the influence which it has on the surface tension at the boundary of different phases in the protoplasm. *By mixing solutions of egg albumin, congo red and a dye called fustic in the presence of alum, the colloidal particles of which each is composed run together to form larger colloidal ag- gregates, which by ultramicrosconic examination can be seen to be composed of a red, a yellow and a green colloidal particle. The attractive force holding the particles together is electric in this case. CHAPTER IX FERMENTS, OR ENZYMES One of the most striking developments of modern research in biochem- istry concerns the nature of enzyme action. So remarkable are many of the facts that have been brought to light that it can not fail to interest every one engaged in the study of life phenomena whatever the nature of that study may be to know something of the main questions at present occupying the attention of investigators in this field. In this chapter a brief survey will be given of some of these questions; no at- tempt will be made at completeness, and only where necessary for the sake of example will reference be made to individual types of enzyme action. The discovery by Buchner that an enzyme can be expressed from yeast cells which is capable of instantly bringing about the alcoholic fermen- tation of dextrose solutions has been responsible for a great deal of the modern advance. Formerly, yeast cells were believed to bring about alcoholic fermentation as a result of their growth: it was believed to be a life phenomenon, or "vital process." Now we know that yeast cells produce an intracellular ferment or endo-enzyme* to which its sucroclastic properties are due and which can act apart from the cells that produce it. It is no great stretch of imagination to think of all chemical reactions mediated by cellular activity as due to a similar mechanism, and this thought has led to the hypothesis that all processes of intermediary metabolism in the animal and plant are caused by enzyme action. Before Buchner 's day we knew only of the extracellular enzymes (such, for example, as the digestive ferments), that is to say, of enzymes, produced indeed by cells, but secreted from them and acting outside their protoplasm; now we must recognize intracellular enzymes acting where they are produced, in the protoplasm of the cell. But we must not permit this conception to carry us too far. Without further investigation we must not imagine that the riddle of life is thus solved. As an example of the role which extra- and intracellular enzymes are supposed to play in the animal economy may be cited the metabolism of protein. Proteolytic enzymes are very widely distributed in the active tissues of the animal and plant. By their agency in animal life, the com- *The terms "ferment" and "enzyme" are synonymous, but the latter is preferable as the noun, leaving the former to be used as the verb. 71 72 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES plex protein molecule is split up to render it absorbable from the intes- tine, and the tissues appropriate from the blood those of the degradation products that they require for the construction of protoplasm, which, later, they decompose so as to utilize the energy which the organism demands. All these processes are believed to be the work of enzymes. The Nature of Enzyme Action The changes brought about by enzymes can also be accomplished by ordinary chemical means, but these have often to be of a very energetic nature to accomplish what the enzyme can so quickly and quietly perform. It is the custom to regard enzymes as catalysts. A catalyst is a sub- stance which accelerates (or retards) a chemical reaction which in its absence could proceed at a different (usually slower) pace. The action of catalysts has been aptly likened to that of a lubricant. A weight placed at the top of an inclined plane, so held that the weight only slowly slips down, has its velocity greatly increased if its under surface be oiled. The oil accelerates the action but does not affect the ultimate result. Catalysts do not combine with the final products of the reaction, these being, as a rule, the same as they would have been had no catalyst been added. Another characteristic is the tremendous amount of chem- ical change which even a trace of catalyst can induce. There are many examples of catalysts in the inorganic world, among which may be cited the action of spongy platinum on hydrogen peroxide. This substance normally tends to decompose into water and oxygen, but if a small amount of spongy platinum is added to it, the decomposition is greatly accelerated: H 2 2 = H 2 + 0. A very good example of the action of an inorganic catalyst is that of the hydrogen ion on cane sugar, or other disaccharides, in the presence of water. It accelerates the hydrolysis. Cane sugar solution at room temperature does not indeed, in sterile solution, undergo any appreciable hydrolysis, but at 100 C. it does, which leads us to believe that, though inappreciable, the action also occurs at room temperature. By adding a little hydrochloric acid, or other acid not having an oxidizing effect on sugar, we greatly accelerate the hydrolysis because of the hydrogen ions present in the acid solution. "Within certain limits the rate of hy- drolysis is proportional to the amount of catalyst present- Enzymes, like other catalysts, produce their action when present in very small amounts (e. g., sucrase can hydrolyze 200,000 times its weight of cane sugar; diastase can convert starch to sugar in a dilution of 1-1,000,000) and there is a distinct relationship between the amount of enzyme present and the rate of the reaction. The final product of the FERMENTS, OR ENZYMES 73 reaction is, however, the same at whatever rate it proceeds, and the enzyme does not appear in the final products. Many enzymes such as diastase can be found unaltered in amount after they have completed their action. This is determined by adding a fresh supply of substrate (that is, of material to be acted on), when the enzymic action proceeds again in the usual way. The same is no doubt true for all enzymes, though as yet it can actually be proved for only a few of them. Enzymes, therefore, may be defined as catalysts produced by living organisms. The Properties of Enzymes Although enzymes are examples of catalysts, they exhibit many proper- ties that appear to differ from those of inorganic catalysts. It will, therefore, be advisable in considering each quality to compare it in catalysts and enzymes, for by this method a much clearer conception of the nature of enzyme action can be gained (Bayliss 19 ). Those properties that are strictly peculiar to enzymes we shall consider later. 1. Most enzymes are remarkably specific in their action, whereas inor- ganic catalysts are very much less so. Thus, in the case of the enzymes which bring about inversion of disaccharides, this specificity is clearly shown. There is a special enzyme for each of the three disaccharides maltose, lactose and cane sugar and one of these can not replace another. Still more strikingly is this specificity of enzyme action demonstrated in the fact that certain enzymes, such as zymase (expressed from yeast), will act only on bodies having a certain configuration, that is, having their side chains arranged in a certain way. Thus, there are two varie- ties of dextrose (a and /?), which differ from each other solely in the fact that the side chains are arranged in different positions with rela- tion to the central chain of carbon atoms. This form of isomerism is called stereoisomerism because the two bodies rotate the plane of polar- ized light to an equal degree in opposite directions. Zymase acts on one of these but not on the other, and there are innumerable examples of the same kind. Indeed, of all bodies that exist in two stereoisomers only one is found in living cells and it is on this variety alone that the enzymes in animals can act. A similar specificity exists between certain drugs and their pharmacological action. Specificity of action is explained by supposing that a union occurs between the substrate and the enzyme, and for this union to take place the enzyme must possess a configuration which corresponds accu- rately with that of the substrate. The process has been compared to a lock and key; the key must be shaped to fit the lock, or it can not operate. The specificity does not, however, in itself disprove the close 74 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES relationship between enzymes and inorganic catalysts, for on the one hand there are several enzymes which do not exhibit this property, and on the other, there are inorganic catalysts which do. For example, lipase, the fat-splitting enzyme of pancreatic juice, decomposes not only fats but to a greater or less degree a number of bodies of the same gen- eral build (esters), and tyrosinase can decompose, not tyrosin alone, but all phenol compounds. Conversely, the hydrogen ion to the pres- ence of which acids owe their catalytic powers can decompose the ordi- nary esters (that is, of acids containing the carboxyl or COOH group) but it has no action on the sulphonic esters. However, enzymes are cer- tainly much more specific in their action than inorganic catalysts. 2. Temperature does not influence catalysis and enzyme action in the same way. As the temperature is raised in the case of inorganic catalysts, the reaction becomes about doubled in rapidity for each rise of 10 C., whereas in the case of enzymes it becomes increased up to a certain opti- mum temperature, beyond which, as the temperature rises, the reaction is first slowed and then disappears altogether. This peculiarity of enzymes as compared with inorganic catalysts need not in itself disprove the analogy between the two, because enzymes do not form true, but colloidal solutions. Colloidal solutions, as we have seen, are really fine suspensions of ultramicroscopic particles ; there is no splitting into ions of the dissolved substance, as is the case with true (molecular) solutions, but the colloid is suspended in the water or other solvent to form a heterogeneous system (page 52), on which account the surface area of the menstruum is enormously increased. Rise in temperature alters the extent of the surface area, and thereby intro- duces an influence which progressively opposes catalysis. Although inorganic catalysts in molecular solution show no optimum temperature but increase in activity in proportion as the temperature is raised, inorganic colloidal catalysts may show an optimum temperature. Thus, spongy platinum shows an optimum temperature in its action on a mixture of hydrogen and oxygen. It has therefore been suggested that it is because they are colloids that enzymes exhibit this property. 3. Inorganic catalysts frequently carry the reaction to a further stage than that attained by the action of enzymes. For example, acid breaks down the protein molecule niuch more completely than -do the proteolytic enzymes. This difference is perhaps explained by the fact that enzymes are retarded in their activities when there comes to be a certain accumu- lation of the products of the reaction present. The final stages in the reaction may become so slow as to be almost inappreciable. This de- crease in activity is partly due to a union between the enzyme and the products of its activity. FERMENTS, OR ENZYMES 75 4. The velocity constant in the case of inorganic catalysts remains un- changed throughout the reaction, whereas in the case of enzymes it be- comes either less or greater as the process proceeds. When a substance is changed by catalytic action, it is, of course, constantly being diminished in concentration so that less and less of it remains to be acted on. This implies that there are fewer molecules present for the same amount of catalyst to act on and consequently that the amount changed in a unit of time is progressively less. At any moment, therefore, the rate of catalysis will be proportional to the amount of substance (substrate) left. To understand this we must refer back to what we have learned about mass action. If we suppose that two substances A and B react to form two other substances C and D, then, by the law of mass action, the reac- tion will not go on to completion but will stop when a certain equilibrium is reached. The reaction can be represented by the equation A + B + C + D, which means that it proceeds at a rate proportional to the reacting molecules. In some cases this reaction goes on until either A or B has practically disappeared (that is, the equilibrium point is very near the right of the equation), as is the case in the inversion of cane sugar: C 12 H 22 O n + H 2 = C 6 H 12 6 + C 6 H 12 6 Taking place as it does in an excess of water, and there being very little tendency for the reaction to go in the opposite direction (cf. re- versible action page 25), the only thing which will influence its velocity is the concentration of cane sugar; in other words, the velocity of the reaction at any moment will depend on the concentration of the cane sugar still left undecomposed. This can be determined by means of an equation.* The value of such an equation is that it gives us a figure K, represent- ing the amount of inversion that would occur in each unit of time if the cane sugar were kept in constant concentration. When, for example, it is stated that K for a particular strength of acid acting on cane sugar *If x be the amount of sugar inverted in time t, C, the concentration of the sugar not yet in- verted, and if we use a figure called a constant (K) to express the fundamental rate of the reaction (which will therefore be different for different reactions), then r= KC. But C can not be the same at any two consecutive periods of time, because the reaction is going on continuously. This renders it necessary to use the notation of the differential calculus, and we have -= = KC. The sign 8 ot indicates that the reaction is a constantly changing one so that 5x and 8t represent such infinitely small amounts that they can not be measured. By methods of integration, however, it can be shown that the above equation may be written: K=-r^r">*- nat - --' thus permitting us to find the value of K (Cj. Cz being the concentrations of cane sugar at the times T! T 2 ). Any two determinations during the course of the reaction can be used for calculating K. These equations apply only to cases in which but one substance 5s changing (monomolecular reaction). When two substances are involved, the equation is more complicated. 76 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES solution is 0.002, this means that when volume, concentration of acid and temperature are constant in a gram-molecular solution of sugar, 0.002 gram-molecule of sugar would be inverted the first minute and 0.002 gram each succeeding minute, provided we could keep the solution con- stantly a gram-molecular one, that is, provided we could add sugar just as quickly as it becomes inverted. At first sight it may appear of little practical importance to determine K. In our present discussion concerning the nature of enzyme action, it is however of great value for, whereas with inorganic catalysis K is really of constant value, with enzyme action it is not so. Thus, when cane sugar is inverted by sucrase an enzyme present in the intestine and in yeast the constant gradually rises; for most other unimolecular reactions mediated by enzymes it gradually falls ; for example, the action of trypsin on proteins. Where there is a great excess of substance to be acted on, in compari- son with the amount of enzyme present, it will be found that a more constant value than K is obtained when we compute the absolute amount of substance decomposed in a given time. In such a case, too, the amount of change in a given time will be proportional to the amount of enzyme present, indicating that some sort of combination between en- zyme and substrate must be the first step in the fermentative process. This fact has been noticed by us in connection with the hydrolysis of glycogen in the liver. When there is an excess of glycogen present, the amounts which disappear in equal intervals of time after death are the same; when, on the contrary, there is not much glycogen, the amount which disappears gradually declines, but, if K be computed by the above equation, it is constant. To make these facts clear it may be well to pause for a moment to consider an illustration. The conditions obtaining when there is a large excess of substrate over enzyme may be compared to those governing the removal of a pile of bricks from one place to another by a number of men. The pile of bricks represents the substrate ; the men, the enzyme. If each man works up to his capacity, it is plain that the number of bricks transferred in a given time will not depend at all on the size of the pile to be transferred. When, however, the pile of bricks gets small, though the same number of men continue to work the number of bricks transferred in a given time falls off, because the men interfere with one another's activities in securing their loads from the pile. When a similar stage is arrived at in enzyme processes, we have to use the velocity con- stant to show how much work could be done by the enzyme if the amount of substrate were maintained of constant amount. In the large volume of recent work which has been done with the FERMENTS, OR ENZYMES 77 object of discovering the cause of these variations in the velocity con- stant in the case of enzymes, four important conditions have been recog- nized: (1) reversibility; (2) gradual destruction of the enzyme; (3) com- bination of the enzyme with products of the reaction; (4) autocatalysis. Of these four influences the only one which could be held accountable for an increase in the activity of the enzyme is autocatalysis; in this process the enzyme by its action produces substances which intensify its own activity. In some cases at least for example, the action of invertase on cane sugar these are acid bodies, a moderate increase in acidity favoring the action of this enzyme. The other influences all tend to retard the reaction and progressively lower the value of K. Negative autocatalysis occurs when the enzyme produces products which interfere with its activity. Gradual destruc- tion of the enzyme and its union with the products of its activity will manifestly also decrease its power. There is plenty of evidence that both of these processes may occur. Reversibility of Enzyme Action But the most important of all the causes of retardation of enzyme activity is undoubtedly reversibility of action, which is an application of the law of mass action (page 25). If we take the saponification of an ester, the equation is: CH 3 CH 2 CH 2 COOC 2 H 5 + H 2 O => CH 3 CH 2 CH 2 COOH + C 2 H 5 OH. (ethyl butyrate) (butyric acid) (ethyl alcohol) The equilibrium point is not so near the position of complete hydrol- ysis as in the case of the inversion of cane sugar; in other words, the tendency for the bodies produced by the hydrolysis to reunite and form the original substances is quite marked, so that the reaction comes to an end before all the ethyl butyrate has been decomposed. For some time before the equilibrium point is reached, there will have existed a progres- sively increasing opposition to the breakdown of the ester, as a conse- quence of which, when enzymes are used to accelerate the reaction, the velocity constant, as determined by the above equation, will gradually fall as the reaction proceeds. Conversely, in a mixture of ethyl alcohol and butyric acid there is very slow synthesis to ethyl butyrate, and here again lipase accelerates the process; it induces a recognizable synthesis within a short time. Ethyl butyrate is usually employed for these ex- periments because, on account of its odor, the ester is readily recognized. Thus, if the alcohol and acid be mixed alone, no ester will be detectable, but if some lipase be added, it will soon become so. Similar synthetic action of lipase has also been demonstrated for mono- and tri-olein. 78 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES It should be clearly understood that pure catalysts, such as the hydro- gen ion, in accelerating a reaction like the above, do so equally in both directions, so that the position of equilibrium remains unchanged. En- zymes may, however, cause this position to change because of their form- ing intermediate combinations. The reverse phase of certain reactions is probably the cause of at least some of the synthetic processes which occur in the animal body. A great difficulty in accepting such a view, however, is the fact that the equilib- rium point of all hydrolytic reactions, in the presence of an excess of water, is so near complete hydrolysis that very little synthesis can be possible. That is true so long as the substance synthesized is soluble, but if it is nearly insoluble in water, or if it is immediately removed from the site of the reaction by diffusion, or in any other way, then it is obvious that it will go on being synthesized by the reaction. Thus, in the intestine neutral fat is hydrolyzed by pancreatic lipase into fatty acid and glycerol, which are absorbed into the epithelium, where they again come under the influence of intracellular lipase. This latter will tend to accelerate the synthesis of neutral fat from the fatty acid and glycerol until the equilibrium point of the system (fat acid + glycerol + neutral fat + H 2 0) is again reached; but this point, although it is near the right hand of the equation, will really never be reached for the reason that the neutral fat, as quickly as it is formed, will become deposited in insoluble globules in the protoplasm and thus be removed from the equation. In support of this view it has been found that lipase is present in intestinal mucosa after all traces of adherent pancreatic juice have been washed away. By similar reactions the fat of the tissues becomes decomposed to fatty acid and glycerol and passes out of the blood when the concentra- tion of fat in this fluid falls below a certain level. Provided one of the substances synthesized is insoluble or can in some other way be removed from the reaction, it is plain that, even though the equilibrium point is very near to that of complete hydrolysis, yet the reversion will be suf- ficient to do all that is required of it. Results such as the above have prompted many to conclude that it is by such reversible action that all synthetic processes occur in the living organism. But the demonstrable synthesis of an ester must not be taken as evidence that all other syntheses are explainable on the same basis. For example, we have seen above that in the case of cane sugar the equi- librium point in the equation is so near that of complete hydrolysis, that no measurable amount of cane sugar is formed when dextrose and levulose are allowed to act on each other, and that cane sugar does not appear when sucrase is added to the mixture. If instead of sucrase we take another of the sugar enzymes namely, maltase, which accelerates the FERMENTS, OR ENZYMES 79 decomposition of maltose into two molecules of glucose there is, how- ever, evidence of synthesis as a result of the acceleration of a reversible reaction. To understand these results we must remember that ordinary dextrose is a mixture of two stereoisomers designated a and j8. "When two molecules of a dextrose condense (that is, fuse togther with the loss of a molecule of water) maltose is formed, but when two molecules of /3 dextrose condense isomaltose results. There is some controversy as to whether maltase is really responsible for the synthesis of a dextrose molecules to maltose, it being claimed by some that this is accomplished by another enzyme, emulsine. If this were true it would materially minimize the importance of reversible action as a factor in cellular syn- thesis. The latest evidence goes to show, however, that it is maltase and not emulsine that is responsible in the above case (cf. Bayliss). Evidence, both direct and indirect, is also steadily accumulating to show that enzymes may accelerate the synthesis of proteins. As pieces of indirect evidence we have: (1) the retardation of the digestive action of trypsin, etc., which sets in after the process has gone on for a time, and (2) the recommencement of a digestive process apparently at an end, if the products of the digestion are removed by dialysis or other means. As direct evidence may be cited the formation of synthetic products when pepsin is added to concentrated solutions of peptone, and the diminution in the number of small molecules, as judged by meas- urements of electrical conductivity, when trypsin is added to the prod- ucts of tryptic digestion of caseinogen. Protamine a simple form of pro- tein has also been found to be produced when trypsin obtained from a mollusc was added to a tryptic digest of the same protamine. The significance of these facts in connection with the metabolism of the amino acids will be evident when we -come to study this subject (page 634).* Specificity of Enzyme Action Although in all of the above features of enzyme action there is nothing to contradict the view that they are catalytic agents, there remains one peculiarity which at first sight seems uninterpretable on such a basis. This is with regard to their often remarkable specificity of action. Thus, as we have seen, maltase can hydrolyze maltose alone (which is com- posed of two a-dextrose molecules), but not isomaltose (composed of ^-dextrose). This means that mere difference in the configuration of molecules is sufficient to alter the influence of enzymes on them. Since such differences could not influence that of inorganic catalysts we must We have been unable in this laboratory to demonstrate any synthesis of cogenase is added to a hydrolysis mixture of dextrine, maltose and glucose produced by the action of glycogenase on pure glycogen. 80 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES explain the cause of the difference. This has been done on the basis either that enzymes are colloids or that the active (catalytic) group of the enzyme is attached to a colloid molecule. Before a substance can be acted on, it must combine with the colloid, which it does by the proc- ess of adsorption (see page 66). This can occur, however, only when there is a harmony between the adsorbing substance and the substance adsorbed. Instances of the specificity of adsorption have already been given. In support of this view it has been found that of the two proteases, a and /?, in the spleen, one is adsorbed but not the other when a solu- tion containing them is shaken with Kieselguhr. Furthermore, when solutions of invertase are shaken with certain inert powders, the in- vertase is adsorbed by some of them but not by others. In strong sup- port of the adsorption hypothesis is also the fact that the same mathe- matical laws as apply in the process of adsorption are obeyed in the ratio which exists between the activity of an enzyme and its concen- tration in the solution. To sum up, then, catalysis as exhibited by enzymes involves three processes: (1) contact between the enzyme and the substrate, which will be dependent on their rates of diffusion; (2) adsorption between them, which will depend on their configurations (cf. the lock and key simile) ; and (3) the chemical change which itself probably takes place in two stages. In connection with the third process, it is probable that an initial com- pound of a definite chemical nature is first formed, followed by the hydrolytic or other chemical change, after which the enzyme group becomes free. It is very significant in this connection to note that in their solubil- ities there exists a distinct relationship between the ferments and the substrates on which they react. Thus, trypsin is very soluble in water and acts on water-soluble proteins; lipase is soluble in fat solvents. Certain Peculiarities of Enzymes Notwithstanding the very strong case that is made out for the cata- lytic hypothesis, there are certain facts which many find it difficult to make conform with such a view. One of these is that dextrose can undergo three distinct and separate types of decomposition according to the enzyme allowed to act on it. These are alcoholic fermentation, butyric acid fermentation and lactic acid fermentation. It is difficult to see how simple catalytic action can be responsible for all three results. The enzyme must not only initiate the changes but also direct their course. Another peculiarity is that when certain enzymes e. g., rennin, pep- FERMENTS, OB ENZYMES 81 sin, etc. are inoculated in animals, they cause specific antienzymes to appear in the blood of the inoculated animal. Thus, when antirennin serum is added to milk it greatly hinders clotting on the subsequent addition of rennin. It is probable that powerful antienzymes are pro- duced in the animal body for the purpose of protecting the tissues from attack by enzymes. It is on account of the presence of antienzymes that intestinal parasites can exist in the intestine, and the immunity from digestion which the mucosa of the gastrointestinal tract enjoys, is believed to be due to the same cause. But there is considerable doubt regarding this claim. Fresh pancreatic juice when injected into the empty intestine digests its walls. When food is present in the intes- tine it evidently prevents digestion of the walls by diverting the enzyme to itself. Types of Enzymes Having learned something about the general nature of enzyme action, we may now turn our attention to certain details that have a practical importance. In the first place, with regard to nomenclature, in the earlier work each newly discovered enzyme received a name which was often quite inappropriate. Many of these names are retained, such as pepsin, trypsin, ptyalin, etc., but it is now customary to name the enzyme according to the substance on which it acts. This is done either by replacing the last part of the name of the substance acted on by the termination -ase (for example, the enzyme which inverts maltose is called maltase), or by merely adding -ase to the name of the substance acted upon (thus, the enzyme which hydrolyzes glycogen is called glycogenase). Most of the enzymes in the animal body accelerate hydrolytic proc- esses and are classified according to the chemical nature of the sub- strate on which they work. Thus, we have: 1. The amylases accelerating the hydrolysis of polysaccharides, e. g., ptyalin (in saliva), amylopsin (in pancreatic juice), glycogenase (in liver), diastase (in malt). 2. The invertases accelerating hydrolysis of disaccharides, e. g., malt- ase, lactase and sucrase (in succus entericus). 3. The proteinases accelerating hydrolysis .of proteins, e. g., pepsin (in gastric juice), trypsin (in pancreatic juice), erepsin, intracellular proteinases. 4. The Upases accelerating disruption of neutral fats, e. g., steapsin (in 'pancreatic juice), intracellular lipases. 5. Arginase accelerating hydrolysis of arginin into urea and or- nithin, (intracellular). 82 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES 6. Urease accelerating hydrolysis of urea to ammonium carbonate (in many microorganisms and in the soy bean). 7. Glyoxylase converting glyoxals into lactic acid (page 698). Other enzymes accelerate oxidative processes and are called oxidases and per oxidases. Others bring about the displacement of an amino group by hydroxyl (desamidases) . Others cause coagulation (coagula- tive ferments), e.g., thrombin, rennin. One of the enzymes present in succus entericus acts by converting the zymogen (trypsinogen) into the enzyme (trypsin). Enzyme Preparations So far it has been impossible to prepare enzymes in a pure state al- though, being colloidal in nature, they are readily precipitated or ad- sorbed along with other colloids. Since most enzymes exist in cells, it is necessary to break up the cells in order to isolate the enzyme. This is done in various ways. By one method the cells are ground in a mortar with fine sand, then made into a paste with infusorial earth (Kieselguhr), the paste enclosed in stout canvas and placed under an hydraulic press at about 300 atmospheres pressure; a clear fluid separates and this contains the enzymes. An- other way is to freeze the tissue with liquid air and grind it in a steel mortar by means of a machine. Still another and less expensive method, and one which we have found most useful for organs and tissues, con- sists in reducing the tissue to a pulp and, after sieving it to get rid of connective tissue, etc., spreading the pulp on glass plates and drying in a slightly warmed, dry air current. The scales of dried material are then ground in a paint mill with toluene, and the resulting suspension filtered ; the powder which remains on the filter, after thorough washing with toluene, is dried and kept for future use. The toluene removes all the fatty substances, so that when shaken with water, etc., the enzymes dissolve. Conditions for Enzymic Activity Reactions brought about by intracellular enzymes are very readily inhibited when there comes to be a certain accumulation of their prod- ucts of action. Thus, yeast ceases to ferment sugar when the alcohol has accumulated to a certain percentage. This action is partially due to a toxic action of the alcohol on the cell, which paralyzes its power of absorbing the substance to be acted on by the intracellular enzyme. If these products be not in some way removed, they will ultimately kill the cell and stop the fermentation. We have seen above how the ac- cumulation of products may interfere with the activities of enzymes in FERMENTS, OR ENZYMES 83 other ways in which the enzyme does not suffer destruction, as is shown by the fact that it resumes its original activities on removal of the products. Enzymes, both intracellular and extracellular, are very sensitive to- wards the inorganic composition of the medium in which they are act- ing. For the intracellular enzymes this is what we should expect when we bear in mind the profound influence of inorganic salts on the heart beat and on cell growth and division. This influence of salts and of reaction (acidity, etc.) on the life of the cell is so pronounced as to lead some observers to believe that abnormal cell multiplication in the body, as in the case of tumor formation, is due to changes in the inorganic composition of the tissue fluids. Extracellular enzymes are also very susceptible to the influence of inorganic salts but more especially so towards the reaction of the solution. In terms of modern chemistry we may say that the concentration of H- and OH' ions has a profound influence on the activities of enzymes. Most of the enzymes of the an- imal body perform their action normally in the presence of a slight ex- cess of OH' ions, that is, in faintly alkaline reaction. Indeed the only exception of importance to this is the pepsin of gastric juice, which nor- mally acts in an acid medium. An excess of either OH' or H- ions inhibits the activity of the enzyme and usually destroys it permanently. The activities of enzymes are also influenced by light, many of them being destroyed by sunlight; cells such as microorganisms are similarly affected. Before being secreted the digestive enzymes exist in the cells which produce them as inactive precursors called zymogens. The granules seen in resting gland cells are of this nature. The activation of the zymogen, or its conversion into the enzyme, occurs after it has left the cell, and this has been considered as another safeguard to digestion of the cell. Sometimes the activation does not occur until the zymogen has travelled some distance along the gland duct, as in the case of the proteolytic enzyme of pancreatic juice. Till it reaches the intestine, this exists as trypsinogen (the zymogen), but it is here acted on by another enzyme- like body produced by the intestinal epithelium and called enterokinase PHYSICOCHEMICAL REFERENCES (Monographs and Original Papers) 3 Bayliss, W. M.: Principles of General Physiology, Longmans, Green & Co., 1915. 2 Philip, J. C.: Physical Chemistry, Its Bearing on Biology and Medicine, Arnold, ed. 2, 1914. 3 McClendon, J. S.: Physical Chemistry of Vital Phenomena, Princeton University Press, 1917. 4 Starling, E. H.: Principles of Human Physiology, ed. 2, 1915, Lea and Febiger. 84 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES sKahlenberg, L.: Jour. Physical Chem., 1906, x, 141. eReid, E., Weymouth: Jour. Physiol., 1898, xxii, Ivi. . ^Wilson, T. M.: Am. Jour. Physiol, 1905, xiii, 150. sHaldane, J. S., and Priestley, J. G.: Jour. Physiol., 1916, 1, 296; Priestley, J. G.: Ibid., p. 304. sQlark, W. M., and Lubs, H. A.: Jour. Bacteriology, 1917, ii, 1 and 109. iHenderson, L. J.: The Excretion of Acid in Health and Disease, Harvey Lectures, J. B. Lippincott Co., 1915, x, 132. nHenderson, L. J.: The Fitness of the Environment, Macmillan, N. Y., 1913. i^Van Slyke, D. D. : Jour. Biol. Chem., 1917, xxx, 289, 347, 363. isLevy, R. L., and Kowntree, L. G.: Arch. Int. Med., 1916, xvii, 525. i4Cullen, G. E.: Jour. Biol. Chem., 1917, xxx, 369. ispalmer, W. W., and Henderson, L. J.: Arch. Int. Med., 1913, xii, 153; and Jour. Biol. Chem., 1912, xiii, 393; xix, 81; xvii, 305; xxi, 37. isSellards, A. W.: The Principles of Acidosis and Clinical Methods for Its Study, Harvard University Press, Cambridge, 1917. I'Lloyd, F. H.: Private communication. isMacallum, A. B, : Surface Tension and Vital Phenomena. University of Toronto Studies, No. 8, 1912; also Ergebnisse der Physiologic, 1911, ii, 598. iBayliss, W. M.: Enzymic Action, ed. 2. Monographs in Biochemistry, Longmans, Green & Co. 2oBayliss, W. M. : Neutralisation, etc. siHaggard, and Henderson, Y. : Jour. Biol. Chem., 1919. 22Christiansen, Douglas, and Haldane, J. : Jour. Physiol., 1914, xlviii, 246. 2^Morawitz, P., and Walker, J. C.: Biochem. Ztschr., 1914, Ix, 395. 2>*Macleod, J. J. K.: Jour. Lab. and Clin. Med., 1919, iv, 1. 25Bradford, S. C. : Biochem. Jour., 1918, xii, 351. 26Dale, H. H., and Evans, E. L.: Jour. Physiol., 1920, liv, 167. 27Evans, E. L. : Jour. Physiol., 1921, Iv, 159. 2Van Slyke, D. D.: Physiol. Rev., 1921, i, 141. sojoffe, J., and Poulton, E. P.: Jour. Physiol., 1920, liv, 129. soMatthews, A. P.: Physiol. Rev., 1921, i, 553. PART II THE BLOOD AND THE LYMPH CHAPTER X BLOOD: ITS GENERAL PROPERTIES (Partly Contributed by R. G. PEARCE) The blood, being the carrier of the nutritive and waste substances of the body's metabolism, must at one time or another contain all the ma- terials which compose the tissues in addition to those which are peculiar to the blood itself. It is a very complex fluid, and all of its constituents are not fully known. Structurally it is composed of water in which are dissolved various gases and organic and inorganic bodies, the corpuscles and platelets. THE QUANTITY OF BLOOD IN THE BODY The volume of blood in the body may be measured by bleeding and subsequently washing out the blood from the vessels and then estimating the amount of hemoglobin in the total fluid (Welcher's method). This method employed in the case of two criminals who had been decapitated gave the weight of the blood as 7.7 and 7.2 per cent of the body weight. Bloodless methods for determining the total volume of blood are based upon the principle of adding a definite quantity of a known substance to the circulation and then estimating its concentration in a sample of blood withdrawn from the body shortly afterward. If the substance can not leave the blood vessels and does not cause fluid to be withdrawn from the tissues, the total quantity of blood in the body can be calculated from the concentration of the injected substance in the blood. The most accurate methods based on this principle are Haldane and Smith's, in which car- bon monoxide gas is inhaled in a given amount and the carbon monoxide hemoglobin subsequently determined colorimetrically ; and Keith, 18 Rown- tree and Geraghty's, 20 which employs vital red, a dye of low diffusibility. The dye remains long enough in the body to be thoroughly mixed with the blood, and its concentration in the plasma is determined colorimetrically by comparing with a suitable standard mixture of dye and serum. These 85 86 THE BLOOD AND THE LYMPH methods give the total amount of blood in the body as from 5 to 8.8 per cent of its weight. Meek has recently developed a method in which gum acacia is used. After mixing with the blood, the concentration of this substance is determined from the calcium content. Being colloid, none of the gum leaves the blood vessels. Further details on methods will be found in the papers of Hooper, Whipple, etc. 22 The vital red method is employed as follows: A 1.5 per cent solution of vital red is preserved in sterile condition in 15-20 c.c. quantities. A needle is inserted in the basilic vein and 6-8 c.c. of blood withdrawn into a dry syringe containing finely pow- dered pot. oxalate. The syringe is removed from the needle, and another syringe con- taining an amount of the vital red solution corresponding to 3 mg. vital red per kilo body weight, is connected with the needle and the dye slowly injected. The blood removed in the first syringe is now transferred to a paraffined centrifuge tube, and in about 10 minutes a third syringe is connected with the needle and 8-10 c.c. of blood removed and transferred to two paraffined tubes. From one of these a specimen is taken for the hematocrit. The tubes are then centrifuged rapidly for some time. The dilution of dye in the plasma is now determined colorimetrically, using as a standard the fol- lowing: 1 c.c. diluted dye (0.5 c.c. of the original dye solution to 100 c.c. 0.8 per cent NaCl sol.). 1 c. c. plasma before injection 2 c. c. 0.8 per cent NaCl. and using as test solution 1 c. c. plasma after dye injection 3 c. c. 0.8 per cent NaCl. The formula for calculation is 200 X c - c - dye injected X 100 = c. c. plasma E Where R per cent reading of test solution. By use of the above method Keith, Rowntree and Geraghty found that in man the plasma normally constitutes 5 per cent or % the body weight, i.e., 50 c.c. plasma per kg. To determine total blood volume the hematocrit must also be used, and it was found that the total volume = = 8.8 per cent or l/11.4th of body weight. Normal individuals therefore have 85 c.c. blood per kg. In pregnancy, before term the blood and plasma volumes are increased, but within a week or so of delivery the volumes return to normal. In obesity the plasma and blood volumes are relatively small. Many cases of anemia exhibit a relatively larger plasma volume. Hypertension may be accompanied by a small blood volume, therefore this condition is not due to large blood volume. The newer methods have shown that the volume of the circulating fluid is maintained fairly constant in spite of influences tending to alter it. The body accomplishes this by drawing upon the reserve fluid in the tissues and by varying the rate of water excretion, particularly through the kidneys. Years ago the doctrine of an increased amount of BLOOD: ITS GENERAL PROPERTIES 87 blood in the body (plethora) gave rise to the therapeutic use of bleeding. Especially was this thought to be useful in conditions which we now recognize as chronic hypertension, and which show no increase in blood volume. Indeed variation in blood volume is not common, although plethora may occur in polycythemia, chlorosis, and anemias, and there may be a temporary reduction in the amount of blood in diseases in which there is a great depletion of water, as in Asiatic cholera, and fol- lowing very severe hemorrhage. While the total quantity of the blood in the body does not vary greatly, the concentration of its various constituents is subject to distinct, change. The volume percentages of the corpuscles and the plasma can be approx- imately determined by allowing oxalated blood to sediment or by cen- trifuging in a graduated cylinder by the use of the hematocrit. Such methods are not very reliable, but may yield some important information. Normally 45 to 50 per cent of the volume of blood is composed of cor- puscles. It varies more or less directly with the number of red blood cells. THE WATER CONTENT OF THE BLOOD Since the blood plasma is essentially a watery solution, some idea of its water content can be obtained by a determination of the specific gravity. The most accurate method for accomplishing this is to deter- mine directly the weight of a given volume of blood and compare it with the weight of the same volume of water. Since this method re- quires a rather large amount of blood, indirect methods using smaller amounts have been devised. One of these (Hammerschlag's) uses a solution of chloroform and benzol of a specific gravity of about 1.050, in which a drop of blood is suspended by delivering it cautiously from a pipette bent at right angles near its tip. If the drop sinks, chloroform is added; if it rises, benzol is added until the drop remains suspended. The specific gravity of the benzol-chloroform mixture is then determined, and this value is supposed to give the specific gravity of the blood. The specific gravity of the blood determined in this way varies be- tween 1.040 and 1.065. It is somewhat less after eating and increases after exercise; it is slightly lower during the day than at night, and the variation in individuals is considerable. The changes which occur in the specific gravity of the blood in disease are chiefly due to variation in the percentage of protein, since the salt content of the blood is rela- tively fixed. It is only when great changes occur in the concentration of the noncolloidal salts that they markedly affect the specific gravity. From 90 to 92 per cent of the plasma and from 59.2 to 68.7 per cent of the corpuscles consist of water. Of the whole blood, from 60 to 70 per cent 88 THE BLOOD AND THE LYMPH by volume or about 55 per cent by weight consists of plasma; and from 40 to 30 per cent by volume or 45 .per cent by weight consists of cor- puscles. THE PROTEINS OP THE BLOOD The plasma obtained by centrifuging the blood rendered noncoagula* ble by oxalates, hirudin or other means (see page 100), contains 5 to 8 per cent of coagulable proteins. These proteins are serum albumin, serum globulin, and fibrinogen. They can be separated from each other by the use of acids and neutral salts. Their proportion varies under dif- ferent conditions, but is approximately as follows : Fibrinogen, 0.15-0.6 per cent; serum globulin, 3.8 per cent; serum albumin, 2.5 per cent. Fibrinogen The least soluble of the blood proteins is fibrinogen. The plasma is almost freed of it by half-saturation with sodium chloride, or with a small amount of acetic acid. It is precipitated as fibrin in the process of blood coagulation (see page 102), and is estimated by weighing the amount of fibrin which it produces. Pibrinogen disappears rapidly and the blood fails to clot when the liver is removed in Eck fistula dogs (see page 651). It also diminishes in CHC1 3 and P. poisoning. If blood is withdrawn and then defibrinated and reinjected fibrinogen reappears in twenty-four hours. Serum Globulin and Serum Albumin Globulins are ordinarily defined as being insoluble in distilled water, and albumins as being soluble. It is, however, impossible to separate serum globulin and albumin satisfactorily in this manner. The globu- lin obtained by dialysis can be returned to solution by the addition of a suitable amount of water, which makes the salt adherent to the pre- cipitate a weak saline solution. In neutral or acid solutions it is coag- ulated by heat at about 75 C. But it does not act as an individual pro- tein, since a portion of it is precipitated by dialysis or by carbon diox- ide. Probably serum globulin really consists of two or more proteins. The serum albumin remaining in solution after saturation with am- monium sulphate likewise does not represent a chemical entity. It is possible by carefully heating the solution of serum albumin to distin- guish three separate coagulation temperatures. This fact has been in- terpreted as meaning that the serum albumin consists of at least three closely related proteins. Since the refractive index of the Mood depends primarily upon the amount of protein present, this has been taken as a means of determining BLOOD: ITS GENERAL PROPERTIES 89 variations in the concentration of the proteins. It has been found that the concentration of the blood proteins varies somewhat; during ex- ercise it is increased probably because of the taking up of water by the tissues, and during profuse bleeding it is diminished because large amounts of fluid are being added to the blood from the lymph, which is relatively poor in proteins. The ingestion of considerable amounts of salts has been found to reduce the concentration of the blood proteins for a short time. In pathological conditions, as in diabetes, when rapid changes in the body weight due to alterations in the diet are oc- curring, changes in the fluid content of the blood are often observed. Likewise in edema caused by faulty renal function, there may be a re- tention of fluid in the blood before there is any indication of edema. The hydremic condition of the blood can therefore be considered as a useful diagnostic aid in determining the water metabolism. The relative concentration of the proteins of the blood is also of some interest, especially since in certain diseases a considerable amount of blood protein is lost. By refractrometric methods it is possible to sep- arate the globulin and albumin fractions. Normally the total proteins range between 6.7 and 8.7 per cent, of which the albumins lie between 4.95 and 7.7 per cent, and the globulins between 1 and 2.54 per cent. In some diseases, as in chronic nephritis, pneumonia, and syphilis, the total proteins of the blood are decreased and the relative amount of serum globulin is increased On the other hand, in many mild infections and chronic septic conditions the globulin fraction may be increased with no change occurring in the total protein content. 3 Serum Proteins disappear from the blood when the animal is bled and simultaneously transfused with washed corpuscles suspended in saline. If the blood be removed in several moieties during the day, it will be found that 1 per cent of the protein will have reappeared next day, after which it will reappear more slowly taking 5-7 days to return to normal. If the bleeding be performed at one period, however, there will be a very rapid return (within 15 minutes) of a part of it, which indicates the presence of a reserve store of proteins somewhere in the body. A slower regeneration of the remaining part then follows. This regeneration proceeds at about the same rate as that at which the liver regenerates after being damaged by CHC1 3 or P., which suggests a rela- tionship of the liver to the regenerative process of blood proteins. This is further supported by the fact that regeneration of blood proteins is decidedly delayed after the liver has been damaged by P. or CHC1 3 . Further evidence of the relationship of the liver to blood proteins is obtained by the fact that pronounced damage to the liver lowers blood protein somewhat and also that Eck fistula clogs regenerate protein more 90 THE BLOOD AND THE LYMPH slowly than normal animals. Regeneration is more rapid and complete on a meat or mixed diet than it is during fasting. Regeneration is also more rapid on a meat diet than on one of bread. Depletion of serum proteins below 1 per cent is fatal. THE FERMENTS AND ANTIFERMENTS OF THE BLOOD The blood plasma contains many of the ferments present in the tissues. The nature of these ferments has been the subject of many investiga- tions in recent years, primarily because it has been found that they are intimately connected with the problems of immunity. Among the ferments the following have been demonstrated in the blood: Proteases are probably present normally in the human blood serum in small amounts, but they are found in large amounts in the white blood corpuscles. A protein foreign to the body if injected into the blood ordinarily produces no untoward symptoms, but a second injec- tion following the first by some days will produce symptoms of poison- ing known as anaphylaxis. This fact has led to the assumption that the injection of any foreign protein into the blood promptly leads to the appearance therein of specific proteolytic enzymes which will digest the strange protein into its derivatives, which are poisonous. This power of the body to produce specific proteases has been the subject of much research and debate, and Aberhalden proposed a test for preg- nancy, for cancer, and for other conditions in which he made use of this phenomenon. He believes that the presence of placenta or tumor tissue causes proteins to be formed which bring about the production of specific ferments whose duty it is to rid the system of these substances. Other investigators fail to find the specificity in proteolytic action claimed by Abderhalden, and believe that proteolytic ferments which are capable of digesting foreign proteins are absorbed from the alimentary canal from the digestive juices (Boldyreff ) . Some investigators fail to confirm the claim that the proteolytic activity of the blood serum is increased under the above conditions. Blood contains an antiferment known as antitrypsin. This can be removed from the blood serum by several substances, among which are kaolin, colloidal iron and starch. Serum thus treated shows strong pro- teolytic activity and autodigestion will occur. In this case there can be no question of the specific origin of proteases. Abderhalden believes that the ferments of the blood of the pregnant woman are able to digest the placental tissue. Human placental tissue has the ability of absorb- ing antitrypsin and it is very questionable as to whether the test pro- posed by Abderhalden is due to the new formation of ferments or to BLOOD: ITS GENERAL PROPERTIES 91 the removal of the antitrypsin and the action of the protease normally present in the blood. Nuclein ferments are capable of decomposing nucleic acid and purins into the simpler bodies. Lipases have been demonstrated in the blood. Amylase. The presence of starch-splitting ferments in the blood was first shown by Magendie in 1841, and later Bernard showed that gly- cogen or starch injected into a vein produced glycosuria. Since then it has been proved conclusively that diastatic enzymes are normally present in the blood and lymph. The source of these enzymes has given rise to much speculation. Some observers believe that they are derived from the amylopsin of the pancreatic secretion, while others believe that they are manufactured by the liver. Ligature of the pancreatic ducts is said to increase the amount of amylase, while removal of the pan- creas may (Carlson and Luckhardt) or may not (Schlesinger) increase the amylase of the blood. In some forms of experimental diabetes the amylase of the blood has been found increased, and this is the case in human diabetes (Myers and Killian). If this is true, a cause for the inability of the diabetic to store up glycogen is found. In impairment of renal function, there is usually an increase in the blood amylase and a decrease in the urine amylase. This has been suggested as being of diagnostic value. The blood contains a feeble glycolytic enzyme capable of destroying glucose. It is claimed that this power is reduced in diabetics (Lepine). Catalase is found in the blood and tissues generally. It has the power of liberating oxygen from hydrogen peroxide without any accompany- ing oxidation process. Its physiological significance is not known. It is said that the amount of catalase is increased during excitement and exercise, and is decreased in conditions where the body's activity is lowered. Its determination is clinically unimportant at present. CHAPTER XI BLOOD: THE BLOOD CELL (Contributed by R. G. PEARCE) THE RED BLOOD CORPUSCLES, OR ERYTHROCYTES The most prominent function of the blood is to carry oxygen to the tissues. It owes this property chiefly to the red blood cells which are present in large numbers (5,000,000 per c.mm. of blood). These cells are biconcave discs, having a diameter of about 7.7 /A. They are con- structed out of a framework composed largely of lipoid material, in the meshes of which is deposited a substance called hemoglobin, to which the remarkable oxygen-carrying power of the blood is due. Nei- ther the manner by which the red cell carries its hemoglobin nor the intimate structure of the cell itself is accurately known. It is com- monly believed that the hemoglobin is held enmeshed in a framework or stroma, or encased in the cell membrane. One thing is certain, how- ever, that the union of hemoglobin with the stroma of the red cell is a fairly strong one, since mere fragmentation of the corpuscle fails to liberate the hemoglobin. The fact that the framework contains a large amount of lipoid substances enables the corpuscles to maintain their shape and is responsible for their characteristic permeability. Hemoglobin is a very complex substance belonging to the group of conjugated proteins. By chemical means it can be broken up into a simple globulin and a pigment hematin, containing iron. When com- pletely saturated, oxygen is present in hemoglobin in the proportion of two atoms of oxygen to one atom of iron (Peters) ; or 401 c.c. of oxygen can be carried by hemoglobin containing one gram of iron, the molecular weight of the molecule being about 16.669, or some multiple thereof (Barcroft and Peters) (see also page 392). At this figure the iron in the molecule would represent 0.34 per cent of the total weight of the molecule. The corpuscular surface area has been estimated to be 3200 square meters. There is therefore a very large surface avail- able for the absorption of oxygen from the alveolar air, as the blood corpuscles pass in single file through the capillaries of the lungs. Since the amount of oxygen which the blood can carry depends upon its hemoglobin content, it is of some importance clinically to have THE BLOOD CELL 93 methods of determining the approximate amount present. The amount of hemoglobin present in a quantity of blood is usually determined colorimetrically by comparing the color of the blood with standard col- ors which correspond to known strengths of hemoglobin. In normal persons the amount of hemoglobin varies greatly at different ages, and in order to determine whether or not a given blood contains more or less hemoglobin than normal, it is imperative to consider the age. The greatest variations occur between birth and the sixteenth year. After the sixteenth year the blood in males usually contains a larger amount than that in females (Williamson 4 ). Instruments used in determining the amount of hemoglobin should be standardized to give the value in grams hemoglobin per 100 c.c. of fluid. The amount of hemoglobin which is present in each corpuscle in terms of normal is therefore of some clinical interest. This relation of the number of red cells to the amount of hemoglobin is known as the color index and is computed as follows: The average red count in man is 5,000,000 to the c.mm., and the average minimal amount of hemo- globin is taken as 13.88 grams in 100 c.c. of blood (=80, Sahli; =90, Miescher; =86, Plesch; and 110, Tallquist methods). These relative values give a color index of one. The percentage of normal red cells divided by the percentage of normal hemoglobin present gives the color index. The Origin of the Red Blood Cells In fetal life the spleen and the liver are generally believed to be re- sponsible for the formation of the red blood cells. In extrauterine life this function is taken over by the red bone marrow. In the primitive condition all red blood cells are supposed to be nucleated. In extra- uterine life the nuclei of the red cells are lost, and nonnucleated forms are alone present in the blood stream. In fetal life and in certain path- ologic conditions, the rate of blood formation is so rapid that some nucleated cells appear in the blood. The normal response of the body to a loss of red blood corpuscles consists in an increased activity of the blood-forming cells of the red bone marrow. It is not easy to follow the course of the regeneration of the red corpuscles or to discover the mechanism of their formation in the bone marrow, since this tissue pre- sents a mixture of cells which are precursors of the varied corpuscles found in the blood and the identity of which can not be determined. Recently new methods of staining blood for microscopic examina- tion have allowed more detailed study to be made on the site and method of blood cell formation. When fresh unfixed blood is treated with solutions of various dyes, such as brilliant cresyl blue, polychrome 94 THE BLOOD AND THE LYMPH methylene blue or neutral red, an otherwise invisible structure appears in some cells in the form of coarse granular particles or threads, which give a reticulated appearance to the corpuscles. These reticulated cells are more abundant in infants' blood and in patients suffering with se- vere anemia or hemolytic jaundice than in normal blood, and may be taken as evidence of the youth of the red cell and not as a degenera- tive process. Since the number of the reticulated cells that are present in the blood is more or less directly proportional to the hemopoietic activities of the bone marrow, enumeration of the reticulated cells is of clinical importance in anemias. In conditions in which animals have been made plethoric by the transfusion of blood, it has been found that the number of reticulated cells is decreased; the bone marrow of these animals also shows a marked reduction in reticulated erythroblasts. The diminished rate of blood cell formation sometimes noted after blood transfusions may be explained by assuming that the stimulus which awakens the formation of red cells in the bone marrow is absent or made subnormal on the injection of red cells into the blood, and thus the formation of red cells is depressed. Small transfusions are there- fore preferable to large ones in cases in which the rate of blood forma- tion is greatly impaired. By means of living cultures of red bone mar- row the different stages of the development of the normoblasts into true red corpuscles may be studied (Tower and Herm 5 ). Some evidence has been gathered from such studies which points to the conclusion that in place of the red cells being cells which have lost their nucleus, as is the current teaching, they are rather cells which develop as a nuclear bud and escape into the circulation as true red cells. The nucleated red cell and the red nucleated corpuscle of the bird are the product of intranuclear activity and are morphologically identical. Rates of Regeneration of Erythrocytes Microscopic examination of the blood during rapid regeneration of red cells shows the presence of nucleated forms. Nucleated red cells in the blood have therefore been taken as an inevitable feature of rapid blood regeneration. The evidence upon which this belief depends, however, is hardly complete, since changes in the manner of red blood cell formation may be responsible for the nucleated forms. The red bone marrow is considered the seat of red cell formation, and it is true that an abnormal increase in the red bone marrow usually accompanies increased red cell formation. The nature of the stimulus which brings about the new formation of red cells is not understood. Oxygen want may be an important factor, since we find the presence of an abnormally large number of red cells in conditions where there is a scarcity of THE BLOOD CELL 95 oxygen in the- inspired air, as in life at high altitudes, or a difficulty in its absorption through the lungs, as in congenital heart disease. The red cells produced following hemorrhage and in simple anemia contain less than the normal amount of hemoglobin, but their shape and size are approximately normal, and few nucleated cells are present. In the regeneration of red cells which is found in pernicious anemia, we find the cells containing an unusually large amount of hemoglobin. The red cells in this disease have abnormal forms, many being large, with or without a nucleus, and containing basic staining granules. This type of blood cell formation is due to degenerative changes. The Fate of the Erythrocytes The length of life of the red blood cell is unknown. Estimates based upon the daily excretion of bile pigments are not reliable, since Hooper and Whipple have shown that the pigments, in part at least, arise from pigments which the liver has made in excess of its needs for the manu- facture of hemoglobin, and which, not being needed, are excreted. 15 There is no question however that every erythrocyte sooner or later undergoes disintegration, a process formerly thought to be ushered in by the ingestion of the red blood cell by a phagocyte in the spleen or in a hemolymph gland, the hemoglobin of the disintegrated cell being set free and carried to the liver, where it is broken up into hematin, which the body stores for future use, and into- bile pigments, which are ex- creted. Rous and Robertson 6 fail to find evidence that this process occurs in man to an extent sufficient to account for the normal destruc- tion of the blood cells. However they have recently found another and unsuspected method for blood destruction in all animals thus far studied namely, the disintegration of the blood cells by fragmentation while they are circulating, without loss of their hemoglobin. These fragmented cells are found most frequently in the spleen. They believe that the small ill-formed cells, known as microcytes and poikilocytes, observed in severe experimental anemias, are due not to the fact that they are produced by the bone marrow, but rather to the fact that the marrow in its anemic condition is not able to produce a resistant ery- throcyte, and fragmentation therefore takes place too readily. A sim- ilar condition may exist in the severe anemias of man and account for the general high resistance of the red cells found in the blood of these patients, inasmuch as the weak cells are generally fragmented very soon after they are formed. Long ago Ehrlich stated that the microcytes and poikilocytes of anemia are the result of fragmentation of the cells in the circulating blood, but he believed that this fragmentation was a 96 THE BLOOD AND THE LYMPH purposeful division in order to increase the total surface of the red cells. The ultimate fate of the red cell fragments is not known. It is reasonable to suppose that the fragmented bits containing hemoglobin are carried to the liver, where the hemoglobin is transformed into hematin and bile pigments. Hemolysis Another method of red blood cell destruction, which, however, does not take place normally, is by hemolysis. The nature of the combina- tion of the hemoglobin with the stroma of the red cell, as already re- marked, is not definitely known. That it is not merely contained in a sac is shown by the fact that the cell may be cut into bits without the hemoglobin being set free. In some manner the hemoglobin is chem- ically bound with the stroma of the red cell, from which it can be freed by a number of physicochemical and chemical agents. This proc- ess is known as hemolysis, and the substances which bring it about are known as hemolytic agents. The manner in which these agents effect the release of hemoglobin from the blood is quite varied. If the osmotic pressure of the plasma is lowered by dilution, the pres- sure within the corpuscle remains high, and water is absorbed by the cell. If this absorption is sufficient, the cell ruptures and the hemoglobin is discharged. For this reason it is necessary in diluting the blood to use solutions of salt having an osmotic pressure equal to that of the blood to protect the red cell from hemolysis. This is obtained by using a 0.9 per cent solution of sodium chloride. Better results are had, however, by using either Ringer's solution (0.9 per cent NaCl, 0.026 per cent CaCl 2 , and 0.03 per cent KC1) or Locke's solution (0.9 per cent NaCl, 0.024 per cent CaCl 2 , 0.042 per cent KC1, 0.01-0.03 per cent NaHC0 2 and 0.1 per cent glucose). In normal corpuscles hemolysis occurs to a small extent in solu- tions containing about 0.42 per cent of sodium chloride. In certain diseases the fragility of the corpuscles may be increased (Butler 7 ). The membrane and stroma of the erythrocyte contain lipoidal ma- terial which is soluble in alcohol, ether, fatty acids, and bile salts. Addition of these agents to the blood brings about hemolysis, presum- ably by dissolving the lipoidal material present. The hemolysis which occurs with saponin is similar in type, since saponins combine with lipoids, the compound being soluble in water. The hemolytic properties of serum, whether they are found to be normally present when the bloods of certain animals are mixed or to be produced artificially by the injection of foreign red cells, furnish a subject of great interest from the standpoint both of immunology and THE BLOOD CELL 97 of clinical medicine. The hemolytic serum produced by the injection of foreign corpuscles owes its activity to two substances. The one called the amboceptor, or immune body, is specific against the type of cell injected and is increased during immunization. The second body is the complement; it is nonspecific, and is not increased dur- ing immunization. Complement is destroyed by heating the serum for one hour at 55 C., leaving the amboceptor alone present. Corpuscles placed in such serum are not hemolyzed until complement either from fresh immune or from nonimmune serum is added. The serum of animals possessing natural hemolytic properties towards the corpuscles of other animals likewise owes its effect to the joint action of amboceptors and complement. Ordinarily the serum from animals of one species does not exhibit hemolytic properties to blood from another animal of the same species. In unusual cases, however, the serum of an animal will produce hemol- ysis of the corpuscles of an animal of the same species. Such sera are said to possess isohemolysins. The fact is of great importance in the transfusion of blood from one individual to another. The cause of the acute hemolysis which occurs in the disease parox- ysmal hemoglobinuria is not known. It is probably due to the presence of a hemolytic substance which unites with the blood corpuscles at temperatures below the normal body temperature, since the attack fol- lows exposure to cold, and blood from patients subject to the condition may be hemolyzed in vitro by cooling and subsequently heating it. LEUCOCYTES There are a number of varieties of white cells in the blood. These are differentiated from one another by their shape, staining properties, and the granules in their protoplasm. We may divide them into two main groups nongranular mononuclear cells and granular polynuclear cells. The nongranular mononuclear cells are termed lymphocytes. Two va- rieties are differentiated, the small and the large. The small mononuclear leucocyte makes up from 23 to 28 per cent of the total leucocytes and the large mononuclear, from 2 to 4 per cent. The polynuclear leucocytes are divided into three groups according to whether their granules stain with basic, neutral or acid stains. The leucocytes that stain with basic dyes, or the basophile cells, are very few, making up less than one per cent of the total count. Likewise the acid-staining granular cells, acidophile, are few, comprising from 2 to 4 per cent of the total count. The most numerous are the neutrophiles, 98 THE BLOOD AND THE LYMPH or the polynuclear leucocytes, with neutral-staining granules. These comprise from 65 to 75 per cent of the total count. Another type of white cell is known as the transitional cell, because it was supposed to represent an intermediate form between the mono- and polynuclear cells. Probably such transitions do not occur, and the transitional leucocyte is related to the mononuclear cells. The polynuclear cells originate in the bone marrow, and for this reason have been termed myeloid cells. They develop from cells in the bone marrow termed myeloblasts, which are nongranular and con- tain a large nucleus. In the course of development the characteristic granules appear, and the nucleus remains round and later becomes lobulated. These intermediate forms are called myelocytes. The mono- nuclear cells originate in the lymphatic tissues of the body. The leucocytes possess the ability to make ameboid movement and to ingest foreign particles which may be presented to them. On ac- count of this latter ability they are commonly called phagocytes. In the process of inflammation the leucocytes assemble at the spot which is the seat of the injury or infection, and remove the foreign organism or necrotic tissue by ingesting and digesting it. It is not definitely known whether or not the lymphocytes func- tion as phagocytes. Other functions besides those as phagocytes have been ascribed to the white cells, but they are not universally ac- cepted. The number of leucocytes in the blood is subject to con- siderable variation. They normally number between 6,000 and 8,000 per c.mm. At the height of digestion and after strenuous exercise there is usually a small increase, and under pathological conditions, especially in infectious diseases, this becomes quite marke'd. Some infections increase the polymorphonuclear cells, while others add to the lymphocytes. The factors governing the type of increase are not fully known, nor are the functions of the various forms differentiated. The Blood Platelets These are small oval particles about 3 /x in diameter, which are found in large numbers (250,000 to the c.mm.) in the blood. They are sup- posed to be formed from particles of protoplasm which are pinched off from the large blood cells in the bone marrow. Their biological and chemical properties are not understood. They probably play a very important role in the coagulation of the blood (see page 104). CHAPTER XII BLOOD: BLOOD CLOTTING On leaving the blood vessels, the blood clots so as to form a plug, which assists in preventing further hemorrhage. The clotting must therefore be considered as a protective mechanism against excessive draining of blood out of the organism. When the wounded vessels are small, the clotting, along with constriction of the damaged vessels and the formation in them of thrombi containing large numbers of platelets, serves to effect complete stoppage of the hemorrhage even though the blood pressure may not have become materially reduced. The greater loss of blood from larger vessels causes the arterial pressure to fall, and this enables the clot to stiffen and seal the wound before the pressure again rises. When the clotting power of the blood is subnormal, life is endangered by even trivial wounds; under these conditions the smallest surface scratch may continue to bleed exces- sively in spite of whatever local treatment is applied. The most ex- treme degree of this condition occurs in hemophilia, a disease which is characterized by a most interesting family history namely, that although it affects as a rule only certain of the male members of a family, yet it is transmitted from generation to generation by the female side alone. The disease has existed in certain of the royal families of Europe for many generations, which has made it possible by con- sulting the genealogical trees to demonstrate the infallibility of this law of inheritance. The clotting of the blood is also either depressed or increased in a variety of physiological and pathological conditions. We shall, however, defer further consideration of these until we have learned something of the nature of the factors which are responsible for the process itself. The Visible Changes in the Blood During Clotting In a few minutes after it leaves the blood vessels, the blood forms a jelly-like clot, which adheres to the walls of the container in which the blood is collected and soon becomes so solid that the vessel may be inverted without spilling any of the blood. Clotting is now said to be complete. The clot soon begins to contract, and as it does so, drops of clear fluid or serum become expressed and float on the surface of the 99 100 THE BLOOD AND THE LYMPH clot or collect between it and the walls of the container, so that after some time the clot breaks away from the container and comes to float in the serum. The latter may be perfectly clear, but usually is more or less opalescent, partly because of the presence of fat, and partly be- cause of leucocytes which have migrated out of the clot on account of their power of diapedesis. If a drop of freshly shed blood is examined under the microscope, it will be observed that the first step in clotting consists in the formation of fine threads radiating from foci, which are undoubtedly the blood platelets. The fine threads are called fibrin. They multiply rapidly, so as to form an interlacing meshwork which entangles the red blood corpuscles and leucocytes. By the use of the ultramicros,cope (page 52), Howell 1 and others have observed that the fibrin (produced by adding thrombin to oxalated plasma) is really deposited in the form of fine crystalline needles "fibrin needles" which become packed together as they increase rapidly in numbers. Although the process of clotting consists therefore in the conversion of a hydrosol into a hydrogel (see page 61), it is a unique process; a solution of the blood protein which is responsible for the formation of the fibrin (fibrinogen) may, like other colloidal solutions, be precipitated in a variety of ways, but it is only when the conditions are favorable for blood clotting that fibrin needles, and therefore fibrin threads, are formed. The blood of invertebrates forms a structureless gel when it clots (Howell). Methods of Retarding Clotting of Drawn Blood To understand the nature of the clotting process and the factors that are responsible for its occurrence, it is advantageous to simplify the conditions somewhat by getting rid of the red corpuscles and most of the other formed elements of the blood and then using the fluid in which these are suspended in living blood namely, the plasma. This separation of blood into corpuscles and plasma is readily effected either by sedimentation or by centrifuging after measures have been taken to inhibit or greatly delay the clotting process. The methods used for this purpose are numerous. A few of the most important are as follows: (1) Keeping the blood at a temperature very slightly above freezing point. This method is, however, not very effective unless the blood is immediately received into narrow vessels placed in ice and the tempera- ture kept most strictly at the low level. In the case of horses' blood and other slowly clotting bloods, the method succeeds without these precau- tions. (2) Receiving the blood through a strictly clean and smooth can- nula, coated with a layer of paraffin or vaseline, into a vessel similarly coated. This method is of practical importance when it is necessary to BLOOD CLOTTING 101 transfuse blood without making a vessel-to-vessel anastomosis. (3) Mix- ing the blood with chemicals that are capable of removing the calcium from solution. Such reagents are potassium or sodium oxalate (in a con- centration of 0.1 per cent after mixing), and sodium fluoride and sodium citrate (2 per cent solution, with one part of the solution to four parts of blood). (4) Mixing the blood with certain neutral salts, particularly the sulphates of sodium and magnesium (one part of 27 per cent solution of magnesium sulphate mixed with four parts of blood). Blood thus treated is known as " salted blood," and the plasma separated by centri- fuging, as "salted plasma/' Clotting is readily induced by adding water to the salted blood or plasma, and in this way diminishing the concen- tration of the salts. (5) The addition to blood of one of a class of sub- stances known as antithrombins. Leech extract or the purified substance separated from it, known under the trade name of "hirudin," and sub- stances present in blood removed from animals after they have been injected with peptone solutions, are examples. The methods which have just been described are those applied to blood after it has left the blood vessels. Another interesting group of anti- coagulants prevent clotting only when injected Mo the blood vessels of the living animal. The most powerful example of this group is snake venom, certain varieties of which can prevent clotting in the dosage of %oo of a milligram for each kilogram of body weight. Similar but much less potent effects are produced by the injection of several proteolytic enzymes, but most attention has been paid to the effect of commercial peptone injected in solution intravenously in the proportion of 0.3 gram to each kilogram of body weight. Blood subsequently removed up to about half an hour or more does not clot, and as we have already seen, if added to blood from another animal, materially retards clotting. This group of intra vitam anticoagulants is particularly interesting, since none of the substances belonging to it is capable of preventing clotting of blood when mixed with this after it has been shed. Their action therefore obviously depends on the production of some substance in the body, probably, as we shall see later, in the liver, since they fail to act after the removal of this organ from the circulation (see page 111). The time of clotting varies greatly according to the conditions under which the blood is collected and the animal from which it is derived. Human blood, for example, received into a test tube from a puncture through the skin may clot at any time within three or ten minutes, five minutes being taken as an average time for blood kept at a temperature of about 20 C. This time may be considerably shortened by increasing the extent of foreign material with which the blood comes into contact, and more particularly by whipping the blood with a bunch of twigs or 102 THE BLOOD AND THE LYMPH wires. In this latter case, however, the clot does not form in the usual manner, but the fine threads of fibrin collect on the twigs or wires, leav- ing behind the blood serum with the corpuscles still suspended in it. The fibrin removed in this way may then be washed free of adherent serum. The serum and corpuscles now form defibrinated blood, which is used for many physiological purposes. Clotting is also greatly acceler- ated by allowing the blood to flow over exposed tissues. Something is evidently added to it from the tissues which accelerates the clotting process, this influence being particularly marked in the case of blood of the lower vertebrates. When the blood of the bird, for example, is received through a cannula inserted directly into a vessel with as little injury to the walls as possible, it very slowly clots if at all, but soon does so if the blood is allowed to come into contact with excoriated tissues, or if it is mixed with tissue extract, such as that of muscle. Clotting is considerably accelerated by warming the blood. The ap- plication of a cloth or tampon well wrung out with hot physiological' saline to a wounded surface is a most efficient means of allaying hem- orrhage from vessels too small to ligate. The Nature of the Clotting Process Plasma obtained by centrifuging blood that has been prevented from clotting by one of the foregoing methods can be made to clot by removing the inhibiting influence; for example, in cooled plasma by warming the blood to room temperature, in salted plasma by diluting it with at least an equal volume of water, and in decalcified plasma by adding a suffi- cient amount of soluble calcium salts to combine with all the added oxalate and leave a small trace of calcium salts in excess. The first question concerns the source of the fibrin, and the answer to it is furnished by comparing the composition of bood plasma with that of serum. Though both of these fluids contain the proteins, albumin and globulin, in approximately the same concentrations, the plasma also contains another protein not unlike globulin in most of its reactions, but distinguished from typical globulin in that it is precipitated by half-saturation with sodium chloride, in which typical globulin is solu- ble, and is more readily coagulated by heat. To produce half-saturation of the plasma with sodium chloride, equal volumes of plasma and satu- rated sodium-chloride solution are mixed together. The precipitate of fibrinogen, as the substance is called, is then collected at the bottom of the tube by centrifuging and is washed several times by decantation with half-saturated sodium-chloride solution. The washed precipitate, dis- solved in weak saline solution (preferably containing a trace of bicar- bonate), will then be found to clot under certain conditions. BLOOD CLOTTING 103 The next question concerns the nature of the conditions that cause the fibrinogen to clot. When a fibrinogen solution is mixed with a few drops of blood serum, a clot usually forms, which however is not the case when plasma is added or when the serum is heated before adding it. Because a small quantity of serum is capable of causing the clotting of a large quantity of fibrinogen solution or plasma, it is supposed that the active substance present in it is of the nature of a ferment fibrin ferment or tKrombin. It must be pointed out, however, that there is considerable doubt whether this active body is really of the nature of a ferment or enzyme. For example, although heated serum does not cause clotting, thrombin, prepared from serum by the method about to be described, in the absence of inorganic salts can withstand even a boiling temperature. Moreover, true enzymes are characterized by the fact that, like other catalytic agents, a very minute quantity can effect a change in an indef- inite amount of substance without the enzyme becoming used up in the process (page 72). When thrombin is allowed to act upon a fibrinogen solution, on the other hand, it is said that only a fixed amount of fibrin can be formed when a small amount of thrombin is added. Neither does this amount increase when the time of reaction is prolonged. Whatever may be the significance of the foregoing facts, it is impor- tant to know that the clotting substance, thrombin, can be isolated from blood serum in a tolerably pure condition. For this purpose blood serum is allowed to stand under a large volume of alcohol for a week or two ; the precipitate is then collected and rubbed up with water, which extracts the thrombin from it, leaving the serum protein in a coagulated state. The resulting watery solution of thrombin may be further pre- cipitated by alcohol, the precipitate washed in alcohol and redissolved in water, yielding ultimately a solution which exhibits very marked co- agulating powers when added to plasma or fibrinogen solution. Throm- bin shows most of the protein reactions but it is not coagulated by heat. As would be expected, a considerable quantity of thrombin remains adherent to the fibrin formed in the process of clotting, and Howell 8 describes a very useful method by which it can be separated from fibrin and preserved in a dry condition. Briefly stated, this method consists in allowing washed fibrin to stand overnight under eight per cent sodium-chloride solution, which dissolves the thrombin. The resulting extract is then mixed with an equal volume of acetone, which throws down a precipitate containing the thrombin. To preserve it, the precip- itate is collected on a number of small filter papers, which are subse- quently opened out and dried by exposure to a current of cold air before an electric fan. When the thrombin solutions are desired, the dried pre- cipitates are extracted with a little water. 104 THE BLOOD AND THE LYMPH Thrombin does not exist in blood plasma, for if a clean and paraffined glass tube is inserted into an artery and the blood collected under al- cohol, the precipitate after standing a few weeks will yield no thrombin when triturated with water. Quite clearly, therefore, the thrombin is produced at the time the blood clots, and the question arises, What is it produced. from? It will be remembered that, when the blood is ex- amined under the microscope during the clotting process, the fibrin threads are seen to start from foci which correspond to the blood plate- lets. It would appear therefore that the thrombin must be derived from some substance that is shed forth from the 'platelets during the disin- tegration which they undergo shortly after the blood is shed. The sub- stance is called prothrombin. The platelets or their precursors, the megacaryocytes of red bone marrow, are probably not its only source, for clotting may occur in the complete absence of platelets, when it appears to come from the leucocytes. Prothrombin appears plentifully in the fluid used to perfuse red bone marrow outside the body (Drinker and Drinker 9 ). To sum up what we have so far learned, it may be stated that the process of clotting starts with the disintegration of blood platelets and probably of leucocytes, as a result of which there is shed forth into the plasma a substance called prothrombin, which immediately afterward becomes activated or converted into thrombin. The thrombin then at- tacks a protein present in plasma called fibrinogen, producing from it in thread-like form the insoluble protein, fibrin. But this does not com- plete the history, for at least two other important factors come into play ; the one is the presence of soluble calcium salts, and the other that of peculiar substances derived from the tissues outside the blood vessels and called thromboplastic substances or thromboplastin (Howell). We must now consider the action of these two factors. The Influence of Calcium Salts. As already explained, the proof that soluble calcium salts are necessary for clotting is furnished by the ob- servation that the process is entirely prevented when the freshly drawn blood is mixed with soluble oxalate. To this proof, however, objection might be made on the score that the oxalate per se inhibited the clotting. That such is not the case is indicated by the fact that, if the oxalated blood or plasma is dialyzed against physiological saline solution till all the soluble oxalate has been removed from it, clotting is still absent but immediately supervenes if some soluble calcium salts are added. The question arises as to how the calcium ion acts. Two possibilities exist: (1) that it is concerned in the conversion of fibrinogen to fibrin, and (2) that it is necessary for converting prothrombin into thrombin. It can quite readily be shown that it is by the second of these processes BLOOD CLOTTING 105 that the calcium acts ; for example, clotting occurs when purified throm- bin is added to dialyzed oxalate blood or plasma or to a pure solution of fibrinogen. Citrates prevent clotting by forming calcium citrate, which although soluble does not ionize in solution. It is the free calcium ions that are important. The action of the fluoride is somewhat mysterious, for it has been found that to produce clotting in fluoride plasma the sim- ple addition of calcium chloride will not suffice; thrombin itself must be added as well. Some authors assert, however, that if the calcium chlo- ride is added cautiously to " fluoride" blood, it will induce clotting (Rettger). In any case it appears that the fluoride does something more than precipitate the calcium; possibly it prevents the breaking up of platelets and leucocytes. The Influence of the Tissues. As already stated, when slowly clotting blood, like that of a bird, is collected through a sterile glass tube into a thoroughly clean vessel and immediately centrifuged, the plasma will often remain indefinitely unclotted. If an extract of some tissue, such as muscle, is added, however, the plasma immediately clots. To a much less degree, the same phenomenon is exhibited by mammalian plasma when it is collected in a similar manner. From these observations the conclusions may be drawn that the tissues furnish some substance as- sisting in the clotting process, and that this substance is also formed from certain elements present in mammalian but not present in avian blood. The absence of platelets from the latter blood suggests that these must be the source of the activating substance in mammalian blood. It is plain that this tissue factor in clotting is of importance in hasten- ing the process when an animal is wounded. Before attempting to formulate an hypothesis that will explain the process of clotting, it is necessary to call attention to one other impor- tant fact. This refers to the presence in blood of a substance that pre- vents clotting and is hence called antithrombin. Antithrombin is pres- ent in normal blood, for a given specimen of pure fibrinogen will clot less rapidly when mixed with serum to which some oxalated plasma has been added than with an equal amount of the same serum correspond- ingly diluted with a solution of soluble oxalate. A striking increase in the concentration of antithrombin in blood can be brought about by rapidly injecting a solution of commercial peptone into the blood ves- sels fifteen to thirty minutes before bleeding. The peptonized blood or plasma will remain fluid for many hours, if not indefinitely. That the failure of this blood to clot depends on the presence of some anticlotting substance, and not upon the absence of one of the necessary clotting sub- stances (fibrinogen, thrombin, etc.), is evidenced by the fact that the addition of some of it to a mixture of thrombin and fibrinogen inhibits 106 THE BLOOD AND THE LYMPH the coagulation, which it does not do, however, if it is first of all heated to 80 C. and filtered free of the coagulated protein. Moreover, the antagonistic action is quantitative in the sense that a fixed amount of the peptone-plasma inhibits the action of a fixed amount of thrombin. The source of antithrombin in the body appears to be mainly at least the liver, for it has been found: (1) that peptone injection into an animal from which the liver has been removed does not cause antithrombin to be formed (Denney and Minot) ; 10 (2) that peptone injections into the portal vein cause antithrombin to appear in the blood much more rap- idly than when the injection is made into a systemic vessel; and (3) that, when the liver is perfused outside the body with a perfusion fluid con- taining peptone, antithrombin accumulates in the perfusion fluid. A fluid containing a high concentration of antithrombin is secreted by the so-called salivary gland at the head end of the leech. The func- tion of the fluid is to prevent clotting of the blood, so that the animal may continue to suck it without interference by clotting. After apply- ing leeches for medicinal purposes it is therefore necessary to wash the wound thoroughly with water so that all traces of the antithrombin may be removed ; otherwise the bleeding may continue for a considerable time. Practical use is made of this effect of the leech to prevent clotting of blood outside the body, or it may be used to inhibit coagulation intra vitam in experiments where clotting would otherwise interfere with their prog- ress; for example, in crossed circulation experiments (page 384) and in experiments in vivid diffusion (page 641). For such purposes the leech head is cut off and extracted either with saline or by treatment with chloroform, which removes other proteins from the saline solution leav- ing a. strong antithrombin, known under the trade name of "hirudin." At temperatures about that of the body the action of antithrombin is greatly augmented. In animals like the mammals in which the content of antithrombin is small, this may be important in maintaining the flu- idity of the blood (Howell). Blood containing antithrombin can be made to clot by the addition of thrombin, and therefore of blood serum. CHAPTER XIII BLOOD: BLOOD CLOTTING (Cont'd) THEORIES OF BLOOD CLOTTING Attempts to link all the foregoing facts together in the form of a simple theory have not so far been entirely successful. All agree that the fibrin is derived from fibrinogen by the action of thrombin, the points in dispute being those which concern the origin of the thrombin and the mode of action of the calcium and thromboplastic substances. The theory most widely accepted in Europe is that of Morawitz, according to which the thrombin exists in living blood in an inactive state 1 called thrombogen (prothrombin), which becomes converted into thrombin by the simultaneous action on it of soluble calcium salts and of thrombo- plastic substances furnished by the tissue cells in general and by the cellular elements of the blood platelets and leucocytes. According to this view the thromboplastic substance, aided by the presence of calcium ions, converts thrombogen (prothrombin) to thrombin. It acts there- fore as a kinase and is called thrombokinase. The fundamental fact of this theory, then, is that kinase is necessary for the union of the cal- cium with prothrombin a fact, however, which is challenged by Howell, who states that prothrombin may be converted to thrombin by the action of calcium ions alone. This investigator believes that the thrombo- plastic substance acts not as a kinase but because it neutralizes anti- thrombin, which is constantly present in the blood, and the function of which is to prevent the calcium from uniting with the prothrombin to form thrombin. HowelPs theory in his own words is as follows: "In the circulating blood we find as constant constituents fibrinogen, pro- thrombin, calcium salts and antithrombin. The last named substance holds the prothrombin in combination and thus prevents its conversion or activation to thrombin. When the blood is shed, the disintegration of the corpuscles (platelets) furnishes material (thromboplastin) which combines with the antithrombin and" at the same time liberates more "prothrombin; the latter is then activated by the calcium and acts on the fibrinogen." Antithrombin can also prevent the action of thrombin on fibrinogen. As already pointed out, the thromboplastin can be de rived from the blood itself in the mammals, but only from the tissues in the lower vertebrates. It is interesting to note that the thromboplastin 107 108 THE BLOOD AND THE LYMPH can be extracted from the tissues by fat-solvents, and that it appears to belong to the class of phosphatids, being indeed closely related to, if not identical with, kephalin (Howell). Intravascular Clotting The practical application of the theory of blood clotting concerns the manner in which the blood is maintained in a fluid condition in the blood vessels, and the disturbance of this function causing intravascular clot- ting. According to the one theory, the blood is maintained fluid by the absence from it of any considerable quantity of kinase, and according to the other, by the presence in it of an amount of antithrombin suffi- cient to prevent the union of calcium with prothrombin. The fluidity is maintained even when large amounts of thrombin or of blood serum, which contains this substance, are injected into the living animal. We can best explain the immunity of the blood to the action of thrombin un- der these circumstances as being due to the instantaneous appearance in it of antithrombin in amounts sufficient to prevent the action of thrombin on fibrinogen, for, as stated above, it is claimed by Howell that anti- thrombin has this influence as well as that of preventing the conversion of prothrombin into thrombin. Intravascular clotting may be brought about by a variety of means: (1) Mechanical damage to the lining of the blood vessels; after the ap- plication of a ligature, for example, the damaged endothelium is soon covered by a clot, which gradually becomes firmer and firmer, and may spread up the vessel to the next branch. (2) The presence of foreign substances in the blood. Emboli, for example, are apt to cause clots to form at the places where they stick, namely, in the smaller vessels. Clotting is also a frequent occurrence when there are local dilatations of the cardiovascular tube, and it may occur under imperfectly understood conditions causing the condition known as thrombosis. (3) An inter- esting variety of intravascular clotting is that caused by the intrave- nous injection of saline extracts of cell-rich tissues, such as the thymus, lymph glands or testes (Wooldridge). By precipitation with acetic acid and digestion with peptone, a residue' can be obtained from these extracts which, when dissol'/ed in alkali, has a very pronounced intra- vascular clotting effect. Since these precipitates are very rich in phos- phorus, it is probable that they are of the nature of phosphoprotein (nucleoalbumin) . Their action must depend on neutralization of anti- thrombin, according to Howell's theory, or because they serve as throm- bokinases (according to Morawitz' theory). As a matter of fact, however, the foregoing observation is not com- pletely explained by either theory. If in place of making one injection BLOOD CLOTTING 109 frequent injections of small amounts of the above material are made, instead of intravascular clotting, a delay in the coagulation time is likely to occur. Indeed, repeated injections of small amounts may en- tirely remove the clotting power of the blood. The readiness with which this so-called "negative phase" appears, seems to depend on the nutri- tive condition of the animal at the time of injection. If a large dose is injected into a fasting dog, for example, thrombosis is confined to the portal area, whereas if it is injected into a recently fed animal, the thrombosis is universal throughout the vascular system. The develop- ment of the negative phase is undoubtedly dependent upon some reac- tion on the part of the living cells of the organism, since it does not occur on the addition of similar substances to blood outside the body. The reaction is, indeed, akin to that by which immune bodies in general are produced. For example, a toxin injected in large amount has a cer- tain toxic effect, but in repeated small doses with intervening intervals it leads to the production of an antitoxin. So with the substance in question; a large dose injected at one time causes a positive effect clot- ting but smaller doses frequently injected, the opposite effect want of clotting. It is probable, as suggested by Starling, that more intensive study of the conditions causing intravascular clotting will throw con- siderable light on the general question of the production of immunity. Measurement of the Clotting Time To measiire the clotting ti/me of drawn samples of blood, several conditions must be observed. These have been tabulated by Addis 1 * as follows: 1. The specimens of blood must always be obtained by exactly the same technic. It would introduce serious errors to compare the clotting time of one specimen of blood received from an incision of the skin (ear lobe) with that of another collected in a syringe by venipuncture. 2. The temperature conditions must always be the same. Probably 25 C. is the best temperature to use. Higher temperatures are unsuitable for two reasons: first, be- cause during its collection the blood will have become cooled to about or below this point, and time would be consumed in raising it higher; and second, because the time of coagulation is more and more shortened for each degree that the temperature is raised, this acceleration becoming especially pronounced for temperatures above 25 C. Quite apart from the liability to incur errors incident to measurement of shorter periods of time, observations at higher temperatures necessitate most rigorous adher- ence to a fixed temperature of the water-bath. Temperatures much below 25 C. are unsuitable, because the clotting sets in gradually and it is difficult to tell precisely when it occurs. 3. The blood must always be collected in the same sort of vessel and come in con- tact with the same kind and amount of foreign material. To this it may be added that the receiving vessel must be scrupulously clean; any trace of old blood clot or of serum is especially to be guarded against. 4. The end point must be sharp. It is here that the greatest technical difficulties are met with in making precise measurements, and it is greatly to be desired that 110 THE BLOOD AND THE LYMPH different investigators should adopt some uniform method. For experimental pur- poses the method of Cannon and Mendenhallis i s no doubt the best, and it has the added advantage of giving a graphic record of the observations. The accompanying figure (Fig. 19) shows the principle of the method. The blood is received through a standard cannula (C) into a tube (T) 5 cm. long and of 5 mm. internal diameter; and a loop (of 2 mm. diameter) at the end of a copper wire (D), which is 8 cm. long and 0.6 mm. in diameter, is allowed to fall gently into the blood at regular intervals. The upper end of the wire is articulated with the short arm of a light lever so counter- poised that when the stop (.K), which ordinarily holds it in a horizontal position, is released, the wire, now having a net weight of 30 mg., falls on the blood in the tube. Fig. 19. Diagram of the graphic coagulometer. The cannula at the right rested in a water bath not shown in this diagram. For further description see text. (From Cannon and Men- denhall.)* The long arm of the lever is provided with a writing point, which is made to inscribe its movements on a drum. So long as the blood is unclotted the loop sinks into it when the lever is released and a vertical line is traced, but whenever clotting occurs the loop sticks on the blood and the writing point does not rise. For clinical purposes where blood collected in a syringe by venipuncture is used, the method of Howell 13 is most accurate. It consists in placing 2 or 4 c.c. of the blood in a wide tube (of 21 mm. diameter) that has been cleaned by a bichromate- acid mixture. The period that elapses between the moment of the entry of fluid into the syringe and Fig. 20. Coagulometer. The drop of blood is placed on the upper end of the glass cone and the air stream is directed against it from the side tube shown by the black dot. The apparatus is placed on the stage of the microscope and the drop observed by the low power. that at which the clot has become firm enough so that the tube can be inverted without spilling any blood, is taken as the clotting time. Since the blood does not come in contact with exposed tissues, it takes from 20 to 60 minutes to clot by this method. For routine clinical examination of blood taken from a skin wound Brodie and Kussel's method** is most satisfactory. This consists, in principle, in observing a drop of blood, under the low power of the microscope, while a fine current of air is gently blown against it at regular intervals in a tangential direction. Until clotting sets in, *Am. Jour. Physiol., May 1, 1914, xxxiv, No. 2. BLOOD CLOTTING 111 the individual corpuscles move freely in a circular direction, but as soon as clotting begins they move in masses which soon tend to become fixed so that, although they move somewhat when the air impinges on them, they immediately return to their original position when the current is discontinued. When clotting is complete, the air current merely presses on the corpuscles at one point. By this method the clotting time averages five minutes. A convenient apparatus for this method is that of Boggs, which is shown in Fig. 20. It consists of a truncated cone of glass, projecting into a moist chamber provided with a tube on the side so arranged that when air is blown into the chamber, it strikes the drop of blood placed on the end of the cone tangentially. Blood Clotting in Certain Physiological Conditions Besides the experimental conditions already enumerated as changing the clotting time in the blood of laboratory animals, special mention must be made of the influence of epinephrine injections, of conditions supposed to cause a hypersecretion of this hormone, of the emotions, and of hemorrhage. Epinephrine added to drawn blood does not affect the clotting time, but if small amounts are injected intravenously or even subcutaneously, a marked decrease occurs (Cannon and Gray; cf. Cannon, loc. cit.). Larger injections may have the opposite effect, and intermediate amounts may cause at first a prolongation and later a shortening of the time. These results with larger doses are related to Howell's observation that repeated doses of relatively large amounts of epinephrine in dogs may so greatly retard coagulation as to make the animals practically hemo- philic. It was further found by Cannon and his co-workers that epineph- rine does not influence the clotting time when injected into animals from which the abdominal viscera have been removed from the circulation by ligation of the inferior vena cava and the abdominal aorta. In the light of the influence which destruction of liver cells (by phosphorus, chloro- form, etc.) is known to have in lengthening clotting time, it would seem reasonable to conclude that it must be through this organ that epineph- rine develops its clotting effects. Stimulation of the splanchnic nerves also shortens the clotting time, and it would appear that this action depends on the resulting hyperse- cretion of epinephrine (page 787), for it is not observed following stimula- tion of the nerves in animals from which the adrenal glands have been excised (Cannon and Mendenhall). The interesting suggestion is made by Cannon that the shorter clotting time observed in animals showing strong emotions of fright or fear may also be due to the hypersecretion of epinephrine which this worker believes accompanies such states. Blood Clotting in Disease With the factors concerned in the process so wrapped in mystery, it is not surprising that the underlying causes responsible for delayed or de- 112 THE BLOOD AND THE LYMPH ficient clotting of blood in diseased conditions or for the formation of intravascular clots (thrombi) are little understood. According to How- ell's theory of the nature of the process, which is the most satisfactory at the present time, abnormal clotting might be due to the following causes: (1) A diminished amount of fibrinogen. This occurs when the hepatic cells are greatly damaged, as in poisoning by chloroform or phosphorus and in such diseases as acute yellow atrophy and yellow fever. In many cases of chronic cirrhosis of the liver, as Whipple, etc., 15 have shown, the blood also clots feebly because of deficient fibrinogen. It should be pointed out that it is not so much the clotting time that is increased in such cases, as the firmness or consistency of the clot that is affected. 2. A deficiency in prothrombin. In the condition known as "melena neonatorum," undoubted benefit is derived from intravenous injections of blood serum or by direct blood transfusions, probably because throm- bin or prothrombin is thus furnished. 3. A deficiency of thromboplastin. Since this substance is derived from both blood cells and tissue cells, it does not seem likely that a deficiency could ever occur. Certain observers, however Morawitz, for example lay great stress on this as an important factor in hemorrhagic diseases. 4. An excess of antithrombin. The undoubted increase in this substance that can be brought about experimentally by injecting hirudin or pep- tone into animals, has stimulated careful search for a similar increase in the blood in clinical conditions in which abnormal blood clotting is one of the symptoms (Whipple 16 ). Antithrombin is said to be increased in septicemia, pneumonia, miliary tuberculosis, etc. 5. A deficiency of calcium ions. Although at one time it was supposed that this might be responsible for the feeble clotting in hemophilia, it has not been found, after very extensive trials, that the exhibition of Ca salts in any way relieves the condition. It is said, however, that the slow coagulation seen in obstructive jaundice is decidedly shortened by treatment with calcium salts. One thing stands out prominently in connection with the whole problem, and that is the close relationship of the blood platelets to the clotting process. From these cells are derived, according to Howell, not only the prothrombin but also, as from other cells, thromboplastin. It is not sur- prising therefore to find that decided alterations in the platelet count occur in cases of faulty blood clotting, and that local accumulations of these elements within the blood vessels, produced by their clumping to- gether or agglutinating, is followed by a formation of local clots, as in thrombosis. BLOOD CLOTTING 113 Hemorrhagic Diseases In many of the so-called hemorrhagic diseases (acute leucemia and aplastic anemia) and in the hemorrhagic varieties of diphtheria and smallpox, the platelet count drops from its normal of between 200,000 and 800,000 per cubic millimeter to well below 100,000, and indeed in these conditions it is frequently difficult to find any platelets. Samples of blood clot outside the body within the normal time, but the clot is soft and usually fails to retract in the normal manner. It is on account of this, rather than slow clotting that the hemorrhage continues, so that in appraising the gravity of the symptom it is best to measure not the clot- ting time but the time that it takes for bleeding to cease from a small skin wound, as in the lobe of the ear. This can be very accurately done by applying blotting paper at regular intervals to the puncture (Duke 17 ). The most interesting and at the same time the most mysterious of all conditions in which blood clotting is interfered with is hemophilia. The clotting time is longer than normal, but even after the clot forms, bleed- ing is likely to continue because the clots are very readily displaced. Both clotting time and bleeding time are increased. So far no change in the clotting factors of the blood has been demonstrated in this disease ; the corpuscles and the platelets are normal in numbers, fibrinogen and cal- cium salts are normal, and, as Howell has shown, there is no excess of antithrombin. One significant fact, however, is that the addition of thromboplastin or of its active ingredient, kephalin, greatly shortens the clotting time of the blood when it is removed by venipuncture. In agree- ment with this observation it has been found that hemophilic blood clots much more rapidly, indeed sometimes in the usual time, if it is allowed to flow over cut or damaged tissue and so become mixed with thromboplas- tin. These facts taken together would seem to indicate that the fault must lie in a deficiency in prothrombin, and since this is derived mainly from the platelets, which however are not decreased in number, we must further assume that these elements have undergone some qualitative change preventing their disintegration. An accompanying defect in their agglutinating properties would at the same time explain their fail- ure in hemophilia to clump together at the site of the hemorrhage so as to block the smaller vessels with thrombi; hence the prolonged bleeding time even after clotting has occurred. Thrombus Formation The first formed portion of a thrombus is paler than those formed later, because it contains excessive numbers of platelets; and it seems clear that it is by agglutination of these into masses, which then stick in the 114 THE BLOOD AND THE LYMPH blood vessels and by disintegrating shed forth prothrombin and thrombo- plastin, that the clotting starts. This platelet agglutination may result from stagnation in the bloodflow, or from roughening and damage to the vessel walls. Stagnation may be due either to failure of the circulation as a whole as in heart disease, or to local physical alterations in the vas- cular tube, setting up conditions in which eddy currents with stagnant pools of blood are formed, such as will occur at places where the vessels suddenly become wider, as in varicose veins, in aneurisms and at the sudden bend of large veins. The first formed (platelet) thrombus is fol- lowed by one of a darker color, which fills the vessel up to the next anastomotic branch. Similar stagnation may also follow the obstruc- tion caused by lodgment of emboli in the smaller vessels (air, foreign bodies in fine suspension, bacteria, etc.). The thrombi in such cases are very small and occur particularly in the capillaries of the liver, spleen, and lungs. The small thrombi often serve as foci from which clotting spreads into the larger vessels, this being often encouraged by an increase in the coagulability of the blood. When the intima is inflamed, it is pos- sible that excessive amounts of thromboplastin are produced and that this neutralizes the antithrombin in blood moving so slowly that it is not replaced by fresh blood before clotting ensues, or it may be that sub- stances derived from the inflamed tissue cause the platelets to aggluti- nate. The increased clotting often observed after the injection of hemo- lytic agencies (foreign sera, snake venom, etc.) may also be due to platelet agglutination. Like the thrombosis following embolism, the clotting occurs at first in the capillaries, the initial thrombi containing masses of platelets along with skeletons of blood corpuscles and cells from the blood-forming organs. CHAPTER XIV LYMPH FORMATION AND CIRCULATION CEREBROSPINAL FLUID GENERAL CONSIDERATIONS Lymphatics are modified veins. They grow from the veins in embry- onic life as buds of endothelium, which are rirsi visible in the human embryo in the sixth week of development. The earliest outgrowth oc- curs from the internal jugular vein, and the endothelial buds soon be- come hollow and join together, forming first a plexus and subsequently a sac, from which again lymphatic vessels made of endothelium grow out to invade the skin of the head, neck, thorax and arm, and partly the deep structures of the head. The sac is ultimately transformed into groups of lymph glands. At a later stage similar nodes develop from certain of the abdominal veins, forming a retroperitoneal sac, from which grow out the lymphatics of the abdominal and, to a certain extent, of the thoracic viscera. A similar pair of sacs also develops from the iliac veins supplying the lymphatics for the skin of the legs and abdominal walls. The retroperitoneal and iliac sacs then become connected with the jugular sac by means of the thoracic duct. In the embryo there are no valves in the lymphatic vessels, so that the whole system can be in- jected either from the thoracic duct or from the skin, showing clearly that the superficial and deep lymphatics are parts of one closed system of vessels. Anatomists have succeeded in tracing the course of the lymphatics in many parts of the body. This knowledge is of great importance in connection with the spread of infections, etc. Lymphatics are abun- dant in the skin, the intestine, and connective tissues, but are absent from the muscle bundles, from the hepatic lobules (though present in the connective tissue between them), from the substance of the spleen, and from the central nervous system. The lymphatics have the same functions as blood capillaries, namely, to absorb substances from the tissue spaces. There is some evidence to show that this absorption may be selective. When injections are made into the peritoneal cavity, the pathway of absorption may be either the blood vessels or the lymphatics, according to the nature of the sub- 115 116 THE BLOOD AND THE LYMPH stance injected. True solutions are absorbed by the blood, but granules are taken up by special large cells showing phagocytic powers, and trans- ferred to the lymphatics for example, those of the diaphragm. A sim- ilar selective absorption is well known in the case of the villi of the in- testine, where fat passes into the lacteals and carbohydrates into the blood. It appears as if lymphatic adsorption, both of solid materials and of solutions, requires the cooperation of phagocytic cells. The newer conception of the lymphatics as a closed system is at vari- ance with the older one, in which they were supposed to get smaller and smaller, and their walls less and less complete until ultimately they faded off into the tissue spaces. These, however, bear no closer relation- ship to lymphatics than they do to blood capillaries. The tissue spaces include all the minute spaces between the fibers and cells of the con- nective tissues and between the parenchyma of the organs and the great serous cavities of the body (pleural, peritoneal), as well as specially developed tissue spaces, forming the subarachnoid spaces of the brain, the scala vestibuli and tympani of the cochlea and the anterior chamber of the eye. The fluids in these spaces the tissue fluids are quite dif- ferent from the lymph in the lymphatics both in composition and in function. Indeed, the tissue fluids are among the most varied of all the fluids of the body. The spaces may themselves become linked to- gether so as to form a circulatory system, which is quite independent of the lymphatics. This is particularly the case in the brain, where the tis- sue spaces surrounding every individual nerve cell extend into the sub- arachnoid area, where they drain into the cerebral sinuses through the arachnoidal villi, which exist as lace-like projections of the arachnoid into the dural sinuses, being covered by a layer of mesothelial cells spe- cially abundant at the tips of the villi, where they form cell nests. Ob- servations of the passage of substances in solution by these pathways have been made by injecting potassium ferrocyanide and citrate of iron into the subarachnoid and subdural spaces and afterwards detecting the presence of the salts by mounting sections in acid media, so as to permit prussian blue to develop. Ordinarily the precipitate is found in or near the villi, but after cerebral anemia it forms in the tissue spaces that surround the nerve cells. There are therefore three fluids concerned in the transference of food materials and gases between the gastrointestinal apparatus and lungs and the tissue cells namely, the blood plasma, the tissue fluids, and the lymph. The tissue fluid, being in contact with the tissue elements, serves as their immediate nutritive fluid, and it is the function of the blood and lymph to maintain it of proper composition. Everything must be trans- ferred to and from the tissue cells through the tissue fluid, making it LYMPH FORMATION AND CIRCULATION 117 therefore in many ways the most important of the fluids of the body. In the tissue cells themselves there is also the fluid in which the various colloids and crystalloids that enter into the composition of protoplasm are dissolved. This can be removed from cells only by mechanical means, such as grinding with fine sand in a mortar and subjecting the mass to a pressure of several thousand atmospheres in a hydraulic (Buchner) press. This is known as the tissue juice. The ultimate exchange of foodstuffs occurs between the tissue fluids and the tissue juices across the cell membrane. The extent and character of this exchange depend on many circumstances, some affecting the cell wall, others, the osmotic and other properties of the two fluids. Obviously, the function of the circulation is to maintain the tissue fluids of correct composition, the blood plasma serving to carry food materials and dissolved oxygen to them (see page 393), but being assisted in the opposite function of re- moval of effete products by the lymph. The lymph is purely a scavenger ; the blood is both purveyor and scavenger. The above description of the lymphatics is not universally accepted by anatomists, certain of whom believe that the lymphatics are developed from tissue spaces and are consequently much more extensive than they appear to be from injected specimens. The above conclusions are based on reconstruction models, made from serial sections of embryonic tissues, in which the lymphatics frequently appear as isolated vesicles without visible connections. The failure of injections to penetrate into the re- moter parts of such a lymphatic system in the embryo is attributed to the discontinuity of spaces, which is, however, removed at later stages of development. The manner of absorption of injected fluids does not, however, sup- port the view that the lymphatics are directly connected with the tis- sue spaces. When all the structures of a part are ligated except the main artery or vein, injected poisons which affect central structures, such as the nerve centers, develop their action as quickly as in the in- tact animal (e.g., strychnine). Similarly, when pigments such as meth- ylene blue are injected into the pleural cavity or subcutaneously, they appear in the urine long before the lymph of the thoracic duct. Such results indicate the pathway of absorption to be the blood rather than the lymph vessels. Through this latter channel absorption proceeds more slowly, but can be greatly assisted by massaging the site of injec- tion. When colored solutions, such as India ink or carmine, are injected subcutaneously, however, a very perfect injection of the neighboring lymphatics may ultimately occur, and through the same pathways mi- croorganisms spread from an infected area. 118 THE BLOOD AND THE LYMPH EXPERIMENTAL INVESTIGATIONS It has proved a most difficult problem to gain any exact knowledge of the production of lymph by experimental means. Starling, some years ago, in repeating many of the experiments of older physiologists in the light of the newer facts of physical chemistry, added much that is of interest, and it is chiefly with his work that we will concern ourselves here. The unequal lymph supply of different regions of the body is strik- ingly demonstrated by comparing the lymph flow from the lymphatics of the leg with that from the thoracic duct. No lymph flows from the former unless the muscles are thrown into activity or the blood is pre- vented from leaving the limb by ligaturing all the veins. Changes in the arterial blood pressure do not affect the flow. On the other hand, a great increase in the flow from the thoracic duct can readily be induced by disturbances in the blood supply. Obstruction of the portal vein, for example, immediately increases the lymph flow four or five times because of venous congestion in the intestinal capillaries, whilst a still greater increase perhaps tenfold is induced by obstruction to the inferior vena cava, which raises the capillary pressure in both the liver and the intes- tines. After ligation of the hepatic lymphatics (at the hepatic pedicle), obstruction of the vena cava no longer causes the outflow of lymph to increase, indicating that the lymph in the last mentioned experiment must have come from the hepatic lymphatics. These results, so far as they go, could be satisfactorily explained on the basis that lymph formation is a filtration process, that is, a process dependent upon difference in mechanical pressure between the blood capillaries and the tissue spaces. The lymphatics would then serve as channels to return this fluid to the blood vessels through the thoracic duct. The difference in the magnitude of the increased lymph flow from increase in capillary pressure in different regions would be dependent on the permeability of the filter, the capillaries of the limbs being much less permeable than those of the intestine, and particularly of the liver. Another fact in conformity with this view concerns the composition of the lymph from the two regions, that from the limb lymphatics being poor in protein, whereas that from the thoracic duct does not fall far behind the blood plasma in this regard. Although filtration may explain the considerable increase in lymph flow produced by extreme changes in capillary pressure, it by no means suffices to explain lymph formation under less abnormal conditions. When a muscle or a gland is at rest, it produces practically no lymph, LYMPH FORMATION AND CIRCULATION 119 but during activity the flow becomes marked. This can not be explained by nitration, but may be accounted for by a physico-chemical process namely, osmosis. The energy required for the activity of the tissue cell is produced by chemical changes, whereby large molecules become broken down into numerous smaller ones. These smaller molecules are then discharged into the surrounding tissue fluids, the osmotic pressure of which they increase, with the consequence that water is attracted by osmosis from the plasma in the blood capillaries (see page 4). This increases the volume of tissue fluid, which is then drained away by the lymphatics. The increase in molar concentration will also affect the tissue juices, tending to make the cell swell up by absorbing water. In gland cells this extra water is immediately extruded to form the water of the secretion (see page 455). An analogous method of lymph formation is not confined to situations where the capillaries are relatively impermeable, for it also occurs in the liver, the lymph flow from which is greatly increased by the injec- tion of bile salts. A similar process no doubt results from muscular activity, although in this case the tissue spaces must form a continuous system of their own, there being, according to most authorities, no lymphatics. Considerable interest has been taken in the stimulating effect which certain chemical substances have on the secretion of lymph from the thoracic duct. These so-called lymphagogues belong to two classes crystalline and colloidal. Of the former, glucose, urea, and sodium chloride in hypertonic solution, are the best known. Starling explains their action as dependent upon an increase in the osmotic pressure of the blood. This attracts water into the blood from the tissue juices, and leads to an hydremic plethora, with a consequent increase in capil- lary pressure. If the blood pressure is lowered by hemorrhage before the hypertonic solution is injected, very little stimulation of lymph flow occurs, because there is no available fluid in the tissue to produce the plethora. This observation does not, however, very strongly support the explanation, because so many other disturbances may result from hemorrhage. The colloidal lymphagogues include watery extracts of the dried tis- sues of leeches, crayfish, and mussels, as well as commercial peptone. They probably act by damaging the endothelium of the capillaries, so that filtration occurs more readily. Although their action is displayed more particularly on the lymphatics of the liver and intestines, it is also apparent on the skin capillaries, producing cutaneous edema and the formation of blisters (nettle rash). 120 THE BLOOD AND THE LYMPH EDEMA With such an imperfect knowledge concerning the physiology of lymph formation, it is not surprising that the causes of excessive accu- mulation of fluid in and between the tissue elements should be little un- derstood. All of the conditions which have been mentioned as capable of causing an increased secretion of lymph such as increase in capillary pressure, hydremic plethora, action of poisons on the endothelium are likely to cause edema if the lymphatics of the part are simultaneously obstructed. To produce in animals edema of the subcutaneous tissues like that observed clinically, it is, however, necessary that the vascular disturbance be accompanied either by local damage to the capillary endothelium, such as is produced by arsenic or uranium; or by a gen- eral toxemic condition, such as is set up by nephritis. When large amounts of saline solution are injected intravenously, extensive ex- travasation of fluid may occur into the liver, peritoneum and intestinal lumen, without any subcutaneous edema. Clinical edemas are of at least three types: 1. The inflammatory edemas, in which the fluid permeates the cells of the inflamed area and does not shift to other parts of the body under the influence of gravity. 2. The nephritic edemas, in which the fluid is more or less loose in the subcutaneous tissues and readily changes its position, and which is accompanied by excess of water in the blood with a corresponding in- crease of sodium chloride ; the percentage concentration of sodium chlo- ride in the blood remains unchanged, but that the other constituents diminished. 3. Cardiac edemas, which are also hypostatic, but are unaccompanied by changes in the relative amount of water and sodium chloride in the blood. The second and third varieties of edema may of course be more or less present together, for the kidneys are likely to become secondarily affected during venous stasis. The salt retention in nephritic edema is very significant. As ex- plained elsewhere, it is revealed by comparing the daily output of so- dium chloride by the urine with the concentration of this salt in the blood. Less salt is eliminated than would be the case in a normal in- dividual with the same percentage of salt in the blood. In many cases also edema can be diminished by withholding salt from the food. Widal and Javal have conclusively shown the relationship of retention of water in the body, as judged by variations in body weight, to the hydremic condition, as judged by the refractive index of the blood serum, and LYMPH FORMATION AND CIRCULATION 121 to the amount of salt in the diet. A very considerable retention of water usually occurs before there is any evidence of edema; indeed, as a result of giving salt, the body weight may increase from five to seven kilograms (10 to 15 pounds) within a day or two without the appear- ance of puffiness. The cause of the edema during salt retention is no doubt closely re- lated to the action of lymphagogues. In a normal person excessive in- gestion of salt is immediately followed by excretion of the excess through the kidney. Where the kidneys are diseased, this excess of salt is re- tained in the blood, raising its osmotic pressure and attracting water from the tissue fluids. This leads to excessive thirst, the imbibed water being used to replace that lost from the tissues. But all the crystalline lymphagogues do not, when present in excess in the blood of nephritic patients, necessarily cause edema; urea, for example, may accumulate considerably without any such effect. The different action is usually attributed to inequality in the diffusibility of the two crystalloids through animal membranes, sodium chloride diffusing much less readily than urea. It is most important to note that the fluid in edema is loose in the tissues and can be drained away by the insertion of tubes. There is absolutely no evidence in support of the claim of Martin Fischer that edema is due to imbibition of water by the colloids of the tissues. This question has been fully discussed elsewhere (page 63). THE CEREBROSPINAL FLUID Considerable attention is now paid to the cerebrospinal fluid which is present in the subarachnoid spaces of the spinal cord and brain. This is because the fluid is readily collected by the method of lumbar puncture, which is performed for the purpose either of relieving increased inter- cranial pressure as in hydrocephalus, delirium tremens, eclampsia, enceph- alitis, etc., or of collecting some of the fluid for diagnostic purposes. The rationale of the removal for the relief of pressure in the brain case is somewhat difficult to understand, as will be evident from a perusal of the chapter on the circulation of blood in the brain (page 254) . There is no doubt, however, that the procedure gives relief. It is more particularly with the biochemical characteristics of the fluid that we are concerned here (of Levinson 19 ). The normal fluid (i.e., obtained from patients not suf- fering from inflammatory processes of the cerebrospinal membranes) is colorless, it contains only from 4-6 cells per cubic mm., its specific gravity varies between 1,000 and 1,008 and it does not clot. The H-ion concentra- tion of perfectly fresh fluid, removed during life, can be accurately meas- ured by the colorimetric method without dialysis (page 32) and has been 122 THE BLOOD AND THE LYMPH found by Levinson to vary between P H 7.4 and 7.6, being therefore practi- cally that of blood. On standing in unstoppered vessels the alkalinity gradually increases so that P H of 8 may be reached in two hours. If the vessel be tightly stoppered, however, P H may remain almost stationary. The reason for this change is that there is as large a percentage of bicar- bonate in the cerebrospinal fluid as in the blood plasma. The so-called alkaline reserve, as determined by the Van Slyke method is therefore the same as in blood plasma (viz., about 60). With regard to organic constit- uents, there is only a trace of protein (0.02-0.04 total N.) but the urea and sugar are present in about the same percentage amounts as in the blood plasma. There is no certain evidence that any enzymes are con- tained in the fluid. The pathological changes observed in the fluid are of two types, systemic and meningitic. In connection with the former, it may be mentioned that in uremia the amount of fluid is usually increased and there is a high per- centage of urea; in diabetes, the sugar is increased; in the various psy- choses, in epilepsy and chorea there may or may not be changes. In hydrocephalus the amount of fluid is increased, but it is normal in its properties. This is also, although less markedly, the case in encephalitis and cerebral tumor. Eegarding conditions in which the meninges are in- flamed (tubercular, meningococcic, pneumococcic) the changes in the fluid are very marked and of decided diagnostic value ; it is somewhat increased in amount, turbid, forms a sediment, shows many cells, contains excess of protein (globulin) and gives a typical culture of the infecting organism when examined by bacteriological methods. Levinson has found that there are significant changes in P H in various diseases and he considers that several tests that have been devised for diagnostic purposes are in- timately associated with the changes in P H . These tests are known as the cataphoresis test, the colloidal gold reaction of Lange and the mastic reac- tion, and they all depend on the manner in which the protein colloidal particles aggregate or become precipitated. BLOOD AND LYMPH REFERENCES (Monographs) iHowell, W. H.: The Harvey Lectures, J. B. Lippincott Co., xii, 272. sStarling, E. H.: Human Physiology, Lea & Febiger, 1915. 3Kowe, A. H.: Arch. Int. Med., 1917, xix, 354, . * Williamson, C. S.: Arch. Int. Med., 1916, xviii, 505. sTower and Herm: Proc. Soc. Biol. and Med., 1916, xviii, 505. 6Eous and Eobertson: Jour. Exp. Med., 1916, xxiii, 219, 239, 549. ?Butler, G. G.: Quart. Jour. Med., 1912, vi, 145. sHowell, W. H.: cf. Harvey Lecture; also Am. Jour. Physiol., 1913, xxxii, 264.. Drinker, C. K., and K. E.: Am. Jour. Physiol., 1916, xli, 5. loBenny and Minot: Arch. Int. Med., 1916, xvii, 101; Am. Jour. Physiol., 1915, xxxviii, 233. LYMPH FORMATION AND CIRCULATION 123 "Addis, T.: Quart. Jour. Med., 1910, iv, 14. i2Cannon and Mendenhall: Am. Jour. Physiol., 1914, xxxiv, 225. isHowell, W. H.: Arch,. Int. Med., 1914, xiii, 80. J*Brodie, T. G.: Jour. Physiol., 1897, xxi, 403. isWhipple, G. H.: Arch. Int. Med, 1912, ix, 365; Jour. Exp. Med., 1911, xiii, 136. isAVhipple, G. H.: Arch. Int. Med., 1913, xii, 637. "Duke, W. W : Arch. Int. Med., 1912, ix, 258. isKeith, N. M.: Eeport 27 Medical Eesearch Committee, London, 1919. isLevinson, A.: Cerebrospinal Fluid in Health and in Disease, C. V. Mosby Co., 1919. zoKeith, N. M., Eowntree, L. G., and Geraghty. J. T. : Arch. Int. Med., 1915, xvi, 547. 2iHooper, C. W., Whipple, G. H., etc. : Am. Jour. Physiol., 1920, li, 205-257. 22\Vhipple, G. H., etc.: Am. Jour. Physiol., 1918, xlvii, 356, 370, 379; and 1920, lii, 54. 238abin, F. E.: Physiol. Eev., 1922, ii, 38. PART III THE CIRCULATION OF THE BLOOD CHAPTER XV BLOOD PRESSURE The object of the circulation is to maintain through the tissues a sup- ply of blood that is adequate to meet their demands for nutriment and oxygen and to remove the effete products of their metabolism. The de- mands vary according to the activities of the tissue, being particularly variable in the case of such tissues as the muscular and the glandular. In studying the physiology of the circulation we have therefore to bear in mind two aspects of the problem: (1) the cause for the continuous bloodflow, and (2) the mechanism by which alterations in this bloodflow are brought about. If we open an artery we shall find that the blood escapes from it under such a pressure that it is thrown to a height of about six feet, that its outflow is proportional to the size of the artery, and that it pul- sates. If, on the other hand, we open a vein, we shall find that the blood wells out without any very evident pressure, and that it flows in a continuous stream, its outflow being the same in a unit of time as that of the artery, provided the two vessels are the only ones supplying the particular area. The general conditions governing the bloodflow are the same as those governing the flow of fluid through any system of tubes. For example, in the city water mains it is known to every one that the rate of outflow from any part of the system depends finally on two factors: (1) the difference in pressure at the beginning and end of the system, and (2) the caliber of the tube at the outlet. We may in- crease the outflow either by raising the pressure at the beginning of the sys- tem, the caliber of the outlet meanwhile remaining constant, or by main- taining the pressure constant but increasing the caliber of the outlet. In the circulation of the blood, the difference in pressure at the be- ginning and end of the circulation is furnished by the pumping action of the heart, and the alteration of the caliber of the outlet is provided for by the constriction or dilatation of the blood vessels. These simple physical principles indicate the direction which a study of the circulation 124 BLOOD PRESSURE 125 should take. They indicate that our first consideration should be of the mean blood pressure, how it is maintained, and how it can be made to vary. After we have learned this, we may then proceed to a more particular examination of the mechanism of the pump that is, of the heartbeat ; then finally .we may proceed to examine the nature of the processes by which the caliber of the arteries is controlled. THE MEAN ARTERIAL BLOOD PRESSURE The first prerequisite to the investigation of the blood pressure, as of any other physical problem, is that we should possess some means by which it can be quantitatively measured. The earliest attempt to accom- plish this was made by the English scientist, the Rev. Stephen Hales, a little over a century after Harvey published his account of the circula- tion of the blood. Hales connected a glass tube nine feet in length with a severed artery of a horse, the connection between the two being made by means of a piece of brass pipe joined to the windpipe of a goose as a substitute for rubber tubing. He found on untying the ligature on the artery that the blood rose in the tube to a height of eight feet and three inches above the level of the left ventricle of the heart, and that when at full height it rose and fell with each pulse through a distance of two, three or four inches. Mercury Manometer Tracings The somewhat crude but very significant experiment of Hales clearly established the existence of the enormous pressure at which the blood is made to circulate through the arteries. To render possible a further investigation of the factors on which this pressure depends, it became necessary to invent some more convenient means for its measurement, but this was not accomplished until a century later, when Poiseuille ap- plied the mercury manometer, which Ludwig subsequently adapted so that tracings might be taken (Fig. 21). Having before us such a tracing as shown in Fig. 22, let us consider how it may be used in the study of blood pressure. The first thing we must do is to measure the average height of the tracing above the line of zero pressure; the mean arterial blood pressure is then equal to this distance multiplied by two, because the distance through which the mer- cury has moved up in the limb of the manometer carrying the writ- ing point is only one-half of its total displacement. Since mercury is about 13.5 times heavier than an equal volume of blood, the above measurement must be multiplied by this figure if we desire to express 126 THE CIRCULATION OF THE BLOOD our result in terms of the height to which the blood pressure could raise a column of blood. In arteries of approximately the same size, the mean arterial blood pressure does not markedly vary in different mammals. Thus, in the carotid artery of the dog it averages about 110 to 120 mm. Hg, in that of the cat about 105 to 115 mm., in the rabbit from 90 to 105 mm., in the sheep about 150 mm., in the horse about 200 mm., and in man Fig; 21. Mercury manometer and signal magnet, arranged for recording the mean arterial blood pressure in a laboratory experiment. The pressure bottle (R) is filled with anticoagulating fluid and is connected by tubing with the manometer (M), the cannula for the artery (U) being connected with the T-piece (J). By this arrangement it is possible to flush out the tubing when clotting interferes with the experiment. (From Jackson Experimental Pharmacology.) where between 120 and 140 mm. The pressure varies in different parts of the vascular system, being greatest in the aorta and least in the small- est arterioles but the fall in pressure the pressure gradient does not become very pronounced until the arterioles have become so small that it is no longer possible to insert a cannula into them; thus, the mean BLOOD PRESSURE 127 blood pressure in the renal or femoral artery is very little less than that in the aorta. If we examine the contour of the tracing which the pressure draws, we shall find that it exhibits two types of wave, small and large ; and if we observe the animal while the tracing is being taken, we shall find that Fig. 22. The arterial blood pressure recorded with a mercury manometer (lower tracing), along with a tracing of the. respiratory movements of the thorax. Note that the beginning of respiration occurs distinctly before the rise in blood pressure. the former are caused by the heartbeats and the latter by the respira- tions an observation which immediately raises the question as to the trustworthiness of the method, for it will be asked, How can it be that the heartbeat produces an effect on blood pressure which is less than that of the respirations? Obviously the tracing must be faulty in re- gard to the relative significance of the waves. 128 THE CIRCULATION OF THE BLOOD Spring Manometer Tracings The cause of this inaccuracy depends on the inertia of the mercury, an inertia which is so great that the sudden changes of pressure produced by each heartbeat are not able to overcome it, whereas the much less significant but more prolonged pressure changes produced by each respi- ration develop their full effect on the mercury. These facts led investi- gators to seek for instruments in which the inertia error is eliminated, with the result that they invented what are known as spring manometers. Fig. 23. Hiirthle's spring manometer. Many forms of this instrument have been devised, but for our pur- pose it is necessary to describe the principle of only the simplest and most efficient the Hiirthle manometer. As shown in Fig. 23, it consists of a variety of tambour, which differs from the ordinary tambour in two important particulars: (1) the chamber is made as small as possible, and (2) it is covered not with an elastic membrane but with one of leather or of thin fluted metal. These two precautions are taken in order to avoid spuri- ous waves set up on account of elastic recoil. Such errors are further reduced by filling the tubing and chamber of the tambour with a fluid so as to eliminate the elastic recoil of air. Fig. 24. Normal curve of arterial blood pressure obtained with spring manometer. (Burdon-Sanderson.) Before the tracing taken with the spring manometer can be em- ployed for quantitative measurements, it must obviously be graduated according to some scale. This is accomplished immediately before or after the experiment by connecting the manometer through a T-piece with a pressure bottle, which can be raised or lowered to a specified height, and with a mercury manometer. The displacement of the writing point of the spring manometer corresponding to each 10 mm. Hg of pressure is then written on the tracing. The tracings taken with such a manometer, as shown in Fig. 24, are quite different from those with the mercury manometer. It will be seen BLOOD PRESSURE 129 that now the cardiac waves are decidedly the more pronounced, the respira- tory, being comparatively inconspicuous. The pressure in the arteries, instead of being fairly steady, undergoes very considerable alteration tion during each heartbeat. Examination of this tracing gives us accurate information regarding the blood pressure both between the heartbeats diastolic, as it is called and during them systolic. It gives us a means of measuring the dead load of the circulation that is, the pressure that is constantly present as well as the live load that is superadded to this by each heart- beat. This difference is often called the pressure pulse, and in man it amounts to somewhere about 35 mm. Hg. If we take tracings with a spring manometer from different parts of the arterial tree, we shall find 160 L ine of SYSTOLIC PRESS UPE Of WEAK PRESSURE Line of DtASTOLIC Pressure PULSE Tfyetf/fference between SYSTOLIC D/ASTOL/C PRESSURE Fig. 25. Diagram based on experiments on dogs to show the magnitude of the systolic, diastolic and mean blood pressures at different parts of the circulatory system. O is the line of zero pressure, and the letters below it indicate the parts of the system to which the curves refer. (From Brubaker.) that, as we travel towards the periphery, the pressure pulse becomes less and less marked, until finally by the time the capillaries are reached it has almost entirely disappeared. This decline in the pressure pulse can moreover be seen to be dependent more largely on a fall in systolic than in diastolic pressure. In other words, the dead load of the circulation the diastolic pressure remains practically constant all along the arte- rial tree, whereas the systolic pressure falls relatively quickly (Fig. 25). Clinical Measurements The methods of blood-pressure measurement in man have recently become so perfected that the results are almost as accurate as those ob- 130 THE CIRCULATION OF THE BLOOD tained in laboratory animals by direct measurement through, the use of cannulse inserted into the vessels. Both the systolic and the diastolic pressure can be measured with equal facility and accuracy. Since the technic for making the systolic measurements was described at a much earlier date than that for the diastolic, it has until recently been the habit with a great part of the medical profession to be satisfied with systolic readings alone. This is most unfortunate, because the knowledge which such information gives us is incomparably inferior to that which can be obtained by gauging the diastolic pressure. Until we have learned more about the dynamics of circulation, it would be profitless to go into any details as to the reasons for this statement, but it will soon become self-evident. Suffice it for the present to state that the diastolic pressure is the more important because it gives us the load ivhich the ves- sels and aortic valves must constantly bear, and the resistance which must be overcome prior to the opening of these valves at the beginning of systole. Moreover, it helps us to gauge the peripheral resistance. The first step in the technique of blood-pressure measurements in man is the placing of an armlet or cuff around the arm or leg. This armlet consists of a rubber bag at least 12 cm. broad and covered on its outer surface by cloth or leather. The bag is connected by tubing with a pres- sure gauge and a pump. The pressure gauge may be either an ordinary mercury manometer (Fig. 26) or one of the numerous gauges built on the aneroid principle that are now on the market. For measuring the blood pressure in the vessels of the upper extremities, the armlet should be applied around the fleshy part of the upper arm and for the lower limbs around the thigh. For accurate reading of both pressures in the arm the following procedure should be followed. Having applied the armlet, the pulse is palpated at the radial artery, and the pressure in the arm- let then raised until the pulse can no longer be felt, at which moment the pressure in the manometer is noted. The cuff is then slowly decompressed and the pressure noted at which the pulse reappears. These two readings of systolic pressure should be close together, but they will not usually agree exactly for reasons which will be explained immediately. They give us the palpatory systolic index, as it is called. The pressure is now lowered about 15 mm. Hg, and a stethoscope is placed in front of the bend of the elbow over the artery and as close up to the cuff as possible. With each heartbeat a distinct sound like a pistol shot will be heard. The decompression is now continued slowly, and as the pressure falls the sounds will be heard to become louder and prob- ably somewhat murmurish in quality. At a certain pressure this loud character of the sound will suddenly become much less marked, and the murmurish quality if present will disappear. This point corresponds to the diastolic pressure, which is now read off from the manometer. BLOOD PRESSURE 131 It must be remembered that below this point, as the pressure in the cuff is further lowered, a sound is still heard in the artery; indeed it does not entirely disappear until the pressure has become quite low. This point of final disappearance is, however, of no significance. The cuff is now entirely decompressed, and should be left so for a moment or more, so that the circulation in the part of the arm below it may return to the normal. The above readings should then be controlled by a second observa- Fig. 26. Apparatus for measuring the arterial blood pressure in man. The pressure in the cuff is raised by means of the syringe until the pulse can no longer be felt at the wrist. This pressure is read off on the mercury manometer (systolic pressure). tion, in which the procedure is slightly modified. With the stetho- scope at the bend of the elbow the pressure in the cuff is run up to a little above the previously determined diastolic pressure, so that the sound is clearly heard. The pressure is then further raised till the sound disappears. This point indicates the systolic pressure; it is called the auditory systolic index. It will be found to give a systolic pressure a little higher than that obtained by palpation of the artery at the wrist. The sound being now absent, the pressure in the cuff is lowered until the sound reappears, and the point at which this occurs should almost 132 THE CIRCULATION OF THE BLOOD exactly correspond to that at which the sound was found to disappear. If the palpatory systolic index is not below the auditory, it indicates that some error has been made in the application of the apparatus, and that the reading of the diastolic pressure will be unreliable. The usual source of error is in the position of the stethoscope, if readjustment of this does not bring the two indices into proper relationship, the auscul- tatory method can not be relied upon for either systolic or diastolic readings. In case of failure of the auscultatory method, we have to fall back upon the palpatory method for measurement of the systolic pressure; and for measurement of diastolic, we must use the method known as the oscillatory, which until recent years was the only one known for gauging the dias- tolic pressure. This consists in observing the oscillation of the indicator of the pressure gauge; as the pressure in the cuff falls gradually from below the systolic pressure, these oscillations will be observed to increase in amplitude, until they reach a maximum beyond which with lower pressure they rapidly decline. The pressure in the cuff at the moment when the oscillations are at the maximum represents the diastolic pres- sure. With a mercury instrument it is obviously difficult to employ this method, but with a modern spring instrument it can with a little practice be used with great accuracy and will serve as a valuable check on the diastolic reading as taken by the auscultatory method. The procedure may be altered in various ways, there being only one pre- caution to bear in mind ; namely, that the pressure in the cuff should not be applied continuously for more than a few moments of time, for if this is done for long periods, not only will it interfere with the accuracy of the reading, but it may cause considerable discomfort to the patient. There are several conditions affecting the accuracy of the readings by each method which it is well to bear in mind. These have been investigated by Mac William,* Leon- ard Hill,2 and Erlanger.3 The most important conditions affecting the systolic pressure are as follows: (1) The compression cuff should be a wide one (12 cm.), and it should never be applied so that there is any chance of its compressing the artery against a bony surface. This precaution is necessary, since it has been found that much less pressure is required to obliterate any perceptible pulse below the armlet when the artery is flattened against some hard structure than when it is uniformly compressed in the tissues in which it lies. (2) Discrepancies are often noted between the systolic readings on compression and decompression of the artery; that is, the pulse may reappear on decompression at a lower pressure than that at which it dis- appeared on compression, the difference being most marked when the decompression is done quickly. This difference is owing to the fact that the full force of the pulse does not reach the forearm until all the vessels have become distended with blood. (3) There are often discrepancies in the systolic readings taken from different limbs; thus, it is not uncommon to find that the systolic pressure in the leg is higher than that in the arm even when the observed person is in the horizontal position. These differences are most commonly observed in patients suffering from aortic regurgita- BLOOD PRESSURE 133 tion or thickened arteries. In aortic regurgitation the pulse is of the water-hammer variety, and the greater systolic pressure observed in the leg vessels in such cases seems to depend on differences in the physical conditions concerned in the transmission of this exaggerated pulse wave to the vessels of the two extremities. The reason for the discrepancies in cases of hardened arteries is no doubt that the hardening is likely to be more pronounced in the vessels of the thigh than in those of the arms. When a hardened vessel is compressed it does not collapse uniformly that is, it does not become completely closed but its walls come together at the middle part while chinks still remain at the sides. The blood continues to pass through these chinks, and a very considerably higher pressure in the cuff is required to obliterate them. That this is probably the correct explanation is supported by the observation that, al- though in such patients the pulse does not disappear in the vessels of the foot at the same pressure as it does at the wrist, a distinct change is nevertheless perceptible in the pulse of the foot at a cuff pressure equal to that producing obliteration in that of the wrist. In a patient showing a systolic pressure of 115 mm. for the upper arm and 198 mm. for the leg, at 116 mm. the pulse in the leg, although not obliterated, became notably cut down in volume. Thereafter it persisted at a small volume with little alteration until the pressure became sufficient to obliterate it. It is said that re- peated compression and decompression of the hardened arteries greatly reduces the dis- crepancy in the systolic readings. Differences in systolic readings are also sometimes observed in normal individuals, particularly after muscular exercise, but for these no satisfactory explanation can be given. While palpating the radial artery, it will often be noticed, as the pressure in the cuff is gradually raised from zero, that the force of the pulse increases perceptibly until a pressure of about 50 mm. is reached. This paradoxical behavior of the pulse can also be demonstrated by the sphygmograph (see page 202). Its cause is not un- derstood, but it is of significance that the greatest augmentations occur at the same cuff pressure as that at which a sound first comes to be heard by listening over the artery at the elbow. With regard to the diastolio pressure, there has been some controversy as to whether it is more accurately gauged by the oscillatory or the auscultatory method. If both methods are employed it will usually be found that the oscillatory gives a higher read- ing than the auscultatory. The consensus of opinion seems to be that the latter method is the more accurate, and certainly it is the easier to apply, for with the oscillatory there is often great difficulty in deciding just exactly when the maximum oscillation occurs. The strongest evidence supporting the conclusion that the auscultatory readings are more reliable than the oscillatory has been gained by experiments with an artificial schema, consisting of a wide glass tube representing the armlet, filled with Ringer's solution and closed by rubber stoppers pierced by tubes, which are connected with a fresh artery, which therefore runs from end to end inside the tube. Through tubing connected with the artery a pulsatile flow of oxygenated Ringer's solution' is made to flow at varying pressures, which are indicated by valved manometers (see page 152) connected with the artery tubing just beyond the compression tube. The pressure in the latter is also measured by a manometer, and it is caused to vary by a suitable compressor. By comparing the behavior of the artery with the pulsating movement of a spring manometer connected with the compression chamber, under different degrees of pressure inside and outside the artery, it has been observed that the maximal oscilla- tion occurs when the artery is actually somewhat flattened between the pulse beats; that is, it occurs at an outside pressure above the diastolic pressure, at which of course the vessel should retain its circular shape. When a stethoscope is applied to the tube 134 THE CIRCULATION OF THE BLOOD leading from the artery just beyond the compression chamber, in the above described model, sounds similar to those in the arm are heard with each pulsation. While the pressure is being gradually lowered from above the obliteration point, these sounds will be found to become first audible as soon as a certain amount of fluid is forced through the compressed area at each pulse (the systolic index), and to become louder and often murmurish in quality as the decompression is proceeded with, until a pres- sure is reached at which they suddenly become less intense and change in character. At this moment it will be observed by' watching the artery that the external pressure is no longer capable of producing any flattening of the vessel between pulses. Evidently, therefore, the change of sound corresponds exactly to the diastolic pressure. With regard to the cause, it should be clearly understood that it is the systolic wave that produces it, although its occurrence and character are dependent upon the intra-arterial pressure existing during the diastolic phase. The cause of the sound has been shown to depend on the pro- duction of a water-hammer in the blood vessels below the compres- sion cuff (Erlanger 3 ). By a water-hammer is meant the pressure changes which are caused by suddenly stopping the flow of water in a tube. These changes in pressure cause the walls to be thrown into vibration and so produce a sound. In the taking of blood-pressure measurements, as above described, when the pressure in the cuff is between the systolic and diastolic, the volume of the compressed artery will in- crease abruptly with each heartbeat and thus permit a considerable volume of swift-flowing blood to enter the rest of the artery underneath the cuff. When this quickly moving column of blood comes into con- tact with the stationary blood filling the uncompressed artery below the cuff, it will become immediately checked, and thus distend the arterial wall with unusual violence and set it into vibration. CHAPTER XVI THE FACTORS CONCERNED IN MAINTAINING THE BLOOD PRESSURE Having become familiar with the principles of the methods by which blood-pressure measurements are made, the next problem is to examine into the causes which operate to maintain the pressure. Two of these causes may be considered as fundamental, since without them no such pressure could exist. These are: jl) the pumping action of the heart, and (2) the peripheral resistance that is, the resistance to outflow of blood from the ends of the arterial system. Less essential though im- portant factors are: (3) the volume of blood in the blood vessels, (4) the viscidity or viscosity of the ^blood, and ^(5) the elasticity of the walls of the vessels. We shall now pro.ceed to examine the experimental evidence which indicates the relative importance of each of these factors. 1. The Pumping 1 Action of the Heart Changes produced in the mean arterial blood pressure by alteration in the pumping action of the heart are most strikingly demonstrated by observing this pressure after cutting or during stimulation of the vagus nerves. As will be explained later (page 222), impulses conveyed through these nerves to the heart make the beats slower and weaker. These impulses are constantly acting in the heart, so that when both vagus nerves are cut, the beats become more frequent and stronger, with the result that the mean arterial pressure rises considerably. A lesser degree of this effect can usually be obtained by cutting the vagus nerve on one side (Fig. 27). If now the peripheral end of a cut vagus nerve is stimulated, as by applying an electric current to it, the heart will either stop beating altogether or become very much slowed, with the result that the mean arterial blood pressure will fall, in the former case almost to zero and in the latter, to a level corresponding to the degree of slowing of the heart (Fig. 28). 2. The Peripheral Resistance To demonstrate the influence of peripheral .resistance on mean arte- rial blood pressure, the most striking experiment is performed by cutting or stimulating the great splanchnic nerve. Through this nerve, impulses, 135 136 THE CIRCULATION OF THE BLOOD Fig. 27. Effect of cutting the vagus nerve on the arterial blood pressure. Fig. 28. Effect of stimulating the peripheral end of the right vagus on the arterial blood pressure. BLOOD PRESSURE 137 which are called vasoconstrictor because they constrict the lumen of the blood vessels, are transmitted to the blood vessels in the abdomen. The vessels are under the constant influence of these impulses so that, when the nerves that transmit them are severed, the vessels dilate and thus offer less resistance to the movement of blood. The result produced on the mean arterial blood pressure by cutting the two splanchnic nerves is therefore a marked and sudden fall, which is im- Fig. 29. Effect of stimulation of the left splanchnic nerve on the arterial blood pressure. Note the primary and secondary rises. mediately recovered from if the peripheral end of one of the cut nerves is stimulated artificially (Fig. 29). In choosing this experiment to prove the relationship between peripheral resistance and the mean arterial blood pressure, it must be remembered that it is not entirely conclusive, since the results observed on the mean arterial blood pressure from cutting or stimulating the nerve may be in part explained as due to variation in the total capacity of the circulation ; more room is created by cutting the nerves, less room by stimulating them. 138 THE CIRCULATION OF 1HE BLOOD 3. The Amount of Blood in the Body This can be altered by hemorrhage or transfusion, and the results of such procedures are of interest not only on account of their physi- ological bearing, but also because of their great practical importance. To appreciate the significance of the results, it is important to bear in mind that the total volume of the ~blood constitutes from 5 to 7 per cent of the weight of the animal (see page 85). The immediate effect of hemorrhage on the blood pressure depends on the rate of bleeding. If a large artery, such as the femoral, is cut across, the pressure will show an immediate but moderate fall, due largely to the fact that we have suddenly decreased the peripheral resistance. If on the other hand only a small artery or a vein is opened, the bleeding will at first produce no effect on the blood pressure, and it is only after some considerable amount of blood has been removed that it begins to fall. To be more exact, we may state that the removal of 5 c.c. of blood per kilogram of body weight does not influence the blood pressure. The re- moval of a second portion of 5 c.c. per kilogram causes the blood pres- sure to begin to fall, the fall of pressure for each subsequent 5 c.c. of blood per kilogram removed averaging about 6 mm. Hg, until after 20 to 25 c.c. of blood per kilogram have been removed, when a more rapid fall in pressure sets in (Downs'*). When the pressure reaches the level of from 20 to 30 mm. Hg, the danger limit is reached, for there now supervenes a train of symptoms known as * ' shock, ' ' and the chances for the animal's recovery become uncertain. That the removal of the first por- tion of blood, if this removal is slow enough, does not influence the blood pressure, indicates that some adjustment has occurred in the vascular system to hold up the pressure in spite of the loss of blood. This adjust- ment is believed to consist in vasoconstriction. Recovery from hemorrhage is remarkably rapid, the original volume of blood being restored within a few hours. The chances of recovery de- pend upon the amount of blood lost. A loss equal to 2 or 3 per cent of the body weight can almost always be recovered from in laboratory ani- mals, and in the case of man there is reason to believe that recovery may occur after as much as 3 per cent of the body weight has been lost. The recovery of blood pressure is brought about partly by a transfer of fluid from the tissues to the blood. This abstraction causes a drying out of the tissues, which soon excites an extreme degree of thirst. The dilution of blood by fluid derived from the tissues occurs very rapidly, as can be shown by comparison of the hemoglobin content, or the number of blood corpuscles, in samples of blood removed immediately before BLOOD PRESSURE 139 and immediately after a hemorrhage. The specific gravity of the post- hemorrhagic blood is also decidedly below normal, indicating that the diluting fluid contains a lower concentration of dissolved substances than the blood plasma. The dilution of the blood is indeed often so great that hemolysis occurs, the plasma being distinctly tinted red. Hemorrhage also slightly raises the hydrogen-ion concentration of the blood plasma, and diminishes the store of reserve alkali, so that the ad- dition of a certain amount of acid to the blood (e.g., carbon dioxide) causes a greater rise in the hydrogen-ion concentration. The deficiency in the blood elements produced by the dilution is recti- fied by the manufacture of new corpuscles in the bone marrow, etc., but this process in a liberally fed animal takes several days for accomplish- ment, and while it is going on microscopic examination of the blood will reveal the presence of immature corpuscles. Careful studies of blood regeneration following the removal on two successive days, of 25 per cent of the blood, have shown that even in starving animals the total amount of hemoglobin (percentage of hemo- globin multiplied by the volume of blood) slowly recovers (Whipple and Hooper). Recovery is greatly hastened by feeding with flesh or even with gelatin. Removal of the spleen or the establishment of a bili- ary fistula does not interfere with the recovery. Incidentally it will be advantageous to consider here the effects of transfusion, These are very different according to the nature of the fluid used for transfusion. Three transfusion fluids have been investigated: (1) blood itself, (2) physiological saline solution (see page 96), and (3) physiological saline solution containing viscid substances such as gum or gelatin. The effects are also very different according to whether the solu- tions are injected into animals with normal blood pressure or into those whose blood pressure has been lowered by preceding hemorrhage. The general effects are shown in the curves of Fig. 30. When blood is injected into animals with normal blood pressure, it will very soon cause the pressure to rise, and as the injection is main- tained the rise may continue until the pressure is perhaps 50 per cent or more above its normal level. If the injection is long continued, how- ever, a sudden fall of pressure occurs, on account of engorgement of the right side of the heart. If the injection is not pushed so far, the increased blood pressure after being maintained for a short time returns to its old level. Injection of saline into a normal animal, if made slowly, has no effect at all on the blood pressure; if more rapidly injected, the pressure will rise slightly, but to a much less extent than that observed when blood itself is injected. Much larger quantities of the saline than of the blood 140 THE CIRCULATION OF THE BLOOD can be tolerated before cardiac embarrassment ensues. After the dis- continuance of the saline injection, the blood pressure returns very rapidly to its old level. The most striking result of such experiments is the enormous volume of saline solution which can be slowly injected without perceptibly affecting the pressure. The question is, Where does the fluid go ? If the urinary outflow is examined, a certain increase will Fig. 30. Composite curves to show effects on blood pressure of hemorrhage, and transfusion with various solutions (N. M. Keith, from Bayliss). The average pressure in the various experi- ments before and after hemorrhage is given on the left in a continuous line. The behavior of the pressure after transfusion varied according to the solution used. BLOOD PRESSURE 141 usually be observed, but never by any means sufficient to account for the disappearance of the injected saline. If we open the abdominal cav- ity, we shall find that a considerable transudation of the saline into the peritoneal cavity has occurred, and that the liver is conspicuously edem- atous. A certain degree of edema is also usually evident in the tissues of the extremities. Still more interesting and important, from a practical standpoint, are the results obtained by injecting the above solutions into animals whose blood pressure has been lowered by a previous hemorrhage. If the blood removed during the hemorrhage is defibrinated (see page 102), and then reinjected into the animal, it will bring the blood pressure al- most but not quite back to its original level, which will then be fairly well maintained. If, on the other hand, saline solution instead of blood is injected, the restoration of blood pressure (with an amount of saline equal to that of the removed blood) will amount only to about three- quarters of the extent to which it had fallen. This partial recovery is, moreover, maintained for a short time only, after which the pressure approaches the level to which it was reduced by the hemorrhage. These observations raise two important practical questions: (1) Why is saline relatively ineffective in the restoration of pressure? and (2) Why is the restored pressure not maintained? The answers to these questions brings us to a consideration of the next of the factors concerned in the maintenance of the blood pressure, namely, the viscosity of the blood. 4. The Viscosity of the Blood The importance of this factor arises from the fact that facility of flow in a tube is inversely proportional to the viscosity of the fluid and directly proportional to the driving pressure to which it is subjected that is, to the difference in pressure between two points in the tube. If therefore the output of the heart remain constant, but the viscos- ity of the blood be decreased by a saline injection, the facility of flow will be increased and the pressure decreased. This fact can easily be shown experimentally in a model by causing gum solutions of various concentrations to be driven through a glass tube by means of a small piston pump delivering a constant amount of fluid into the tube with each movement. Although the outflow from the narrow end of the tube must remain constant, the pressure in the tubing will vary in proportion to the viscosity of the gum solution (Bayliss 5 .) Transferring these results to an animal whose blood pressure has been lowered by hemorrhage, it has been found that if saline solutions con- 142 THE CIRCULATION OF THE BLOOD taining a sufficient amount of gum acacia or gelatin to make the viscos- ity about equal to that of blood, are injected, the original level of blood pressure is recovered as well as it would have been had blood itself been in- jected. A 7 per cent solution of gum acacia almost fulfills these require- ments, but unfortunately this solution contains a slightly greater amount of calcium than it is safe to inject into an animal. The excess of calcium may, however, be removed by exactly neutralizing the gum solution with sodium hydroxide, neutral red being used as an indicator. Most of the calcium becomes precipitated as phosphate. The mucilage of the British Pharmacopeia, diluted five times with water, makes a 7 per cent solu- tion of gum acacia. A 6 per cent solution of gelatin, after being heated to 100 C., gives a viscosity similar to that of blood, but on account of the possible presence of tetanus spores such solutions must be very care- fully sterilized before injection, and the process of sterilization causes a decrease in viscosity. The injection of a quantity of one of the above solutions equal to that of blood lost by a hemorrhage will usually bring the blood pressure back to its original height and hold it there for an hour or so. Viscosity is, however, not the only property of such solutions upon which their desirable effect depends. The osmotic pressure of the colloids also comes into play. By injecting saline solution containing a sufficient amount of a colloid such as soluble starch, which gives it the correct viscosity but has no osmotic pressure, the blood pressure, although it temporarily recovers after transfusion, does not maintain its recovery in the same way as with solutions containing gum or gelatin. The difference between a starch solution and one of gum or gelatin is that the former has no osmotic pressure. This property influences the rate at which water passes through the walls of the capillaries as can be shown by observing the outflow of urine from the ureters during the injection into animals of saline alone or of saline containing either starch or gelatin (Knowl- ton 6 ). Diuresis is produced with either of the first two, but not with the gelatinous solution. The reason that the osmotic pressure of certain colloids prevents passage of water from the blood into the uriniferous tubules is that the development of this pressure on the blood side of the renal epithelium tends to counteract the filtration pressure by which the urine is formed (see page 547). Although the urinary factor will not in itself explain the efficiency of the colloids in recovering the blood pressure, the conditions controlling it reveal the mechanism by which the passage of fluid from the blood vessels into the tissues is prevented when solutions of correct composi- tion are injected. Normally the protein content of the blood plasma is higher than that of the tissue lymph, so that there is a continual attrac- BLOOD PRESSURE 143 tion of water from the tissues to the blood an attraction which is nor- mally balanced by nitration going in the opposite direction. When the nitration pressure in the blood vessels exceeds the difference existing between the osmotic pressure of their contents and that of the tissue fluids, water will pass into the tissue spaces. When the blood is diluted, as by the injection of saline solution, the osmotic pressure of the colloids in a given volume becomes lowered and, the nitration pressure remaining constant, fluid passes into the tissue spaces. Of course these explanations rest on the assumption that the walls of the blood vessels consist of a membrane which is permeable to crystalloids but impermeable or nearly so to colloids. A further account of the use of the solutions for transfu- sion in cases of surgical shock will be found on page 311. Another important property of the transfused saline solution to con- sider is its hydrogen-ion concentration. This value increases in the blood left in the body after hemorrhage, and injection of sodium chloride solu- tion aggravates the acidosis; addition of NaHC0 3 so as to make a 0.2 M solution restores the correct P H , and at the same time restores the lost buffer influence (Milroy 7 .) These observations are of interest in the light of the recent discovery of Cannon that a condition of acidosis, as judged by the C0 2 -combining power of the blood, is present in shock, and that the development of this condition can often be guarded against by bicarbonate injections. 5. Elasticity of Vessel Walls The elasticity of the vessel walls is essential to the maintenance of the diastolic pressure. If the walls possessed no elasticity but were rigid, blood pressure would fall to zero between the heartbeats. This fact can very readily be shown by a simple physical model consisting of a pump, to represent the heart, connected through a T-piece with two tubes, one of which is elastic, the other rigid. The free end of each tube is con- tracted to a narrow aperture representing the peripheral resistance, and either tube may be shut off from the pump by means of a stopcock (see Fig. 31). Each tube should also be connected with a mercury manom- eter. If now the stopcocks are arranged so that the fluid passes into the rigid tube while the pump is in action, it will be found that with each stroke of the pump the pressure in the tube rises considerably, but that it falls to zero between the strokes. If now the stopcocks are turned so that the flow is through the elastic tube, the action of the pump being meanwhile kept up, it will be found that the pressure between the strokes is maintained at a height which is dependent on: (1) the rate at which the pump is operating, and (2) the resistance to outflow from the tube. 144 THE CIRCULATION OF THE BLOOD The quicker the action of the pump and the higher the resistance, the lower the fall of pressure between the beats. The physical explanation of this result is clearly that the fluid within the elastic tube when the wave of pressure travels into it from the pump distends the walls of the tube, so that when the pressure from the pump ceases to act, the stretched elastic walls recoil on the column of fluid and so maintain the pressure. We may say that the elastic fibers in the vessel walls store up some of the systolic pressure and then transmit it to the blood during diastole. Fig. 31. Diagram of experiment to show that the diastolic pressure depends on the elasticity of the vessel wall. The pulse (produced by compressing the bulb B) disappears when fluid flows through an elastic tube (F) when there is resistance (g) to the outflow. A, basin of water; B, bulb syringe; C and E, stopcocks; D, rigid tube; F, elastic tube; G, bulb filled with sponge. These considerations would lead us to expect that patients with hard- ened arteries should exhibit a lower diastolic pressure than normal per- sons, which, however, is not usually the case, since such patients also suffer from an increase in the resistance to the flow of blood in the periph- ery. The pressure pulse in these patients is, however, very marked. On the other hand, when the vessel walls become more extensible and elastic, as in certain cases of aneurism, the pressure pulse in the vessels below the aneurism is distinctly less than that observed in normal ves- sels of the same patient. CHAPTER XVII THE ACTION OF THE HEART Having studied the methods for measurement and the main factors con- cerned in the maintenance of the arterial blood pressure, we may now pro- ceed to study in greater detail the two most important of these ; namely, the action of the heart, and the peripheral resistance. The heart action has to be studied from two viewpoints, the physical and the physiological. From the physical viewpoint we have to study the heart as the pump of the circulation. We must see how it acts so as to raise the pressure of the blood within it, and how the valves operate so as to direct the bloodflow always in one direction. We must also ex- plain the causes of certain secondary physical phenomena, such as the heart sounds which accompany the heart action, and of certain secondary changes in pressure produced in the other thoracic viscera by each heart- beat. From the physiological viewpoint we must investigate the conditions responsible for the constant rhythmic activity of the heart and the con- trol to which this is subjected through the nervous system. THE PUMPING ACTION OF THE HEART When the heart is viewed in the opened thorax of an animal kept alive by artificial respiration and lying in the prone position, it can be noted that with each contraction the ventricles become smaller and harder, that the apex tends to rise up a little, so that if the thorax were intact it would press more firmly against the walls, and that it rotates slightly from left to right, but does not move nearer the base of the heart. If the auriculoventricular groove is carefully observed, it will often be noted that it moves slightly toward the apex with each systole, whereas the base of the heart itself, where it is attached to the large vessels, re- mains fixed. The auricles can often be seen to contract and relax before the ventricles. The most noteworthy results of this inspection are that during sys- tole the apex of the heart does not move toward the base, but that the auriculoventricular groove moves slightly toward the apex. That these same movements occur in the intact animal can be shown by the very simple experiment of pushing two long steel knitting needles 145 146 THE CIRCULATION OF THE BLOOD through the thoracic walls into the heart walls, one of them so placed that it pierces the apex of the ventricle, the other so that it pierces the base. The needles then act as levers with their fulcra at the chest wall, and if the movements of their outer free ends, produced by the movements of the heart, are observed, they will be found to confirm the observations made on the exposed heart. More particular investigations of the changes occurring in the shape of the heart cavity during systole and diastole have been undertaken by making measurements of sec- tions across the heart in one or other of these conditions. For such purposes the heart in diastole is easily obtained, but for the heart in systole it is necessary to use the some- what artificial means of injecting the heart with hot chromic acid solution just be- fore the death of the animal. The chromic acid causes the cardiac muscle to contract and maintains it in this condition. The outcome of these investigations is, however, not of much practical importance. Although it is now common knowledge that the direction of the flow of the blood is from the veins to the arteries, yet it may be of interest to consider for a moment the general principle of the methods by which William Harvey succeeded in making this discovery. His evidence was partly anatomic, partly experimental. He pointed out that the walls of the veins, and of the auricles to which they lead, are very thin, whereas those of the arteries and ventricles are very thick, and he concluded that in the veins the blood must flow gently from the tissues toward the heart, to which the valves in the veins direct it, and that in the arteries it must be propelled by pulses with each systole through the arteries towards the tissues by the contraction of the walls of the ventricles. The experimental support for this hypothesis he furnished partly by clamping the large vessels, veins and arteries leading to or from the heart, and observing the resulting distention or collapse of the vessel; and partly by cal- culation of the amount of blood which must be expelled from the ventricles in a given period of time. Harvey's discoveries concerning the events of the cardiac cycle were not much added to until experimental methods were devised by which the pressure changes occurring in the various cavities could be measured and compared. Until such measurements were elaborated, it was impos- sible to investigate the mechanism by which the various valves between the heart cavities and the vessels connected with them perform their function, or to describe with any degree of accuracy the events occurring in the heart chambers during the various phases of the cardiac cycle. It is for the purpose of ascertaining the exact time relationship of these changes that intracardiac pressure curves are studied. Intracardiac Pressure Curves The earliest method for taking such curves consisted in introducing into the cardiac chambers and the blood vessels of the horse, so-called cardiac sounds. These consisted of a more or less rigid tube furnished at one end with a little elastic bag or ampulla and connected at the other with a tambour, by means of rubber tubing. One of these little bags THE ACTION OF THE HEART 147 was placed in one of the ventricles, another in the auricle or aorta, the tube being inserted in the former case through one of the large veins at the root of the neck ; in the latter case through the carotid artery. The intracardiac pressure curves obtained in this way marked a great ad- vance over the methods that had previously been used to study the events of the cardiac cycle, but they were so faulty in comparison with tracings taken by more modern methods that it is not worth while considering them any further here. The physical errors involved in the use of the older instruments were due mainly to the elastic recoil of the membranes, etc., used in their construction. A great improve- ment in technique was afforded by the use of the spring manometer of Hiirthle (see page 128), which was connected with one of the heart cavities by a cannula filled before Fig. 32. Diagram of Wiggers' optical manometer. The wide glass tube (A) (connected with the ventricle, etc.) is connected with a brass cylinder (B) provided with a stopcock (C), the lumen of which comes in apposition with a plate (a) having a small opening in it. The freedom of communication between B and a is regulated by the position of the tap. Above a is a segment capsule (&) 3 mm. in diameter and covered by rubber darn. This carries a small mirror (C) fastened so that it pivots on the chord side of the capsule. Above the capsule is arranged an inclined mirror, from which a strong beam of light is reflected on to the mirror (c) on the capsule. This beam then travels back and the mirror () is adjusted so that it impinges on a moving photographic plate. The slightest movements of the small mirror (C) are thus greatly magnified. insertion with some anticoagulant fluid. The cavity of the tambour was made as small as possible, and either left empty or filled with the anticoagulating fluid. A searching investigation into the physical principles involved in taking records of sudden changes in pressure by such instruments has, however, shown that considerable errors are incurred, the inertia of the moving mass of fiuid in the tubing and the necessity of using levers in order to secure records being responsible for most of them (cf. Wiggers). Their elimination has recently been achieved by using a so-called optical manometer, one of which (Wiggers') is shown in the accompanying figure. It consists of a wide glass tube A, connected above with a hollow brass cylinder B, pro- vided with a stopcock C, the lumen of which tapers from below upward till it assumes the same diameter as an aperture in the segment capsule fc, above it that is, a capsule 148 THE CIRCULATION OF THE BLOOD cut away at one end which is 3 mm. in diameter and covered with rubber dam. By adjustment of this stopcock the pulsations of the fluid in A and B can be damped to a greater or less extent before they are transmitted into the segment capsule. A small piece of celluloid carrying a tiny mirror rests on the rubber dam, being pivoted on the chord side of the capsule. A mirror is attached to the capsule with its plane so ad- justed that the image of a strong light placed at some distance from it is focused on the little mirror carried by the celluloid. The ray reflected from the little mirror and again reflected from the larger mirror is adjusted so as to impinge upon a moving photographic plate travelling at a uniform rate in a suitably constructed photographic apparatus. By the use of such an apparatus the chief errors encountered by the use of the older instruments are eliminated, because there is no moving mass of fluid and there are no levers to set up spurious vibrations. Curves secured by the use of this instrument are shown in Fig. 33. V Fig. 33. Optical records of intraventricular pressure; a-b, auricular systole; b-d, presphygmic period; d-f, sphygmic period; after f, diastole. Instruments of varying degrees of sensitiveness were employed in taking the curves. ('From Wiggers.) Two objects must be kept in view in analyzing the curves: (1) Curves obtained from the different cavities may be compared in order to de- termine the exact moment during the cardiac cycle at which such pres- sure changes occur as must serve to produce opening or closing of the various valves; and (2) the contour of the curves obtained from each cavity may be examined in order to find out exactly how the pressure in that particular cavity is behaving. Comparison of the Curves Before using the curves for ascertaining the relative pressure in the different cavities, they must be graduated according to some scale, for it is clear that by the use of instruments like those we have been describ- ing, the absolute pressure value of each curve will vary according to the construction of the instrument (thickness of membrane, etc.), and in- deed instruments of varying degrees of resistance must be employed in taking curves from places having such different pressures as exist in the auricles and ventricles. The graduation is, however, a very easy matter, and consists, as already explained (page 128), in connecting the THE ACTION OF THE HEART 149 instrument by means of a T-piece with a mercury manometer and a pres- sure bottle and then marking on the tracing, the points corresponding to each 10, 20 or 50 millimeters of increase of pressure, as the case may be. To ascertain the time relationship between the opening and the closing of the auriculoventricular valve, the tracings should be taken simultaneously from the right auricle and the right ventricle, and to ascertain the same with regard to the semilunar valve, from the left ventricle and the aorta.* By comparing the curves it is now an easy matter to ascertain the exact moment at which the pressure in the one cavity comes to equal that in the other. This moment, read on the accompanying time tracing, will obviously indicate that at which the particular valve is just about to open or close. From the results of such experiments, the curves may be superimposed as in Pig. 34. In the first place let us compare the curves from the right auricle and ventricle. The curves begin at the very end of diastole, and they show that a distinct increase in pressure is occurring in both auricle and ven- tricle and lasting about 0.1 second. This is of course caused by auric- ular systole, and since it occurs in both cavities, it indicates that the passage between them, the auriculoventricular orifice, must be open. The ventricular curve then suddenly shoots away beyond the auricular because of the onset of systole in the ventricle, and the point at which the two curves begin to separate indicates the moment at which the auriculoventricular valves close. From this time on until ventricular systole has given place to diastole, (0.3 second), the auricle is therefore shut off from the ventricle. The exact moment in diastole at which the two cavities are again brought into communication i.e., the auriculoventricular valves open is indicated by the curves coming to- gether. Having thus determined the exact moments of opening and closing of the auriculoventricular valve, we may now proceed to compare the intraventricular pressure curve with that taken from the aorta. After the necessary calibration corrections, this curve has been placed in Fig. 34 in its true relationship to the ventricular curve. Beginning again at the end of diastole, we find that the aortic pressure is very considerably above that of the ventricles, indicating that the semilunar valves must be closed, and it will be observed that the intraventricular pressure at the beginning of systole does not rise sufficiently to open them until an appreciable interval (0.02 to 0.04 second) after the closure of the auric- uloventricular valves (lines 2 and 3) ; that is to say, there is a period at the beginning of ventricular systole during which the ventricle is a closed cavity. *The connections with the heart may be made by pushing long cannulae down the large veins or arteries, or in the case of the ventricles by inserting a cannula with a sharp point directly through the wall of the ventricle. 150 THE CIRCULATION OF THE BLOOD It is a period during which the ventricle by its contraction is getting up a sufficient amount of pressure in the fluid contained in it to force open the semilunar valves against the resistance of the pressure in the aorta, and it has been popularly called "the period of getting up steam," or, in physiological language, the isometric, or the presphygmic, period. We shall use the last-mentioned term in our further discussion here. After the aortic valves have been opened, it will be observed that the pressure in the ventricles is just a little above that in the aorta, and that Fig. 34. Superimposed pressure curves from aorta, ventricle and auricle, along with electrocar- diogram and phonocardiogram. A, aorta. V, ventricle. Aur, auricle. EL, electrocardiogram. PH, phonocardiogram. Showing first and second sounds. it continues so during the whole of ventricular systole. When diastole sets in, the pressure in the ventricles quickly falls, and a point is soon reached at which equality of pressure in ventricle and aorta is again attained. This, as we have seen, corresponds to the moment of the closure of the semilunar valves. The pressure in the ventricle, although now rapidly falling, takes a little time before it has fallen low enough to permit the auricular valves to open. Here again, then, the ventricle is a closed cavity, and we have what is known as the postsphygmic period (lines 5 and 6). CHAPTER XVIII THE PUMPING ACTION OF THE HEART (Cont'd) THE CONTOUR OF THE INTRACARDIAC CURVES The Ventricular Curve From an analysis of the contour of each curve, further interesting points are brought to light. The intraventricular pressure curve recorded by older methods was shown as having a flat top or plateau. By the use of the more modern, optically recording, instruments it has been shown that this plateau becomes displaced by an arch if every precaution is taken to prevent dulling down of the pressure changes in the instrument, as by opening wide the stopcock in the instrument (Fig. 33). We may therefore describe the contour of the ventricular curve during the sphygmic period as consisting of a rising portion, almost continuous with the curve during the presphygmic period, a summit and then a declining portion, which is usually slower than the ascending. On the rising portion there is usually a notch at the moment the semilunar valves open. The cause for this is not satisfactorily explained. The practical value arising from a study of the curves lies in the insight which they give us into the nature of the stroke of the cardiac pump. They show us that the impulse which the ventricle gives to the moving mass of blood in the aorta rises quickly, attains a peak and then more gradually falls until the aortic valves close when the fall becomes much more sudden. The pressure is maintained long enough to overcome the inertia of the heavy load of blood in the large arteries. Wiggers has shown that the exact contour of the curve during the sphygmic period depends partly on the degree of sensitiveness of the op- tical manometer used and partly on the tension existing in the ventricle just before contraction. When the tension is low the arch becomes a plateau. In the case of the right ventricle the contour of the curve also depends on the degree of resistance to the bloodflow through the pul- monary circuit. The top of the curve becomes broader when the initial tension is high, and more rounded when there is a high pulmonary resistance. By the use of the older methods of observation (spring manometers) it was believed that the intraventricular pressure fell below the zero line in the early stage of diastole, thus indicating that a negative or 151 152 THE CIRCULATION OF THE BLOOD suction pressure existed. This conclusion seemed to be confirmed by using a mercury manometer furnished with a valve so arranged that only suction pressure could cause a movement of the mercury (a minimum manometer). The more recent methods, in which optical manometers are used, do not indicate the presence of such a suction pressure and there can be no doubt that the results of the earlier methods were erroneous : those by the spring manometers because of elastic recoil, and those of the minimum manometer because of the inertia of the mercury, the valve catching it, as it were, on the rebound. Fig. 35. Diagram to illustrate optical method for recording pressure curves from auricles. A, Auricles; C, Ventricles: B, Aorta; D, Photographic surface. (From Wiggers.) Fig. 35 is a diagram taken from Wiggers to demonstrate the optical method of recording simultaneously the pressure changes occurring within the auricle and ventricle and in the aorta. A, B, and C are small chambers filled with fluid and covered with rubber membranes. They are inserted through the walls of the auricle and ventricle and aorta. Each membrane responds to any change in pressure by a minute move- ment which is magnified by reflecting a narrow beam of light from a mirror fastened to its surface. The light beams are focussed upon a moving photographic film. The volume changes are recorded by slip- ping the ventricles as far as their A-V junctions into a glass oncometer THE PUMPING ACTION OF THE HEART 153 which is covered by a rubber diaphragm and connected with a large tambour. The absence of any suction pressure in the heart shows that its filling cannot depend, as does that of a mechanical pump, on aspira- tion between each beat. This does not mean that there may not be a slight suction pressure in the ventricle, as well as the auricle, during the beginning of each inspiratory movement in the unopened thorax, but since this pressure inside the ventricle must always be less negative than that in the thoracic cavity it can participate in the filling of the heart only to the small extent indicated on page 324. The rate of filling of the ventricle depends primarily on the rate of flow in the great veins leading to the heart (page 217). The Auricular Curve Examination of the intraauricular pressure curve is of particular in- terest because of the relationship which it has to a tracing taken of the movements in the jugular vein at the root of the neck (see page 285). This jugular pulse curve, as it is called, is produced mainly by the changes of pressure occurring in the auricle, from pressure curves of which it differs only in the relative height of the various waves. By graduating the intraauricular pressure curve by the method described above, we can tell exactly the magnitude in the changes of pressure oc- curring during each cardiac cycle. This obviously cannot be done with a tracing taken from the jugular vein, although qualitatively the tracings reflect the changes that are occurring in the auricle. On examining the auricular pressure curve (consult Fig. 34), we find that after the wave of presystole, which of course corresponds exactly with that on the intraventricular curve, a second wave occurs culminating in a peak shortly after the beginning of the presphygmic period. The curve rapidly descends during the first part of the sphygmic period and then slowly rises throughout the rest of ventricular systole, until the moment of opening of the auriculoventricular valve, when it descends again and thereafter runs parallel with the ventricular curve. There are therefore three positive waves on the curve, the first being caused by auricular systole or presystole ; the second, by the closure and possibly by bulging into the auricle of the auriculoventricular valve ; and the third, by the gradual filling of the auricle from the veins. The second wave corresponds to the wave C of the jugular pulse (page 286), which would seem to show that this is a reflected auricular wave. The depression following the second wave corresponds to wave X of the jugular pulse and the lowering of pressure which it indicates is probably '&ne to the cooperation of three forces, all tending to increase the auric- rtlur capacity: (1) The diastole of the walls of the auricle; (2) the 154 THE CIRCULATION OF THE BLOOD descent of the auriculoventricular groove, thus tending to open out some- what the folds in the walls of the auricle; and (3), most important of all, the tendency of the thin-walled auricles to become dilated as a result of the sudden diminution in intrathoracic pressure which is produced at each heartbeat by the discharge of blood from the heart and intra- thoracic blood vessels into those of the rest of the body. Under these conditions all thin-walled structures in the thoracic cavity, the auricles included, will expand to take up the extra room thus created in the thoracic cavity. Similar negative heart pulses, or cardiopneumatic move- ments, as they are called, can be observed accompanying each cardiac systole in the air passages and in the esophagus. This is done by con- necting sensitive tambours through tubing, either with one nostril (while the other nostril and the mouth are closed and the breath held) or with a thin rubber bag lying in the esophagus. Cardiopneumatic movements can sometimes be seen in the intercostal spaces of emaciated persons, and they often become pronounced in cardiac hypertrophy. THE MECHANISM OF OPENING AND CLOSING OF THE VALVES When physical valves open and close as a result of the changes in pres- sure on their two surfaces, a certain amount of fluid must succeed in passing the valve flaps before these become perfectly closed. But there is every reason to believe that such is not the case in the heart, the flaps of both the auriculoventricular and the semilunar valves being already completely closed before pressure conditions entailing a possible regur- gitation of blood through them become established. Auriculoventricular Valves During diastole the flaps of the auriculoventricular valves are hanging down into the ventricle and floating in a half-open position in the blood, which is meanwhile accumulating in the chamber. This position is de- pendent upon the operation of two opposing forces on the valve flaps: the pressure of the blood flowing from the auricle on their upper aspects, and reflected waves of pressure from the walls of the ventricle on their under aspects (centripetal reflux). When presystole occurs, the pres- sure of the auricular stream momentarily increases, thus slightly dis- tending the wall of the meanwhile relaxed ventricle and after a moment's delay causing the reflected wave to become more pronounced. At the same time the muscular fibers in the valve flaps (Kiirschner's fibers) contract and make the flaps shorter, the total effect of the two factors being that the valve takes up a position nearer that of closure. When presystole suddenly stops, the reflected waves will persist for an instant THE PUMPING ACTION OF THE HEART 155 of time longer than the auricular wave which causes them, because of the elastic nature of the ventricular wall, so that the valve flaps close with perfect apposition not merely at their edges but also for a con- siderable distance along their upper surfaces. When ventricular systole starts, the only effect of the high pressure which is brought suddenly to bear on the under surfaces of the already closed valves is to cause them to vibrate and to bulge into the auricles, being meanwhile anchored down and prevented from flapping into the auricle by the chordae tendineae. There is reason to believe that the musculi papillares to which these are attached begin to contract at the very outset of ventricular systole indeed slightly to precede it (see page 274), and thus keep the chordae taut. As systole continues the contraction of these muscles becomes more and more pronounced, and the resulting tightening of the chordae serves to draw down the valve flaps, so that progressively larger proportions of their upper aspects tend to become opposed. Meanwhile the auriculoventricular orifice is also be- coming narrowed down on account of the contraction of the musculature of the auriculoventricular groove. Semilunar Valves The mechanism involved in the operation of the semilunar valves is somewhat different. It has been shown that, when fluid is flowing in a tube, the pressure and velocity are not equal in the axial and peripheral parts of the stream. In the axis the velocity is greater than in the layers of fluid next to the walls, but the pressure is less. This can be seen by observing through a wide glass tube the flow of water in which are sus- pended lycopodium spores. By placing within the wide tube small bent tubes so arranged that one open end lies near the periphery and the other near the axis, the differences in pressure between the axial and peripheral streams can be seen to cause the fluid to flow in the narrow tubes from pe- riphery to axis (centripetal eddies). If the tube should suddenly expand the eddy currents become still more pronounced just where the wider portion starts. In the conditions obtaining at the beginning of the large arteries of the heart, the orifice into the ventricles being constricted, a centripetal vortex must be set up, tend- ing to throw the valve flaps into a closed position, which, however, is pre- vented by the blood rushing between them from the ventricles. They thus take up a mid-position and vibrate in the stream. "When the efflux from the ventricle stops at the end of systole, the reflux, lasting for a moment longer and being now unopposed, immediately closes the valves, in which position they are then maintained by the greater pressure on their aortic surfaces. 156 THE CIRCULATION OF THE BLOOD The position of the valves relative to the events of the cardiac cycle is shown in Fig. 36. The Venous Reservoir of the Mammalian Heart In birds and mammals there are no valves between the venae cavae and the auricles such as there are in lower animals at the sino-auricular junc- tion. Coincident with the appearance of the diaphragm, the sinus is merged with the auricles which are left open to a large venous cistern consisting of the venae cavae, the innominate, iliac, hepatic and renal veins. This cistern is shut off from the remainder of the venous system by six pairs of valves at the femoral, the subclavian and the jugular veins. Not only is it capacious, being capable of holding at least 400 c.c. of blood in man, but it is subject to considerable alteration in capacity because of the collapsible nature of its walls. This cistern is moreover in free communi- cation with another large cistern, capable of holding when full about 1000 Fig. 36. Diagram to show the positions of the cardiac valves: 1, during diastole; 2, during the presphygmic period; 3, during the sphygmic period. c.c. of blood, represented by the liver and portal system. Alterations in thoracic and abdominal pressures will greatly affect the emptying of this cistern into the auricles which explains the marked influence of the tho- racic movements and of abdominal compression on the filling of the heart. A most important function of this cistern is to furnish an immediate re- serve of blood to fill the heart during the performance of a great muscular effort. THE HEART SOUNDS During certain phases of the cycle distinct sounds, the heart sounds, can be heard by applying a stethoscope to the thoracic wall. The first occurs at the beginning of ventricular systole and is best heard over the apex beat; the second occurs at the beginning of diastole and is heard best at the second right costal cartilage or in the second left intercostal THE PUMPING ACTION OF THE HEART 157 space. A third sound, much less distinct, is sometimes heard in diastole a short time after the second. To study the exact time relationship of the sounds the vibrations which they set up can be recorded graphically alongside cardiac tracings by means of a microphone attachment to the electrocardiograph (see page 270). Causes of Sounds It has been found that the first sound consists of two distinct elements, one high pitched and the other of a dull character. The former element is believed to be the result of vibrations set up in the flaps of the auric- uloventricular valves, and therefore in the blood in the heart, by the sudden rise in systolic pressure. The dull element on the other hand is undoubtedly of muscular origin. The evidence for these conclusions is as follows: (1) When the auriculoventricular valves are prevented from closing properly either by disease or by pushing a loop of wire down the large veins, the high pitched quality_disam)ears < and nothing but a rush- ing sound accompanies the dull bruit produced by the contracting muscle. (2) In a heart that has been rendered bloodless by an incision near the apex, or even in an excised but still beating heart, the dull element of the first sound still continues to be heard for a short time. That con- tracting muscle produces a sound is a well-established fact. There are, however, many obscure phenomena connected with the causation of the first sound, but we can not go into such controversial matters here. A close inspection of the electrophonographic tracing shows that the sound starts at the beginning of the presphygmic period, and that it lasts with gradually declining but variable intensity until well into the sphygmic period (Fig. 37). The second sound occurs accurately at the beginning of diastole and can readily be shown to be caused by the sudden shutting and stretching of the semilunar valves, which throws them, the blood in contact with them, and the neighboring walls of the aorta into vibration. Proof of this conclusion is furnished by the following facts: The second sound immediately disappears if the blood is let out of the heart by opening the apex, and it is replaced by a rushing "bruit" if the flaps are pre- vented from closing as a result of disease or of hooking them back by passing a wire down the carotid artery. The third sound, although audi- ble only in some individuals, can nevertheless be shown to exist by the electrophonograph, and since it occurs at the time when the auriculo- ventricular valves open, it is believed to depend upon the sudden inrush of blood from auricles to ventricles. The greatest importance of the sounds is in the clinical diagnosis of val- vular and other lesions of the heart. When a valve leaks, for example, 158 THE CIRCULATION OF THE BLOOD the blood escapes past it under great pressure, and is ejected into a mass of blood at low pressure, these being conditions which are well known to create sounds or bruits. By examining the exact relationship of such bruits to the normal heart sounds, deductions can be drawn concerning the condition of the various valves. Record of Heart Sounds The heart sounds have been graphically recorded by transmitting them through a stethoscope to a microphone placed in circuit with a string galvanometer (electrophonograms,). Through this circuit passes a cur- rent the strength of which depends on the resistance offered by the microphone, and consequently to the number and amplitude of the vibrations of the sounds transmitted to it through the stethoscope. There are several objections to this method. One of these is depend- ent on the varying distance of the heart from the chest wall, which causes many of the sound vibrations to be lost before they reach the stethoscope; another, on adventitious sounds arising from contracting muscles, the impact of the heart against the chest wall, etc., and still another on unequal resonation by the air in the neighboring portions of lungs. To investigate the problem more thoroughly, Wiggers, 37 using anesthetized animals, has recorded the sounds by carefully stitching to the heart (exposed through a small opening in the pericardium) a lever, the end of which was attached to a "transmitter" consisting of a small capsule covered with rubber dam. The transmitter was connected by rubber tubing to a * ' recorder ' ' consisting of another small capsule carry- ing on its membrane (made of rubber cement) an eccentrically placed small mirror, on to which a beam of light was thrown. The movements of the beam of light reflected from the mirror, and caused by the sound vibra- tions, were photographed. Mechanical vibrations set up in the apparatus itself were largely eliminated by a side opening on the recorder, and the effect of outside sounds minimized by surrounding the recorder by a ventilated glass housing. Although this apparatus is not free from faults due to inherent vibra- tion frequency and resonance, the records secured by it are valuable in showing the exact relationship of the sounds to the events of the cardiac cycle. The vibrations from the two ventricles are alike, but differ from those taken from the aorta. The first ventricular sound consists of from five to thirteen irregular vibrations, usually in three groups, the first composed of two small vibrations, the middle one of several large vibra- tions, and the third of a varying number of small vibrations. The duration of the sound is from 0.05 to 0.152 seconds, and the periodicity from 0.004 to 0.054 per second. When compared with an intraventricu- THE PUMPING ACTION OF THE HEART 159 lar pressure curve, the initial vibrations occur 0.01 second prior to the rise in pressure, the main vibrations reaching their greatest amplitude before the sphygniic period begins, and the final vibrations occurring during the early part of the sphygmic period and therefore just before the aortic 11 I f ,5 -5 mm fir Mf. ; ....: B. f, f 1 ** 3 '/\ C. Fig. 37. Electrophonograms along with intraventricular pressure curves from three dif- ferent experiments. In A the uppermost curve shows the pressure, the middle one the sounds of the right ventricle, and the lowermost one those of the aorta. P indicates the relative posi- tion of the curves. M is due to mechanical oscillations. $2 indicates the second sound, and /, 2, 3, and 4 the corrected time relations of the first sounds. In B, the pressure and sound curves are both from the left ventricle (letters same as in A). In C, the aortic and pulmonary arterial sounds are shown (letters same as in A). (From Wiggers and Dean.) pressure has reached its height. The main vibrations therefore occur during the descending limb of the R wave of the electrocardiogram (be- ginning 0.01 second before its completion), the small preliminary vibra- 160 THE CIRCULATION OF THE BLOOD tions occurring during the ascending limb. When taken from the aorta, the record of the first sound is somewhat different, there being no initial vibrations and the main ones being of greater frequency and reaching their maximum earlier than those taken from the ventricle. The sub- sequent vibrations are also larger, especially when the aortic pressure is high (Fig. 37). The record of the second sound at the ventricle is much simpler and usually of less amplitude than the first, consisting of two to six vibrations lasting 0.015 to 0.056 second. They begin a short time after the ventricu- lar pressure begins to fall, approximately at the dicrotic notch of the aortic curve, being completed in from 0.015 to 0.025 second after the bottom of the notch. Their relationship to the T wave is variable. Taken from the aorta, the record of the second sound shows vibrations of greater amplitude and of a greater frequency than that from the ventricle. The absolute and the relative intensity of the heart sounds is of clin- ical value in forming a judgment of the dynamic state of the heart muscle and of the tension or pressure in the aorta and pulmonary artery. Ac- centuation of the second sound, for example, is considered to indicate a high pressure in the aorta. Wjggers 46 has recently confirmed this by ex- perimental methods. He recorded the sounds graphically by attaching receivers to the shaved skin of the thorax over the apex and pulmonary artery in anesthetized dogs, the receivers being connected with sound- recording capsules. After taking normal tracings, various alterations were brought about in the pumping action of the heart or in the blood pressure, and the effect was noted on the amplitude and number of vibrations en- tering into the sound tracings. It was found that the intensity of the first sound varied with the tension which was developed within the ventri- cles during systole, particularly during. the presphygmic period. When the vagus was stimulated for example, the first sound diminished because of the lower diastolic pressure to be overcome during the presphygmic period. Increase in the diastolic pressure without change in heart rate (by reflex vasoconstriction, compression of aorta, etc.) caused the second sound to become accentuated, and decrease of pressure (by nitrites) caused it to become less. Of course in both these experiments the first sound was also affected. It was possible to show, further, that when the pressure changes were more marked in one circuit of the circulation than the other, the sounds were more decidedly altered in the circuit in which the changes predominated (see also Lewis 47 ). CHAPTER XIX THE NUTRITION OF THE HEART THE BLOOD SUPPLY In cold-blooded animals, such as the frog, the heart muscle is nourished by blood soaking into it from the heart chambers, which indeed do not form definite cavities as in the mammalian heart, but exist as an inter- lacement of muscular tissue. In the hearts of higher animals, the muscu- lature is supplied by special arteries (the coronary), although a certain amount of blood may still pass directly from the cardiac cavities into the musculature through the veins of Thebesius. The relative importance of the various branches of the coronary artery in maintaining an adequate nutrition of the heart has been studied by observing the effect of occlusion of one or more of them (W. T. Porter 9 .) Occlusion of the circumflex branch of the left coronary artery caused arrest of the heartbeat in about 80 per cent of cases, the arrest being usually accompanied by fibrillary contraction. Occlusion of the right coronary arrested the ventricular contraction in about 20 per cent of the cases. Smaller branches may be occluded without any evident change in the heartbeat. These results indicate that the capillary areas supplied by the branches of the coronary artery do not freely anastomose with one another. They are more or less terminal arteries ; that is, each branch supplies a distinct region of the cardiac muscle. If one of the smaller branches of the coro- nary is occluded, although there is no immediate stoppage of the heart- beat, yet after some time the area supplied by that branch usually under- goes necrosis, again indicating that collateral circulation can not have become established. It is interesting, however, to note in this connection that anatomic studies have shown that a certain amount of anastomosis does occur between different branches, although it is evident, from the above observations, that an adequate collateral circulation does not im- mediately become established through this anastomosis. PERFUSION OF HEART OUTSIDE THE BODY In order that the blood supply through the coronary arteries may adequately maintain the normal nutrition of the cardiac muscle, certain 161 162 THE CIRCULATION OF THE BLOOD conditions must be fulfilled. The recognition of these conditions has been accomplished by observations on the excised heart, for it has been found that if they are fulfilled the mammalian heart can be made to beat in perfectly normal fashion for several hours after its removal from the animal's body. Indeed certain mammalian hearts, such as that of the rabbit, may be made to beat for several days outside the body. We may consider the essential conditions of the blood supply under four headings: (1) the temperature; (2) the oxygen supply; (3) the pressure; and (4) the chemical composition. Successful perfusion may be performed with artificial saline solutions (e. g., Locke's), but it is simplest in investigating the relative importance of the above conditions to start the heart per- fusion with defibrinated blood. After bleeding an anesthetized animal, such as a dog or a cat, until no more blood can be removed, the blood is defibrinated and filtered through gauze to remove the fibrin. The thorax of the dead animal is then quickly opened, ligatures placed around the main arteries springing from the arch of the aorta, a cannula with its end pointing toward the heart inserted into the descending thoracic aorta, and the latter cut across below the point of insertion of the cannula. The heart is then quickly removed from the thorax and an artificial saline solution (Locke's) allowed to run into the aortic cannula through a side tube, until all the blood has been washed out from the coronary vessels. Dur- ing this operation the heart may develop a few beats even though the solution is quite cool. The aortic cannula is now connected with a bottle containing the defibrinated blood diluted with Locke's solution and brought to body temperature by immersion in a water-bath. By means either of gravity or of a suitably regulated air pressure exerted on the surface of the diluted blood in the bottle, this is forced into the aortic cannula under pressure. The fluid thus finds its way into the coronary vessels; for in passing toward the heart in the aorta it will close the semilunar valves and force its way under pressure into the coronary vessels, subsequently escaping by the coronary sinus into the right auricle. Very soon after the perfusion is started the heart begins to beat vigorously and regularly, thus offering a suitable preparation upon which to test the first three mentioned conditions necessary for the nutrition of the cardiac musculature. If the temperature of the solution is allowed to fall considerably, the beat becomes much slower, and if the cooling is proceeded with, the heart will after a while cease beating altogether. If the pressure is lowered, the beat will not necessarily become slower but very much feebler, and will soon cease. In general it may be said that the temperature of the THE NUTRITION OF THE HEART 163 solution affects the rate of the beat, and the pressure affects its strength. It is, however, obvious that in perfused preparations changes in pres- sure are likely to cause alterations in rate as well as in force, unless great care is taken to keep the heart itself as warm as the perfusion fluid. The importance of an adequate pressure in the coronary vessels has been clearly brought out in certain experiments in which the beat has been maintained for a short time by establishing a pressure in the cor- onary vessels by means of indifferent fluids or gases. Thus, if oxygen gas is allowed to pass through the vessels under pressure, the heart will beat for a short time, and the same result has been observed even when mineral oil or mercury has been perfused under pressure (Sollmann). The necessity for an adequate oxygen supply is very readily demon- strated. If the darker blood ejected from the right auricle with each heartbeat is transferred immediately to the perfusion bottle, the heart- beat will soon become feeble and irregular, to be readily restored to normal when this dark blood is shaken up with air or oxygen. By artificial perfusion in the manner above described, the automatism of the heart may be restored many hours after death. Partial restora- tion, confined to the auricles or to that part of the ventricles lying im- mediately adjacent to the large blood vessels, can also be accomplished in the heart of man several days after death, provided death has not been caused by some acute toxic infection such as diphtheria or septice- mia. The Russian physiologist Kuliabko, has succeeded in restoring for over an hour the normal beat of the heart of a three-months-old boy twenty hours after death from double pneumonia, but here again the pulsation returns only in certain parts of the heart. As will be pointed out, the remarkable resistance of the heart muscle displayed in these experiments has been taken as an argument in favor of the myogenic hypothesis for automatic rhythmic power of cardiac muscle, the argu- ment being that nervous structures could not live so long a time after death. The fallacies in this argument are discussed elsewhere. Heart-Lung Preparation. Although the isolated heart is most useful for the investigation of many of the functions of this organ, particularly for the study of the effect of alterations in the chemical composition of the perfusion fluid and the action of drugs, it is not so suitable when blood instead of artificial plasma is to be perfused or when the effects of altera- tions in the physical conditions of the inflowing or outflowing blood are under investigation. In such cases it is better to use the heart-lung preparation, which moreover has the added advantage that it requires less blood, the proper aeration of which is affected through the lungs (cf. 48 ). 164 THE CIRCULATION OF THE BLOOD The arrangement for pcrfusion of the heart and lungs is shown in Fig. 38. The blood prevented from clotting by the addition of hirudin (page 101) is discharged from the left ventricle into a cannula connected with the innominate artery, all other branches of the aorta being tied off. A side tube, v, connects with an air cushion, afforded by an inverted test tube to take the place of the resilient arterial walls, and the tube then connects with a resistance B, which is furnished by a thin-walled rubber tube (rubber finger stall) enclosed in a glass cylinder into which air is pumped so as to Fig. 38. Arrangement of apparatus for heart-lung preparation of mammalian heart. (Knowlton and Starling.) compress the tube. Beyond this resistance the blood flows into a wide test tube con- nected with a siphon tube which discharges whenever the blood has reached a certain level in the test tube. The blood discharges into a reservoir, from which it flows through a spiral immersed in a water bath, and thence to a cannula tied into the superior vena cava. A tambour is connected with a lateral tube coming off the tube of the siphon, and it records a movement every time the discharge occurs. The blood is pumped by the right ventricle through the lungs, where it is oxygenated, artificial THE NUTRITION OF THE HEART 165 respiration being maintained throughout the experiment. The chief source of difficulty with the preparation is edema of the lungs. This occurs much more readily with the lung of the cat than that of the dog. Analysis of the alveolar air in the preparation gives information of great value regarding the nature of the gaseous metabolism of the heart (consumption of oxygen and respiratory quotient), and by altering the CO^ eon- tent of the inspired air, the influence of changes in H of the perfusion fluid can be ob- served (Evans '* 9 ). RESUSCITATION OF THE HEART IN SITU A suitable intracoronary pressure is a sine qua non for the mainte- nance of the heartbeat, and this is a fact of great clinical significance, for it indicates that any attempts to resuscitate a dead animal are cer- tain of failure unless the method is such as will bring a nutrient fluid under a certain pressure to bear on the coronary arteries. Injection of fluid, even of defibrinated blood, into a vein will obviously fail to ful- fill this condition, for the perfusion must be made into an artery so that the fluid is carried down the aorta and thence into the coronary arteries. The practical question, in so far as resuscitation of the heartbeat is concerned, is therefore, How can we get the necessary fluid under pres- sure into the beginning of the aorta? Even if we were to transfuse fluid under considerable pressure into the aorta through the carotid artery, it would mainly follow the large vessels leading away from the heart, only a fraction of it reaching the beginning of the aorta. To compel the fluid to pass towards the heart we must introduce some obstruction to its passage peripherally. This can be done by the injection of a consid- erable dose of epinephrine (adrenaline) in normal saline solution through the needle of a hypodermic syringe inserted into the tubing leading from the burette or pressure bottle to the cannula in the carotid artery. As the perfusion fluid is running in, the epinephrine injection is quickly made, artificial respiration and cardiac massage being meanwhile prac- ticed. In the majority of animals it will be found that complete res- toration of the normal blood pressure can be effected by this method. Indeed by performing the resuscitation under aseptic conditions, some animals may be permanently resuscitated so far as the circulation is concerned, although the nervous structures, even after a few minutes of " death," never reacquire their normal condition. The epinephrine acts mainly by constricting the small arterioles and thus directing the bloodflow towards the heart, but partly also by a direct I stimulating action on the cardiac muscle. It does not, however, con- tract the coronary vessels; on the contrary, it is said to cause these slightly to dilate. 166 THE CIRCULATION OF THE BLOOD THE RELATIVE IMPORTANCE OP THE VARIOUS CONSTITUENTS OF THE PERFUSION FLUID We can study the chemical conditions necessary for resuscitation of the heartbeat by observing the beat of an artificially perfused heart while solutions of different chemical composition are being perfused through the coronary vessels. At the outset we are impressed with the fact that for successful resuscitation the organic constituents of the nutrient fluid are of trivial importance compared with the inorganic constituents. With a solution containing the proper proportion of in- organic salts, and of course an adequate supply of oxygen, the heart of a rabbit, for example, may be made to continue beating for several days. It is true that it will beat longer if some of the organic con- stituents of the blood plasma, particularly carbohydrate, are present, but on the inorganic constituents alone its ability to beat is truly remarkable. Observations on Cold-Blooded Heart The earlier experiments for the investigation of the chemical condi- tions necessary for the maintenance of the heartbeat were performed on the heart of the frog or turtle. By perfusing either of these hearts with physiological sodium-chloride solution, it was observed that though the beat might continue for some time, yet it gradually grew feebler and feebler, until at last it ceased altogether with the heart muscle in a condition of extreme relaxation or diastole. If small proportions of potassium and calcium salts (as chloride) were added to the sodium- chloride solution, the beat was much better maintained. Sidney Ringer proved that the optimum concentration to produce efficient and prolonged contraction for the heart of the frog or terrapin is as follows: potassium chloride, 0.03 per cent; calcium chloride, 0.025 per cent. The effectiveness of the solution was also found to be increased by the addition of 0.003 per cent of sodium bicarbonate. This acts as a buf- fer substance (page 36), holding the hydrogen-ion concentration at a constant level. More recent work has shown that the hydrogen-ion con- centration of the perfusion solutions is of considerable importance in determining the efficiency of the beat, but the optimum is not the same for the hearts of different kinds of animal, and indeed it may differ for different parts of the same heart. The question naturally arises as to the relative importance of each of the above salts; or rather, we should say, cations, since the anion, chlorine, is the same for all of them. The function of the sodium chlo- ride in the solutions is twofold: (1) to endow the solution with the THE NUTRITION OF THE HEART 167 proper osmotic pressure (see page 4) ; and (2) to perform the special role of the sodium ion in the origination and maintenance of the auto- matic beat. The latter function of Na can be shown by observing the behav- ior of strips cut out from the ventricle of the turtle heart and placed in solutions of correct osmotic pressure but containing no sodium chlo- ride isotonic solutions of cane sugar, for example. They soon cease to beat, but if a small amount of sodium chloride is added to the cane sugar solution, rhythmic contractions return. The role of the calcium ions is almost entirely a pharmacological one. If a strip of turtle ven- tricle which has been made to cease beating by immersion in isotonic sugar solution is placed in a weak solution of calcium chloride before it is transferred to sodium chloride solution, the spontaneous contrac- tions will return earlier and continue for a longer time. On the other hand, if more than the correct amount of calcium salt is present in the solution, the beats will soon be found to become smaller and smaller in amplitude, because relaxation does not properly occur between them, and ultimately they will cease altogether with the ventricle in a condition of extreme contraction, called calcium rigor. The importance of calcium may also be shown by attempting to perfuse a turtle heart with blood serum from which the calcium has been removed by the addition of sodium oxalate (which precipitates it as insoluble calcium oxalate). The heart soon ceases to beat, but can readily be made to do so again by adding a slight excess of calcium chloride. The potassium ions do not appear, like those of calcium and sodium, to be absolutely essential for the maintenance of the heartbeat; at least the heart of the turtle will beat for a long time when perfused with a solu- tion containing only sodium and calcium salts. The explanation of this result need not, however, necessarily be that potassium is an unessential constituent of the perfusion fluid, for it may well depend on the fact that there is a sufficient store of potassium locked away in the muscle fiber to supply the requirements of the heart muscle for this ion for at least as long as the beat would continue under any circumstances. In any case, we know that potassium has a profound influence on the heart- beat, for when the proportion of it in the perfusion fluid is increased, the beat becomes very slow and the tone of the heart is greatly diminished that is, it becomes^ extremely relaxed between the beats; and if the amount is further increased, will very soon come to a standstill in a greatly dilated or diastolic position. The striking antagonism displayed by these inorganic cations upon the heartbeat has led some investigators to suggest that the stimulus re- sponsible for the rhythmic activity of the heart depends on some sort of chemical union occurring between the inorganic cations and the con- tractile substance of the heart. Union of calcium with the contractile 168 THE CIRCULATION OF THE BLOOD substance will lead to systole or contraction, whereas union of sodium or potassiumjadll-lead to relaxation or diastole. Observations on Mammalian Heart Investigation of the efficiency of various saline solutions on the iso- lated mammalian heart has shown that the proportion of the above salts must be somewhat different from that used for the cold-blooded heart. As might be expected, the most efficient proportions are those present in the blood serum of the particular animal whose heart is being per- fused. Basing his proportions upon the results of analyses of the inor- ganic constituents of mammalian blood serum, Locke found that an inorganic solution of the following composition is most efficient: so- dium chloride, 0.9 per cent; calcium chloride, 0.024 per cent; potassium chloride, 0.042 per cent; and sodium bicarbonate, 0.01 to 0.03 per cent. When " Locke's solution," as it is called, is perfused, with oxygen in it, under pressure through the isolated mammalian heart at body tempera- ture, efficient beating can be maintained for many hours. More recently a solution known as Tyrode 's is commonly used. It contains a small amount of magnesium and of phosphates. Although undoubtedly superior for some perfused preparations, such as the intestine, it does not seem to be in any way superior to Locke's for the perfusion of the heart. The bicar- bonates and phosphates in these solutions endow them with a hydrogen-ion concentration near that of the blood (slightly on the alkaline side of neutrality), and at the same time they act as buffer substances. As already pointed out, the organic constituents of such perfusion fluids do not appear to be relatively of nearly so much importance as the inorganic. Nevertheless it appears that a small percentage (0.1 per cent) of glucose does materially improve the nutritive qualities of the solution, and it has moreover been shown that after a while the con- centration of glucose in the perfusion fluid distinctly decreases. This does not of itself necessarily mean that the glucose is .actually utilized by the heart muscle: it might be stored away in it as glycogen. That some consumption of carbohydrate does however occur in the heart has been demonstrated by measuring the intake of oxygen and the output of carbon dioxide through the lungs of an isolated heart-lung prepara- tion perfused outside the body with defibrinated blood. By experiments of this type the attempt has been made to show that the heart of diabetic animals loses the power of burning glucose as compared with the. hearts of normal animals. While the experiments are very suggestive, the results do not as yet justify us in claiming that in the latter disease the power of burning glucose in the tissues has been materially depressed. The concentration of hydrogen ions in the perfusion fluid has an im- portant influence on cardiac efficiency. We also know that the most THE NUTRITION OF THE HEART 169 convenient method for changing the hydrogen-ion concentration of such fluids is by altering their tension of carbon dioxide (see page 371). In a heart-lung preparation (page 163), such alteration in carbon-dioxide tension can very readily be brought about by altering the percentage of this gas in the air with which the lungs are ventilated. To measure the efficiency of the heartbeat in such an experiment, it is convenient to enclose the organ in a cardioplethysmograph, the tracing of which will tell us the degree to which the heart is contracted or relaxed, as well as the output of blood per minute. By increasing the tension of carbon dioxide, it has been found in such experiments that the dilatation of the ventricle is encouraged, so that the heart with each beat discharges a larger quan- tity of blood (Fig. 39). When defibrinated blood is used the optimum pressure or tension of carbon dioxide has been found to lie between 5 and 10 per cent of an atmosphere. Fig. 39. Volume curve of ventricles of cat (lower curve) in a heart-lung perfusion prepara- tion. The air used to ventilate the lungs was replaced between the arrows by a mixture con- taining 20% CO 2 and 25% O 2 . This caused dilatation of the ventricles along with feebler beats and a tendency for the arterial pressure to fall (upper curve). The after effect was an im- provement of the beat. (From Starling.) That the effect of carbon dioxide in encouraging the relaxation of the heart between beats is in part at least dependent upon the change in hydrogen-ion concentration of the perfusion fluid has been shown by securing similar results in experiments with perfusion fluids to which different quantities of weak nonvolatile acids have been added. These observations are of practical importance because of the light which they throw on the cause of cardiac failure following upon conditions in which there has been excessive removal of carbon dioxide from the blood, as in forced ventilation of the lungs. Yandell Henderson has suggested that surgical shock may be, partly at least, due to cardiac failure fol- lowing the "washing out" of carbon dioxide from the blood by the dyspnea so often incident to the administration of anesthetics in surgical operations. CHAPTER XX THE PHYSIOLOGY OF THE HEARTBEAT THE ORIGIN AND PROPAGATION OF THE BEAT THE PHYSIO- LOGICAL CHARACTERISTICS OF CARDIAC MUSCLE The origin and propagation of the heartbeat are studied on the excised heart of a frog or turtle, or on the mammalian heart by perfusing it under suitable conditions, which have already been described. The results obtained on the cold-blooded heart apply more or less directly to the warm-blooded. In the first place it is clear that the rhythmic contrac- tility of the heart is not at all dependent upon the central nervous sys- tem, for if it were so, the excised heart could not continue beating. This fact does not, however, necessarily imply that the beating power is in- dependent of nervous structures, for in the heart itself an extended net- work of nerve cells and connecting nerve fibers can readily be demon- strated. It might quite well be the case that the rhythmic beat is de- pendent upon the transmission to the muscle fibers of the heart of impulses generated in the nerve cells and transmitted along the nerve fibers of this local nervous system. Such is the neurogenic hypothesis of the heartbeat. On the other hand, it may be that these nervous structures are not at all responsible for the origination of the beat, but serve merely as sta- tions on the pathway of the nerve impulses, transmitted to the heart from the central nervous system along the vagus and sympathetic nerves, for the purpose of altering the rate of the heartbeat so as to adjust it to the requirements of blood supply in the various parts of the body. In such a case the rhythmic power would reside -in the muscular tissues of the heart that is, each cardiac muscular cell would have the power, not merely like skeletal muscle of contracting in response to a stimulus transmitted to it, but also of originating that stimulus within itself. This is the myogenic hypothesis. Much controversy has raged around these two hypotheses and although space will not permit a detailed study of the question, it will be necessary, on account of the great importance of the subject from the physiological standpoint, briefly to review the main arguments of each school of thought. There is no piece of evidence offered by the advocates of either the neurogenic or the myogenic hypothesis that can, taken singly, be con- 170 THE PHYSIOLOGY OF THE HEARTBEAT 171 sidered as absolutely conclusive. Although some of "the proofs*' may at first sight appear to be conclusive, yet each of them breaks down when subjected to a closer scrutiny. It is only after we have collected all the evidence for and against each view that we shall be in a position to come to any conclusion, and even then it will be plain that our conclusion can be only tentative. Myogenic Hypothesis Taking first of all the evidence in support of the myogenic hypothesis, the following stands out most prominently: 1. The heart beats in the embryo chick before any nerve cells have grown into it, and not only this, but if portions of heart muscle are re- moved from the embryo and placed in blood plasma, they will continue beating for many days It has also been observed that cells may wander off from this mass of cardiac muscle and undergo multiplication and differentiation, so as to produce isolated muscle cells which exhibit rhythmic contractility. The rebuttal on the part of the neurogenists of this apparently unassailable evidence is to the effect that, although em- bryonic muscle cells may exhibit the power of rhythmic contraction, this does not mean that the fully developed muscle cells will necessarily have such power. In the eary stages of embryonic development, it is of course evident that the functions which in the fully developed animal are del- egated to various special organs and tissues must be performed by cells having several such functions in common. The muscle cells of the heart, for example, may to start with be possessed of the power not only of con- tracting but also of initiating the contraction. In early embryonic life they may be partly nervous in character, a property which they gradually lose as nerve cells and nerve fibers make their appearance. 2. The nervous structures in the heart may be damaged either by me- chanical means or by drugs without apparently interfering with the power of rhythmic contraction; for example, in the heart of large tur- tles it is possible to dissect out a considerable amount of nervous tissue without any disturbance of the beat, and in all animals the administration of atropine, which paralyzes the postganglionic fibers of the autonomic nervous system (see page 231) found in the heart, does not affect it. 3. The apex of the ventricle in such hearts as that of the turtle can be shown, by careful histological examination, to contain no nerve cells, and although a few nerve fibers may be found, these are of course functionless without nerve cells. This virtually nerveless piece of heart muscle can be made to contract rhythmically by perfusing it with suitable saline 172 THE CIRCULATION OF THE BLOOD solution under pressure and starting the beating by application of elec- trical stimuli. Isolated strips of ventricular muscle, in which also no nervous element can be demonstrated, may under favorable conditions be caused to beat quite regularly if supplied with proper nutrient fluid. The rebuttal of this evidence is twofold: In the first place, skeletal mus- cle itself under certain conditions, such as exposure to solutions con- taining an excess of phosphate (Biedermann's), may exhibit rhythmic contractility, especially on cooling, which indicates that exhibition of rhyth- mic power in isolated portions of cardiac muscle need not mean that under ordinary conditions such power is responsible for the normal heartbeat. In the second place, it is pointed out that although we can not reveal their presence by present-day histological methods, this is not conclusive evidence that the heart-muscle fiber may not possess some nervous struc- tures capable of functioning as nerve cells. The heart even of mammals can be made to continue beating for sev- eral days after excision from the body. The nerve cells, as we know them in the central nervous system at least, can not, on the other hand, be made to functionate for more than a few hours after death. Therefore, it is argued, the heartbeat in surviving mammalian hearts can not de- pend on the nervous structures. The argument is however easily refuted: on the one hand, we do not know that the nerve structures situated peripherally in the heart muscles are of the same viable nature as those composing the central nervous system; and, on the other, the survival of the heart may in itself be sufficient to maintain around the nerve cells embedded in it a nutrient environment which is much more physiological than that which we can supply in artificial perfusions of surviving nervous tissues. 4. Circumstantial but nevertheless strong evidence is furnished by the fact that many other varieties of involuntary muscle are endowed with rhythmic contractility; thus, the muscle of the intestines, of the ureters, of the bladder, of the uterus, of the blood vessels of certain animals, and of the lymph vessels in the so-called lymph hearts, main- tain rhythmic contractility after isolation from the animal body. The rhythmic power seems in certain of these cases to be independent of nervous control. Neurogenic Hypothesis In favor of this hypothesis the following evidence is offered: 1. The heart of certain animals of Limulus, the king-crab, for exam- ple is definitely dependent for its rhythmic contractility upon neigh- boring nervous structures. The heart of this animal is a tubular sac- culated organ, and along its dorsal surface there runs longitudinally a THE PHYSIOLOGY OF THE HEARTBEAT 173 nerve cord containing ganglion cells and giving off fibers which proceed in part directly to the heart and in part to lateral cords (Fig. 40). Re- moval of this median nerve cord is followed by total abolition of the heartbeat; the heart becomes perfectly quiescent like an unstinmlated skeletal muscle. In appraising this evidence at its true value, it must be noted that although contraction of the heart can be produced by stim- ulation of the nerve fibers, the contraction is like that of a skeletal mus- cle it is not rhythmic; and moreover and this is most important if the various physiological properties of muscle as described below be stud- ied (page 176), it will be found that in all of them the quiescent heart muscle behaves, not like the heart muscle of other animals, but like that of skeletal muscle. This evidence, therefore, while indisputably showing that the heart of Limulus depends for its rhythmic power upon neigh- boring nerve structures, does not justify the assumption that this will be the case in the heart of animals having different physiological properties. 2. The disposition of the nervous structures in the heart, especially of the frog and turtle, exactly corresponds to the degree of development of Fig. 40. Heart and cardiac nerves of Limulus polyphemus. (Carlson.) aa, anterior ar- teries; la, lateral arteries; In, lateral nerves, nine, median ganglionic chain; os, ostii or afferent stomata, each pair of which corresponds to one of the segments into which the Limulus heart is divided. the rhythmic power of the different parts of the heart ; thus, the greatest rhythmic power is manifested by the sinus and the least by the tip of the ventricle at the bulbus arteriosus. In the former position the nerve structures are very prominent ; in the latter, no nerve cells and but few nerve fibers can be detected. This proof is, however, easily assailed. In the first place, it may merely be a coincidence that the disposition of the nerve structures and the development of rhythmic power correspond. The unequal rhythmic powers may depend primarily on a difference in structure of the muscle fibers themselves, such differences having been shown to exist between the muscle cells of the sinus and those of, say, the ventricle. The former cells, for example, have much less developed crossed striation and their protoplasm is much more gran- ular ; in short, they are much more embryonic in type than the cells from the tip of the ventricle. If a jury had to return a verdict from evidence of so conflicting a char- acter, it would no doubt be equivalent to that of the Scottish court "not 174 THE CIRCULATION OF THE BLOOD proven." But it is likely that the majority of the jury would vote in favor of the myogenic hypothesis. Probably the safest viewpoint to take at the present time is that the power of rhythmic contraction is inherent in the cardiac muscle fibers, being most highly developed in those of the venous end of the heart, and least developed in those of the arterial end. Such a conclusion does not deny to the nervous struc- tures of the heart the power under certain conditions of also assuming rhythmic activity. In one case at least namely, the hea'rt of Limulus we know that this is so. For some reason in this animal the cardiac muscle fiber has lost its inherent rhythmic power, and is now dependent for its activities upon rhythmic nervous discharges transmitted to it from the neighboring nerve cords, a condition which is paralleled in the higher animals in the innervation of the respiratory muscles. The respiratory center rhythmically discharges impulses to the muscles, which are quiescent in the absence of these impulses. The Pacemaker of the Heart and Heart-block In a volume of this nature, devoted primarily to the practical appli- cation of physiology, the discussion of these problems may seem a little out of place, but that this is not the case is seen when we consider that the experiments upon which the various points of evidence depend bring to light facts of the very greatest importance in the study of the physiology of the heartbeat. One fact which stands out prominently is that the greatest rhythmic power resides in the basal portion of the heart that is, in what corresponds, in the more primitive hearts, to the sinus venosus. Although the muscle of the entire heart possesses rhythmic power, it does not do so to an equal degree; in the sinus the rhythmic power is extraordinarily developed, while in the bulbus arteriosus it is scarcely recognizable. This observation suggests the possibility that the sinus may dominate the heartbeat that it may be the "pacemaker" for the heart as a whole. The most natural method for demonstrating such a possibility would be to observe the effect on the heartbeat of some Nock between the sinus and the rest of the heart. Such a block can be intro- duced in the heart of cold-blooded animals by local compression around the various junctions. If a thread is tied around the sinoauricular junction, the sinus will go on beating uninterruptedly, but the auricles and ventricles that is, the greater part of the heart below the ligatures will cease beating, sometimes entirely (Stannius' ligature). After a while, however, the heart below the ligature will usually begin to beat, but at a rhythm which is slower than, and independent of, that of the sinus. THE PHYSIOLOGY OF THE HEARTBEAT 175 The experiment can be still better performed by using a wedge- shaped clamp. (Gaskell's clamp.) If this is applied so that the heart can be pinched either at the sinoauricuiar junction or at the auriculo- ventricular, it will be found that, as the cardiac tissue is gradually Fig. 41. Heart-block produced by applying clamp at a-v junction. The clamp was tightened at a. (From Brubaker.) pinched, the portion of the heart below fails to beat as quickly as that above the clamp (Fig. 41). This is known as partial heart-block, and the degree of the block is indicated by the numerical expression 2 to 1, 3 to 1, 4 to 1, etc., meaning that the sinus is' beating either twice as quickly as the ventricle, or three times, or four times as the case may Fig. 42. Tracing of contraction of ventricle, showing the effect of the local application of heat to the auricle at /, and to the apex of the ventricle at 2. Note that the rate in- creased in the former case. be. Similar conditions of heart-block may also be produced by cutting the cardiac tissue partly across at various places in the heart. Further evidence that the sinus dominates the beat in the heart of 176 THE CIRCULATION OF THE BLOOD cold-blooded animals is furnished by observing the effects of local heat- ing or cooling of the various parts of the heart. In all rhythmically acting structures it is well-known that heat increases the rate of the rhythm and cold depresses it. If we locally warm the region of the sinus, as by holding a heated wire near it the whole heart will immedi- ately beat quicker; but if we locally heat the tip of the ventricle, no alteration of rhythm will be observed to occur (Fig. 42). The establishment of the fact that the sinus dominates the heartbeat that it is the pacemaker of the beat raises the question as to how the impulse originated at this place is transmitted over the rest of the heart, and here again a neurogenic and a myogenic hypothesis have to be considered. Before going into this question, however, it will be well for us to consider briefly the manner of response of cardiac muscle fiber to a stimulus, because the behavior of cardiac muscle under such conditions is considerably different in many regards from that of skel- etal muscle, and it is to these differences that many of the peculiar alterations in the beat observed after interfering with the conducting structures between the sinus and the rest of the heart, are to be ex- plained. The Physiological Characteristics of Cardiac Muscle It is necessary to bring the heart into a quiescent state in order to investigate the properties of its musculature. This is accomplished by Fig. 43. Frog heart showing the position of the first and second ligatures of Stannius (Hedon): /, auricles; 2, sinus; j. ventricle. It is the first ligature which brings the heart to a standstill. the application of the Stannius ligature between the sinus and the auri- cles (Fig. 43). After tightening the ligature the auricles and ventricles become quiescent, and by observing the effects following the applica- tion of electric or other stimuli we can compare the behavior of the cardiac muscle with that of skeletal mttscle similarly stimulated. This comparison is made because of the assistance which it offers in compre- hending the properties of cardiac muscle. As a matter of fact, recent investigations have shown that the differences between the two types of muscle are not fundamental, since under certain conditions the one may THE PHYSIOLOGY OF THE HEARTBEAT 177 be made to behave like the other. They are dependent upon the pres- ence or absence of anastomosis between the muscle fibers. 1. When electric stimuli of varying strengths are applied to skeletal muscle, the contraction produced by each stimulus is proportional to the strength of the latter until this has become of such a strength that the maximal response is elicited. In cardiac muscle, on the other hand, an entirely different result is obtained, for the weakest stimulus, if it produces any response at all, produces one that is maximal; that is, the height of contraction is the same as it would have been had a much stronger stimulus been applied. Expressing this result in general terms, we may say that in cardiac muscle a minimal stimulus produces a maxi- A. Skeletal Muscle B. Cardiac Muscle Fig. 44. Effects of stimuli of increasing strength on skeletal and cardiac muscle to illustrate the "all or nothing" principle in the latter. (From Practical Physiology.) mal effect, whereas in skeletal, the effect, as measured by the height of contraction, is proportional to the intensity of stimulation. This is some- times known as the "all or nothing phenomenon" (Fig. 44). 2. If maximal stimuli are applied successively and at short intervals of time to skeletal muscle, a slightly higher response results from each succeeding stimulus, until about ten stimuli have been applied, after which for some considerable time the same height of contraction follows each stimulus. If each contraction is recorded, it will be seen that the first few contractions give a staircase effect or treppe; that is, if a hori- zontal line is drawn from the top of each contraction to the next one, the 178 THE ClftCULATlOK OF TSE BLOOD effect of an ascending staircase with gradually diminishing steps will be produced. If we repeat this observation with cardiac muscle, we shall find that it is much more pronounced than in skeletal muscle; and more- over, in obedience to the all or nothing principle, the treppe is obtained in cardiac muscle whatever may be the relative strengths of the stimuli applied to the heart, provided always that all of them are effective; whereas in the case of skeletal muscle it can be demonstrated only pro- vided the stimuli are of equal strength (Fig. 45). 3. If an effective stimulus is applied to a skeletal muscle while in process of contraction, as in response to a preceding stimulus, the second stimulus prolongs the contraction produced by the first one. If, however, the second Skeletal muscle Cardiac muscle Fig 45. The effects of successive stimuli on skeletal and cardiac muscle to show the prominence of the staircase phenomenon, or treppe, in the latter. (From T. G. Brodie.) stimulus is applied during the latent period* of the first one, it will have no effect that is, the muscle during this period is refractory, f From these results it follows that stimuli succeeding each other during the contraction period will, in the case of skeletal muscle, cause a continuous contraction, or tetanus, as it is called, because the contraction produced by each stimu- lus will add itself to that of its predecessor before any trace of relax- ation has set in. If, however, the second stimulus is applied so late in the contraction period of the first that time is not available for the latent *By "latent period" is meant the period after the moment of application of a stimulus during which no effect of that stimulus is observed. |By "refractory period" is meant the time following the application of a stimulus during which a second stimulus develops less than its full effect or no effect at all. THE PHYSIOLOGY OF THE HEARTBEAT 179 period of the former to be expended, then obviously a slight relaxation will have occurred before the effect of the second stimulus develops itself, and tetanus will be incomplete. These facts will be evident from the accom- panying tracings (Fig. 46). Skeletal muscle Stannius' heart ic effects of successive stimuli and of tetanizing The small vertical marks show when the stini from tracings published by T. G. Brodie and Leonard Hill.) Fig. 46. The effects of successive stimuli and of tetanizing stimuli on skeletal muscle and cardiac muscle. The small vertical marks show when the stimuli were introduced. (Compiled In the case of cardiac muscle the above described properties are quite different, for the refractory phase extends throughout the whole period of contraction; that is, a second stimulus applied during the contraction produced by a previous stimulus has no effect whatsoever; it does not 180 THE CIRCULATION OF THE BLOOD have one until the muscle has reached the full extent of its contraction and is about to relax. Since a latent period must supervene upon the application of this second stimulus, it follows that no complete fusion of the contractions is possible. Complete tetanus therefore, does not occur in cardiac muscle, however frequently the stimuli may be applied (Fig. 46). The refractory phase is a property of extreme importance in under- standing many of the peculiar irregularities observed in cardiac action. If we observe the effect of stimuli applied at varying periods after the Fig. 47. Myograms of frog's ventricle, showing effect of excitation by break induction shocks at various moments of the cardiac cycle. The line O indicates the commencement of all the beats during which the shock is sent in. It will be noted that in /, 2 and 3, the heart is refractory to the stimulus. The signals indicate the moments at which the stimuli were ap- plied. From 4 to 8 the heart reacts by an extrasystole, after a delay, which is progressively less the later in diastole the stimulus enters, as shown by the sections shaded obliquely to make them more conspicuous. The extrasystolcs increase in height from 4 to 8, each being followed by a compensatory pause. (From J^uciani's Human Physiology.) termination of the refractory phase of a previous stimulus, we shall find that the height of the extra contraction is directly proportional to the time after the end of the refractory period at which it is applied. If a stimulus is applied at the very beginning of diastole, the extra contrac- tion will be small, whereas if it is applied at the end of diastole, the extra contraction will be at least as high as that of the preceding. It may be higher because of the treppe. THE PHYSIOLOGY OF THE HEARTBEAT 181 These observations enable us to interpret the results obtained by ap- plying electric shocks (extra stimuli) to the beating heart during different phases of systole and diastole. During systole, the muscle being refrac- tory, no effect is produced by the extra stimulus, but during diastole extra systoles which are progressively more pronounced the later in diastole they occur, follow the application of each stimulus. These re- sults are so far exactly like those obtained with a quiescent heart. But another phenomenon now becomes evident; namely, that following each extra systole there is a compensatory pause in the action of the heart, of such duration that, when the next natural beat occurs, it does so practically at the same time as it would have occurred had no artificial stimulus been applied. This will be apparent from the accompanying diagram (Fig. 47). It should be noted that the refractory period is greatly diminished by raising the temperature of the heart. Indeed, under these conditions and with strong stimulation it may be possible to produce an almost complete tetanus. The importance of knowing the above facts is that we are thereby enabled to explain the peculiar manner in which the ventricle responds to stimuli transmitted to it from the sinus and the auricle. The muscu- lature of the auricle and ventricle of the mammalian heart is not one continuous sheet, but is separated by a space at the auriculoventricular junction, across which, in specially organized structures, the beat of the auricle is transmitted to the ventricle. Sometimes the stimuli are so frequent that the ventricular muscle is unable to respond to each stimu- lus transmitted to it, with the result that marked irregularities in con- traction occur (see page 293). In this way certain of the cardiac irregu- larities observed in man can be explained. By slowing the auricular beats as by giving digitalis these irregularities often disappear. When the extra systoles occur in the ventricle itself either because of disease or of the action of drugs, the contraction may be so feeble that it fails to open the semilunar valves with the result that the second sound is not heard although the first sound is and the arterial pulse shows a missing wave. In such a case the pause in the arterial pulse curve equals the distance between two normal waves. When the extra systole is just strong enough to open the semilunar valve a feeble second sound is heard and a low wave appears on the pulse curve, followed by a com- pensatory pause. CHAPTER XXI THE PHYSIOLOGY OF THE HEARTBEAT (Cont'd) THE ORIGIN AND PROPAGATION OF THE BEAT IN THE MAMMALIAN HEART As has been shown in the preceding chapter, there is no doubt that in the cold-Hooded heart the beat originates at the sinus venosus, whence it spreads to the rest of the heart. Very strong evidence has also been presented to indicate that the beating power is inherent in the muscle fiber itself and independent of nervous structure. This would suggest the further possibility that the structures through which the beat is propa- gated are the muscle fibers and not the nerve fibers in other words, that the propagation of the heartbeat, like its origination, is myogenic rather than neurogenic. Direct proof of this hypothesis is readily fur- nished by numerous experiments, among which may be mentioned mak- ing interdigitating cuts across the heart, or excising a ribbon of ven- tricular muscle by an incision simulating the walls of Troy. In both these cases the beat will be found to travel from one end of the muscular band to the other, although it is evident that all the nerves proceeding from base to apex of the heart must have been severed. Of course this evidence is not irrefutable, for it might be argued that there are nerv- ous structures disposed in the form of a plexus continuously all over the heart, and that some branches of the plexus remain uncut in the above experiments. It is only in the heart of Limulus that undoubted evidence exists that the beat is transmitted by nerves, but as we have seen, this heart in all its properties is probably the proverbial exception which proves the rule. The balance of evidence stands in favor of the view that the propagation of the beat over the cold-blooded heart is myogenic and not neurogenic. CONDUCTING TISSUE IN MAMMALIAN HEART When we attempt to investigate the problems of the origin and propa- gation of the beat in the warm-blooded heart, many experimental diffi- culties of course face us. In overcoming these, the first thing we must do is to establish the structural relationship between cold-blooded and warm-blooded hearts. In the embryo of both classes of animals the 182 THE PHYSIOLOGY OF THE HEARTBEAT 183 heart arises as the so-called cardiac tube. As development proceeds, diverticula grow out from the walls of this tube to form the auricles and ventricles. In the comparatively simple heart of the turtle these dispo- sitions of the auricles and ventricles in relationship to the cardiac tube are more or less evident even in the fully developed heart, particularly in the case of the auricles (Fig. 48) ; but in the heart of the higher mammalia it is impossible by superficial examination alone to show any remains of the primitive cardiac tube. More careful anatomic investiga- tions during recent years have, however, shown that it exists in the form of certain definite structures composed of tissue histologically quite dif- ferent from that of the rest of the heart, and disposed in such a manner TH TH Fig. 48. Heart of tortoise as suspended. B, body of tortoise; TH. threads to levers; CL, clamp holding aorta; A, auricle; C, coronary nerve; S, sinus; V, ventricle. (From Gaskell.) as would indicate not only that it is derived from the primitive cardiac tube, but also that it is the main pathway along which the beat is transmitted. This primitive cardiac tissue is much better developed in certain re- gions than in others, the first portion of it to be discovered being that known as the auriculoventricular node, or the node of Stanley Kent* (Figs. 49 and 50) . This structure is found at the base of the interauricular sep- tum on the right side and near its posterior margin. It exists as a collection of peculiar small primitive cells and fibers, and is continued forward and downward as a bundle of the same peculiar tissue to the interventricular septum, where, a little in front of the attachment of the septal valve, it *The discovery of this node is often erroneously attributed to His, and called after his name. 184 THE CIRCULATION OF THE BLOOD bifurcates into right and left branches which run down each side of the septum immediately underneath the endocardium. Each main branch ultimately divides up into an intricate system of smaller branches, which become reflected over the inner surface of the ventricles where their exist- ence has been known for some time as the so-called Purkinje fibers. The right bundle remains undivided until after it has run along the moder- Fig. 49. Dissection of heart to show auriculoventricular bundle (Keith) ; 3, the beginning of the bundle, known as the A-V node; 2, the bundle dividing into two branches; 4, the branch run- ning on the right side of the interventricular septum. (From Howell's Physiology.) Fig. 50. Photograph of model of the auriculoventricular bundle and its ramifications, con- structed from dissections of the heart (Miss De Witt). All of the branches in the left ventricle are not included. (From Howellj ator band or its representative, but the left bundle divides early. The fibers ultimately end in close association with the papillary muscles. The node and main bundle and the two branches before they have begun to divide are surrounded by a sheath of fibrous tissue, and they seem to have a liberal blood supply. It is of interest that they con- tain a high percentage of glycogen. In the human heart the auriculo- THE PHYSIOLOGY OF THE HEARTBEAT 185 ventricular node and bundle measure about 15 mm. in length and about 2 mm. in width. The rest of the tissue between the auricles and ventricles is fibrous in nature, although other connections like those of the auriculoventricular bundle have been described by Kent. One of these, called the right lat- eral connection, runs between the right auricle and the external wall of the right ventricle. Another, but much smaller, mass of similar embryonic cardiac tissue has more recently been discovered by Keith and Flack in the parts of the auricle which correspond anatomically to the sinus venosus of the heart of cold-blooded animals that is, in the area lying between the openings of the venaa cavae and around the coronary sinus. To be more explicit, this tissue lies "in the sulcus terminalis just below the fork formed by the junction of the upper surface of the auricular appendix with the superior vena cava. " This sinoauricular node, as it is called, is more or less club-shaped, the blunt end of the club being above, as shown in the accompanying figures (Pigs. 51 and 52). It is important to note that there is no direct connection visible between the sinoauricular and auriculoventricular nodes (Fig. 52). Another anatomic fact seen also in the accompanying figure, concerns the disposition of the muscular fibers of the auricle. These radiate in bundles in a peculiar fan-shaped manner from a point which lies im- mediately below the sinoauricular node to all parts of the superficies of the right auricle. This point has been called the concentration point. At the termination of the venae cavae, the muscle fibers are arranged more or less circularly. Having become familiar with the disposition in the mammalian heart of the primitive cardiac tissue, along which in the heart of the lower animals we know that the heartbeat spreads, we may now proceed to examine the evidence showing that this tissue is also responsible for the origination and propagation of the beat in the heart of mammals. With regard to the origin of the beat in a normally beating mammalian heart, it is of course impossible to see where this takes place. If the heart is excised, however, it will continue to beat for a few moments, and as it dies it will be observed that the power of contraction remains in the au- ricular region, and particularly at the bases of the venae cavae, for a con- siderable time after the ventricles have ceased to beat. This part the ultimum moriens is situated in most hearts somewhat lower than the sinoauricular node. That it is the last part of the heart to cease con- tracting does not necessarily mean that it is the part of the heart in which the beat ordinarily originates; it means simply that this is the part of the auricle in which the power of contraction remains for the 186 THE CIRCULATION OF THE BLOOD longest time after death. Although, the observation does not enable us to determine exactly where the heartbeat originates, yet it makes it very probable that this is somewhere in the auricles ; a conclusion which is borne out by many other pieces of evidence, such as those obtained by Fig. 51. Diagram of an auricle showing the arrangement of the muscle bands; the concen- tration point (C.P.); and the outline of the S.A. node (S.A.N.). The diagram is to scale, and illustrates by the circles and connecting dotted lines the method of leading off by paired contacts and the subsequent orientation. (From Thomas Lewis.) I Auricular appendage HR- node -Auriculoventricular node -Aurlculoventricular bundle Right & left ventricular * bundles -Musculi papillares Fig. 52. Diagram to show the general ramifications of the conducting tissue in the heart of the mammal. It will be observed that there is none of this tissue between the sinoauriculo- and auriculoventricular nodes. the study of polysphygmograms (page 285), of electrocardiograms and of observations on the heart during heart-block (page 270). Our problem therefore narrows itself down to determining the exact point of the right auricle at which the beat originates. THE PHYSIOLOGY OF THE HEARTBEAT 187 SITE OF ORIGIN OF THE BEAT The working hypothesis from which we may proceed to attack this problem is that the beat originates in the sinoauricular node, and to put this to the test, various methods have been employed: (1) Warming or cooling or injuring the node and noting the effect on the heartbeat. Such procedures greatly affect the rate of the heartbeat, whereas they produce no change when applied to other parts of the heart. (2) De- termination of the comparative rhythmic power of strips cut out from different regions of the auricular walls. It is greatest in those taken from the region of the node. (3) Determination by the use of galvan- ometric curves of the relation of the node to the seat of origin of cardiac impulse. By all these methods the results indicate clearly that the beat originates in the sinoauricular node, but on account of the great im- portance in connection with the interpretation of electrocardiograms in man, it is particularly with the result of the third group of experiments that we will concern ourselves here. Evidence Furnished by Studying the Current of Action Which Accompanies the Heartbeat To start with, it is essential that we make ourselves familiar with the principles of the methods employed. These principles are briefly as follows: When a wave of contraction passes along a muscle, it is im- mediately preceded by a change in electrical potential, which can be detected by means of a galvanometer connected with the muscle through so-called nonpolarizable electrodes. The galvanometer employed must be extremely sensitive, and must not vibrate after the current has ceased to pass. The form generally in use today is known as the string galva- nometer of Einthoven. It differs from the galvanometer ordinarily em- ployed in physical laboratories in that the current instead of passing through a coil of wire surrounding a magnetic needle, passes through a silverized quartz thread suspended in the strong magnetic field which exists between the two opposing poles of a horseshoe electromagnet. The string is thus surrounded on all sides by innumerable lines of force extending between the two poles of the magnet. When a current, how- ever small, passes along the string, it will generate lines of force of its own, and these by reacting with the stationary lines of force of the field will cause the string to move. The string is placed in the pathway of a strong beam of light, and its shadow, after being magnified by lenses, is projected on a moving photographic plate or paper arranged in a suitable holder. The nonpolarizable electrodes referred to are employed in place of ordinary electrodes in order to obviate the generation of elec- 188 THE CIRCULATION OF THE BLOOD trie currents set up by the contact of metal with the saline constituents of the muscle juices. If we connect a galvanometer by means of nonpolarizable electrodes with two parts of a denervated muscle (the curarized sartorius of the frog), it will be found that a current is set up whenever a wave of con- traction passes over the muscle from one end to the other. The part which first contracts becomes electrically negative to the rest of the muscle, but as the wave of contraction passes along it, the " negativity" de- creases at the end at which the wave started until, when the wave has reached the middle of the strip, neither end of the muscle shows any difference in potential, so that the string comes back to a position of rest. However, as the contraction wave reaches the farther end of the muscle, this lead in turn becomes negative, and the string swings in the ^ Fig. 53. Diagram to illustrate the development and spread of the wave of negativity in a strip of muscle (curarized sartorius) when stimulated at the end (P). The shaded portions show the position of the negativity. The portion of the curve drawn by the deflections of the galvanom- eter at each stage are shown at the right (a, b, c, and d). (After Lewis.) opposite direction (Fig. 53). From this comparatively simple experiment it can be seen that a muscular contraction wave arises at the electrode which is negative first, and that the movement of the string of the galvanometer is most marked that is, the deflection is greatest when the two electrodes are applied at the extreme ends of the muscle. When they are brought closer together, the initial deflection becomes much less marked ; in other words, the amplitude of the negative wave is greatest when the time interval between the receipt of the excitation at the two contacts is greatest. The application of these facts to the study of the initiation of the beat in the auricle requires that we should consider another proposition: namely, if a pair of contacts are arranged in the center of a circular sheet of muscle and the edge of this sheet is stimulated at different THE PHYSIOLOGY OF THE HEARTBEAT 189 points, the amplitude of deflection of a galvanometer connected with the pair of contacts will be most pronounced when these are radial to the points of stimulation, for under these conditions it is evident that the greatest possible difference will exist between the intervals required for the wave to reach each contact. Bearing these principles in mind, we may now proceed to examine the evidence pointing to the origin of the heartbeat at the sinoauricular node: (1) When two electrodes are applied at different points of the au- ricle, the amplitude of movement of the string of the galvanometer produced by each heartbeat is greatest when the line joining the elec- trodes converges on the sinoauricular node. To make this clear the movement of the string must be photographed in the manner above described, the resulting tracing being called an electrocardiogram. From the experiments with the circular sheet of muscle alluded to it is evident that the stimulus to produce this result must have arisen in the neigh- borhood of the node. (2) If one electrode is placed on the sinoauricular node and the other electrode is moved about from place to place on the auricle, the deflection being noted at each new position, the electrode on the node will always be found to be negative to the other electrode.* No such consistency will be manifest if both electrodes are moved about on other parts of the auricle. (3) As we shall see immediately, the current of action of the beating heart may be recorded by connecting a galvanometer with various parts of the body; for example with the right fore limb and the left hind limb. On the electrocardiogram thus obtained are several waves, one of which, called the P-wave, can easily be shown to correspond to the contraction of the auricle (see Fig. 272). If now we compare such electro- cardiograms with those obtained during contractions of the auricle caused by applying electrical stimulation to various parts of it, it will be found that the electrocardiogram of the artificial beat simulates the normal curve only when the stimulated part is in the neighborhood of the sinoauricular node. In other words, it is only when the stimulus is applied to the sinoauricular node that a characteristic P-wave is obtained. When the appendix or the superior vena cava is stimulated, the P-wave is distorted although the other waves of the electrocardiogram may be normal. (4) By taking electrocardiograms from various direct leads placed on the auricle and comparing the records with that of a standard limb lead taken simultaneously, we shall find by exact measurement that the time of onset of the excitation wave of the auricle, as measured in relationship to the P-wave on the standard electrocardiogram, is shortest *The connections between the electrodes and galvanometer are always arranged so that any upward movement of the shadow of the string above the line of equal potential at the two electrodes indicates electric negativity. 190 THE CIRCULATION OF THE BLOOD when one electrode is over the upper end of the sinoauricular node, and that in other regions of the auricle it always appears at a later interval. Further details on this subject will be found in the papers by Eyster and Meek 8 and in Lewis monographs. Frequently, in taking electrocardiograms from different parts of the auricle, it is found that certain of the curves show small waves of positivity below the line of equal potential preceding the main wave of negativity. These initial deflections are most marked when both the electrodes are far removed from the sinoauricular node for ex- ample, when they are placed on the auricular appendix ; but they are never present when Fig. 54. Simultaneous electrocardiograms to show the cause for extrinsic deflections. The upper curves are from the appendix and the lower ones from lead II. The chief or intrinsic deflection (Tn) is seen to disappear in the right-hand appendix electrocardiogram, because the base of the appendix has been crushed. The extrinsic deflection (Ex) remains, as do the ven- tricular deflections (V 1 F 2 ), (From Lewis.) one of the electrodes is placed on the sinoauricular node itself. In other words, curves taken from leads at a distance from the sinoauricular node are more or less composite in form, being made up partly of the main deflection due to the arrival of the excitation and partly of the secondary deflections dependent upon extrinsic influences acting on the electrodes; that is, the electrode picks up electric discharges from distant areas of muscle while these are in a condition of contraction (Fig. 54). From these considera- tions it follows that the intervals between the intrinsic and extrinsic deflections should be longest in leads that are farthest from the node, and gradually become less as one of the contacts approaches the node, until over this structure the ex- trinsic deflection is no longer recorded. Such has been found to be the case. (Lewis.) CHAPTER XXII THE PHYSIOLOGY OF THE HEARTBEAT (Cont'd) THE ORIGIN AND PROPAGATION OF THE BEAT (Cont'd) FIBRILLATION Mode of Propagation in the Auricles From the mass of evidence we have little doubt that the heartbeat originates in the sinoauricular node, and the question now presents itself as to how the beat is propagated over the remainder of the auricles and into the ventricles. Regarding the propagation of the beat over the auricles, two possibilities exist: (1) it may spread uniformly over the muscular tissue of the auricular wall until it reaches the auriculoventric- ular node, or (2) there may be laid down between the sinoauricular and the auriculoventricular node a special strand of highly conducting tissue. It is no argument against this second possibility that we should so far have been unable by histological methods to differentiate any such struc- tures. There is considerable practical importance attached to the solution of these questions, particularly with regard to the cause of certain types of cardiac arrhythmia, such, for example, as that known as nodal rhythm. Thus, it is evident that if the beat is transmitted uniformly over the muscular tissue of the auricle, then the whole auricle will have con- tracted before the beat has reached the auriculoventricular bundle, by which it is then transmitted to the ventricles. On the other hand, if the beat should travel between the two nodes by special conducting tissue, then the impulse will have arrived at the auriculoventricular node be- fore the auricle has contracted. As a matter of fact, it is not quite settled yet as to which of these two views is the correct one, although the balance of evidence seems to favor the former that is, that the wave is transmitted uniformly over the muscular tissue of the auricle. (Lewis.) The methods employed in attacking the problem have been essentially the same as those described above. One of them may be called the direct, the other the indirect. In the former, a series of pairs of contacts is placed on the auricle, each pair being in a radial direction to the sino- auricular node. The time at which the excitatory process arrives at that contact of each pair which is proximal to the sinoauricular node is accu- 191 192 THE CIRCULATION OF THE BLOOD rately determined from the galvanometric record. The exact distance be- tween the contact and the sinoauricular node is then measured and from the data the average transmission time is estimated. From his results Lewis concludes that the transmission rates are uniform from the node to all parts of the auricle, with the exception of the superior vena cava, in which the rate is considerably lower. One thousand millimeters per second represents very fairly the average rate at which the excitation wave travels. On the other hand, Eyster and Meek 8 state that the wave is propagated throughout the sinus node, and that it spreads to the contiguous venae cavse and to the auriculoventricular node with con- siderable rapidity, reaching the mouth of the superior vena cava in 0.01 second, whereas its passage to the auricle itself takes 0.02 second. There is therefore a delay in the passage of the wave to the auricle, which indi- cates that the excitation must spread to the auriculoventricular node be- fore involving the right atrium. These authors conclude that "this leads to the inevitable conclusion that the cardiac impulse spreads to the ven- tricle and to the right auricle by different paths, and does not pass to the ventricle through the auricle, as ordinarily stated." In the second, or indirect, method, the onset of the negative wave from different leads in the auricle is compared against a standard. For the standard Eyster and Meek have used the record of the mechanical sys- tole of the auricle, but the interpretation of the result is extremely dif- ficult on account of the rate at which the changes are occurring. Lewis, on the other hand, has used the standard electrocardiogram for purposes of comparison. Mode of Propagation of the Beat to the Ventricles After reaching the auriculoventricular node, the beat is transmitted to the ventricles along the auriculoventricular bundle a fact which has been most clearly demonstrated by the experiments on heart -block. "We have al- ready seen (page 174) that although each chamber of the heart of a turtle or frog has a rhythm of its own, this is much more pronounced at the venous end of the heart, and when the transmission of the beat to the ventricles from the auricles is obstructed or blocked, as by compression or partial cutting at the auriculoventricular junction, the ventricles, after coming to a standstill for a time, subsequently contract with a rhythm which is entirely independent of that of the auricles. In the mammalian heart the same results may be obtained by arrang- ing a clamp so that it compresses practically nothing but the auriculo- ventricular bundle (Erlanger.) If the compression is extreme, the rhythm of the ventricles is quite independent of that of the auricles, but if it is only partial, the ventricular systoles follow regularly every sec- THE PHYSIOLOGY OF THE HEARTBEAT 193 ond, third, or fourth auricular contraction. If after such a complete or partial heart-block has been instituted, the clamp is removed, it will usually be found that the heart-block disappears and the auricular and ventricular contractions fall back into their usual sequence. The im- portance of this discovery, apart from its physiological interest, rests in the fact that it is exactly duplicated in clinical experience. If the pulse tracing of the radial artery is compared with that of the jugular vein in certain types of heart disease, it will be found that the auricle is beat- ing two or three times more quickly than the ventricles. In many of these cases it has been found on autopsy that definite lesions often syphi- litic in nature involve the auriculoventricular bundle. In other cases, however, such lesions have not been discovered. Sometimes the bundle is so severely diseased that the block is complete, the ventricles con- tracting quite independently of the auricle. In such cases it is assumed that the beat originates in the uninjured part of the bundle below the seat of the block. It should be pointed out here, however, that all cases of slow pulse in the arteries are not necessarily dependent upon heart-block, but may depend upon a slow beat of the auricle itself. This is called Sometimes after complete destruction of the auriculoventricular bun- dle the beat continues to be transmitted to the ventricle, and conversely this transmission has sometimes been observed to be upset by lesions not affecting the bundle. The explanation of both of these exceptional re- sults almost certainly is that the right lateral connection described above (page 184) is serving as the main pathway of transmission for the beat. The facility of conduction through the auriculoventricular bundle is subject to alteration by the impulses passing to it along the vagus nerve, particularly the left vagus. It can also be altered by certain drugs, especially digitalis and strophanthin. The clear demonstration that it is along this bundle that the beat is transmitted is strong evidence in favor of the myogenic hypothesis (page 171) concerning the transmission of the heartbeat, but it does not necessarily disprove the neurogenic hypoth- esis, for histological investigation has shown that the bundle is closely surrounded by an intimate plexus of nerve fibers. Spread of the Beat in the Ventricle After the impulse has been transmitted by the bundle into the ven- tricles, it spreads along the many branches into which, as we have seen, the two main divisions of this bundle separate. The first part of the ventricular musculature to contract is therefore located near the ter- mination of these branches, at the papillary muscles. That these should contract before the rest of the muscle of the ventricles, has an obvious 194 THE CIRCULATION OP THE BLOOD significance in connection with their function of tightening the chordae tendinese so as to prevent any bulging of the flaps of the auriculoven- tricular valve into the auricles when, at the beginning of the presphygmic period, the high intraventricular pressure is brought to bear on their under surfaces. After starting at this point in the ventricle, the con- traction wave seems to spread farther through the ventricular muscle at a fairly uniform rate. Investigation of this problem by means of the galvanometer has been technically a very difficult matter. According to the researches of Lewis and his associates, however, it appears that, when a series of nonpolarizable electrodes are placed at various parts of the outer Fig. 55. Diagram of experiment by Lewis showing the times at which the excitation wave appeared on the front of the heart relative to the upstroke of R in lead II. R.A., right appen- dix; D.B.L., descending branch of left coronary artery. (From Thomas Lewis.) aspect of the ventricle, and comparison made of the moments at which the cardiac impulse arrives, as judged by the appearance of the excita- tion wave relative to R. in a standard electrocardiogram, it has been found that the time of arrival bears no relationship to the anatomical ar- rangement of the muscle bundles of the ventricle. It arrives early and simultaneously over an area of the surface near the anterior attachment of the wall of the right ventricle to the septum. It arrives late at the base of the right ventricle and in the part near the posterior intraventricular groove. The actual rate of spread is moreover much greater than the transmission rate through ventricular muscle. His- tological examination has shown that the branches of the right division THE PHYSIOLOGY OP THE HEARTBEAT 195 of the auriculoventricular bundle are most closely connected with the place where the wave arrives earliest. Somewhat different results are obtained from the left ventricle, but again they are dependent upon the relationship of the part to the Purkinje fibers (Fig. 55). A further dis- cussion of the manner of spread of the impulse in the ventricles will be found in the chapter devoted to interpretation of the electrocardiogram (page 270). FIBRILLATION OF THE HEART Ventricles The even spread of the wave of contraction over the heart depends on the uniform excitability of the muscular fibers. If certain of the muscu- lar fibers, or bundles of fibers, have a greater or less excitability than others, then, when the stimulus to contract arrives, it will not produce a uniform contraction of neighboring bundles, and coordinated action of the cardiac musculature will give place to a confused movement in which each part of the heart is contracting independently of the rest. This fibrillation, or delirium cordis, as it is called, can be produced by a large variety of experimental methods, such, for example, as by stimulating the ventricles with induced electric shocks, or by ligation of a large branch crfrtfre-coronary artery, or by the injection of lycopodium spores into the coronary circulation, or by mechanical stimulation of the heart in the region of the auriculoventricular bundle. Fibrillation of the ventricles is undoubtedly a common cause of death in man, for of course the confused movements make the ventricles in- capable of contracting on the contents of the heart. It is a condition which can probably never be recovered from in the higher animals, but it is of interest that the ease with which it is set up as the result of the application of an electric stimulus varies to a marked degree in differ- ent animals, and that in those hearts in which fibrillation can be elic- ited only with difficulty, recovery can usually be effected either by stop- ping the heart by means of cold and then allowing it to beat again, or by the administration of epinephrine. Of the hearts investigated in this way, that of the rat has been found to be most resistant to stimula- tion; then in order come those of the rabbit, the cat, the dog, and the horse. There is good reason to believe that the heart of man is readily affected. Fibrillation of the ventricle is undoubtedly the main cause of death in most cases of electrocution. Curiously enough, however, it has been stated that, whereas, a current of ordinary intensity (2300 volts alternating current) produces ventricular fibrillation in the heart of cer- tain of the lower animals, at least in that of the horse, a very much stronger current does not do so, and may indeed cause ventricular fibril- 196 THE CIRCULATION OF THE BLOOD lation produced by a more moderate voltage to disappear. Unfortu- nately, however, these stronger currents produce irreparable damage in the central nervous system, so that the method of applying stronger cur- rents, even were it feasible to do so quickly enough, would be of no therapeutic value in removing fibrillation. The disappointing results that have followed the repeated attempts to resuscitate persons killed accidentally by electric shocks is undoubt- edly dependent upon the fact that in the heart of man it is impossible to bring back the normal beat after the ventricles have been thrown into fibrillation. Fibrillation of the ventricle is also the cause of the sudden cardiac failure occurring when blood clots or emboli cause a blockage of the coronary circulation (it is sometimes the cause of angina pec- toris, for example). It must also be remembered in clinical practice that mechanical stimulation of the ventricles may produce fibrillation, so that in attempted resuscitation by cardiac massage care should be taken not to apply this too vigorously. Auricles Although ventricular fibrillation is seldom recovered from, it has been clearly shown in recent years that fibrillation of the auricles is relatively common and that it is by no means immediately fatal. Indeed it is one of the most common of the chronic cardiac disorders in man. Auricular fibrillation can be produced experimentally by the application of a strong electric stimulus to the auricles. If, however, a weaker stimulus is applied, the auricles do not go into typical fibrillation, but come to beat at a very rapid and regular rate, perhaps three or four hundred a minute. This condition is called "auricular flutter," and is quite fre- quently observed in the clinic. The cause of flutter might be either a paroxysm of extrasystoles or a so-called circus movement. The latter possibility was demonstrated by Mines 13 in rings of tissue cut from the auricle of the ray fish; when a stimulus was applied at one point a con- traction wave was set up which went round and round the ring, so that a series of contractions were the result of one stimulus. If such circus movements were associated with depressed conduction of the muscular fibers the excitation wave would be irregularly transmitted and account for fibrillation. Garrey 59 and Lewis both subscribe to these views. The influence of auricular fibrillation and flutter on the beat of the ven- tricle is an extremely important one in connection with the irregular- ities of the heart observed in man, and this influence in most cases is explained by considering (1) the narrowness of the path (in the auric- uloventricular bundle) along which the impulses have to travel, and (2) THE PHYSIOLOGY OF THE HEARTBEAT 197 the varying conditions of excitability of the ventricular muscle, depend- ing upon the existence of the refractory phase (page 178). In auricular flutter, when three or four hundred impulses per minute are passing along the bundle to the ventricle, the contraction produced by the first one will scarcely have started before the second and imme- diately succeeding ones arrive, so that the ventricle will beat at a rate that is much less than that of the auricle, and a condition simulating heart-block will become established. The characteristic feature which distinguishes this from true heart-block, however, is the fact that the ventricular rate is above normal, whereas in true heart-block the rate is much below normal. By means of the electrocardiogram or by polysphygmographic tracings, it can also be shown that the auricle is beating with perfect regularity although very rapidly. In auricular fibrillation the ventricles obviously will respond at a very irregular rate to the impulses transmitted to them, and the auricular contractions, if examined by the methods above described, will show no regular sequence. Further details of the method of eliciting these signs will be described later (page 285). CHAPTER XXIII THE BLOODFLOW IN THE ARTERIES THE PULSES Returning to the physical laws that govern the circulation of the blood, we may now consider the temporary changes produced in the bloodflow in the arteries by each systolic discharge. These changes go under the general term of the pulses, of which three may be distinguished: (1) the pressure pulse, or the pulsatile increase of pressure produced by each heartbeat (see page 129) ; (2) the velocity pulse, or pulsatile accel- eration of velocity; and (3) the palpable pulse, or the pulsatile expansion of the walls of the blood vessels produced by the sudden change of blood pressure in their interior. The general characteristics of the three pulses are the same, certain features being however more pronounced in one than in another. General Characteristics Rate of Transmission of Pulse Wave. The rate of transmission of the pulse wave can be determined by taking simultaneous tracings of the pulses from two far distant parts of the arterial system along with accurate time-tracings. From records (cf. Fig. 95) taken from the apex or the carotid and radial arteries we can determine how long it takes for the beginning of the pulse wave to travel to the radial artery from the point in the aorta from which the carotid artery springs. We shall find that it takes about one-tenth of a second, which, considering the length of the artery involved, would work out as a transmission velocity of about seven meters per second or about seventeen miles an hour. The pulse therefore travels along the blood vessels at a much greater speed than the blood itself is moving, this being, as we shall see immediately, about 0.5 meters per second in the larger blood vessels. The pulse is a wave of sudden increase in pressure and velocity pass- ing along a stream which is flowing in the same direction with a cer- tain more permanent pressure and velocity. A simple physical experi- ment may serve to make this clear: If the first of a row of billiard balls be tapped with the cue, the end balls will fly off while the others are moving slowly along in the direction of the stroke. Each ball becomes accelerated by the ball behind it, and imparts its influence to the ball 198 THE BLOODFLOW IN THE ARTERIES 199 in front. In other words, a pulsatile acceleration of velocity is produced by a pulsatile change in pressure between each two balls. The existence of a pulse wave going in the same direction but quicker than a moving column of fluid can also be illustrated by observing the waves traveling down a stream when a stone is thrown into it. The length of the pulse wave is such that the beginning of it has ar- rived at the periphery of the arterial system before the end has disap- peared from the beginning of the aorta. This is important to remem- ber, for it is a common mistake to think of the wave as being a local one. The determination of the length of the pulse wave depends upon the following equation: L = VT, where L equals the length of the pulse wave, V its velocity of transmission, and T its duration at a given point in the artery. Under ordinary circumstances L would usually work out from 3.25 to 4.5 meters. The rate of transmission of the pulse wave varies according to the rigidity of the walls of the arteries. To understand why this should be so, it will be well for a moment to consider the physical conditions upon which the pulse wave depends. If we connect a piece of rigid tube with the nozzle of a large syringe, with each movement of the pis- ton a wave of pressure will be transmitted to the fluid in the tube, along which it will travel at such a high velocity that it will arrive at the free end of the tube almost instantaneously, and incidentally the out- flow of fluid from the end of the tube with each compression of the pump will be exactly equal to that represented by the movement of the piston. If, on the other hand, an elastic tube is employed, it will be found that the sudden increase of pressure produced by each stroke of the pump causes a distention of the walls, which travels along the tube as a wave at a readily measurable velocity, which is slower the more extensible the tube. Moreover, the outflow of fluid from the free end of the tube will continue for some time after the cessation of the move- ment of the pump. What happens in the tube with each discharge of the fluid is that the portion which is immediately adjacent to the pump undergoes distention and, being elastic, tends immediately afterward to recoil and thus exert a recoil pressure on the fluid contained in the tube. As a result, pressure w T aves are set up in the fluid in all directions. Those that travel back come to a stop because of the piston, while those that travel distally act on the fluid in front of them so as to accelerate it and by temporarily raising its pressure distend the next segment of the vessel wall, until the end of the tube is reached. From this considera- tion it is clear that the more extensible and elastic the wall of the tube is, the longer will it take for the wave of pressure to travel from one end to the other. 200 THE CIRCULATION OF THE BLOOD Alteration in the rate of transmission of the pulse wave in the ar- teries of man depends entirely upon an application of these principles. When the arteries become hardened in old age, the rate of transmission of the pulse wave is markedly increased. The pulse is also transmitted more rapidly in the vessels of the lower extremities than in those of the upper, since in the former the blood vessels are somewhat more rigid. Delay in the transmission of the pulse wave is further observed as one of the signs of aneurism in a vessel; as is well known, aneurism of the subclavian artery on one side causes a delay of the pulse on that side that is perceptible to the fingers. The Contour of the Pulse Curves For more particular study of the exact contour of the pulse wave, and especially for determining the time relationships of the secondary waves, Fig. 56. Diagram of Chauveau's dromograph. a, tube for introduction into the lumen of the artery, and containing a needle or vane, which passes through the elastic membrane in its side and moves by the impulse of the blood current; c, graduated scale for measuring the extent of the oscillations of the needle. a large variety of methods of varying degrees of accuracy have been elaborated for each kind of pulse. Those devised for measuring the pressure pulse have already been de- scribed (see page 128), and for the other pulses they are as follows: Velocity Pulse. Much ingenuity has been displayed in the elabora- tion of methods for recording the velocity pulse. In one of these the artery is cut across and the ends attached to a tube, into the lumen of which projects a paddle or vane articulated with a light lever, which passes through its wall (see Fig. 56). The vane floats in the blood stream, and the outer end of the lever to which it is attached is con- nected with some device to record its movements, which vary with the velocity of bloodflow (hemodromograph). Another method depends on the application of the instrument known as Pitot's tube used by phys- icists. This consists of a horizontal tube having two side tubes, each of THE BLOODFLOW IN THE ARTERIES 201 which is connected at its outer end with a manometer and prolonged inside the horizontal tube, where they are bent at opposite right angles, so that the inner end of one of them the proximal tube points up D Fig. 57. Fig. 58. Fig. 57. Diagram to show principle of Pilot's tubes for measuring velocity pulse. In both tubes the fluid will rise because of lateral pressure, but in the proximal (left-hand) tube it will rise higher than in the distal, because it will also be affected by the velocity of flow. Fig. 58. Diagram to illustrate the principle of Cybulski's Photo-hematotachometer. The fluid in C stands higher than that in D in proportion to the velocity of flow of the blood along Fig. 59. Dudgeon's sphygmograph. (From Jackson.) stream, and records not only the lateral pressure but also the pressure produced by the sudden increase in velocity of the flow, while the 202 THE CIRCULATION OF THE BLOOD other the distal tube being bent down stream, records merely lateral pressure. A photographic record of the movement of the fluid in the two tubes gives the velocity pulse (see Fig. 57). For physiological pur- poses the form of apparatus used is constructed as shown in Fig. 58. Palpable Pulse. To secure a record of the palpable pulse, the so- called sphygmograph is employed, although a tambour having a button in the center which is made to press on the artery may also be em- ployed. The commonest form of sphygmograph is that known as Dudgeon's (Fig. 59). It consists of a small button connected with a spring, the movements of which are transmitted and magnified by means of a system of levers connected with a writing point arranged so as to inscribe its movements on a moving surface. The Analysis of the Curve The general contour of the pulse waves taken by any of the above methods are in general very much the same. The pressure and velocity Fig. 60. Pulse tracing (sphygmogram) taken by sphygmograph. a d, the period of the pulse curve; b, the primary; c, the dicrotic wave. Time marked in fifths of a second. (From Prac- tical Physiology.) pulse curves are, however, not usually taken for the purpose of observ- ing the contour of the wave but rather for measuring the difference in pressure or velocity actually produced during each pulse. It is a record of the palpable pulse that is usually employed for studying the contour of the wave and the presence of secondary waves. A record of the pal- pable pulse wave (Fig. 60) shows two separate waves on the descending limb of the main wave. If a large number of similar pulse curves are taken from different individuals or from the same individual under different conditions, it will be found that of these two waves the secQnjj one is by far the more constant ; and if the waves are timed in relation ship to the heart sounds, this second wave always occurs immediately after the second sound, allowance, of course, being made for the time required for the pulse to be transmitted from the heart to the artery from which the pulse tracing is being taken. If the observation is made very carefully, it can indeed be shown that the second sound cor/ responds exactly to the notch which precedes this wave. The waves that THE BLOODFLOW IN THE ARTERIES 203 precede this notch can not be related to definite changes occurring in the heart. Evidently, then, the secondary pulse waves are not all of equal significance, by far the most important being that which occurs immediately after the second sound, called the dicrotic wave (c), the notch in front of it being called the dicrotic notch. Any secondary waves occurring before the dicrotic are called predicrotic, or if they occur on the ascending limb of the main pulse wave, as they sometimes do, they are called anacrotic. Waves occurring after the dicrotic are called postdicrotic. The relative importance of the dicrotic, in comparison with the pre- dicrotic and postdicrotic waves, is further evidenced by the fact that it alone is seen on a so-called hemataugram, which is the tracing ob- tained by allowing a fine stream of blood, escaping from a pinhole made in the wall of an artery, to impinge upon a moving sheet of white blot- ting paper. That such a tracing shows a dicrotic but no secondary wave, indicates that only the former is present in the blood stream itself, and that the other secondary waves must be produced by some condition arising either in the elastic tissue of the walls of the blood vessels, or in the elastic properties of the instruments used for taking the pulse tracing. The Dicrotic Wave. Because of its obviously greater significance, we shall first of all consider the exact flanse of the di^rotic wave and of the notch preceding it. Theoretically, two possible causes might explain the wave: either it is due to some secondary wave set up at the heart, or it is dependent upon waves reflected from the periphery of the cir- culation back along the blood stream, just as secondary waves are re- flected from the walls of a tub of water when a stone is thrown in the center. In considering this second possibility, we are of course making the assumption that at the ends of the arterial system there is a sudden resistance to the onward movement of blood. The frequent branching which occurs when the arterioles open into the capillaries no doubt of- fers many opportunities for the reflection of pulse waves back to the heart, but these waves must be reflected at such varying distances along the arterial system that there can be little opportunity for them to be- come added together so as to form a wave of sufficient magnitude to make itself perceptible in the blood flowing in the larger arteries. These waves are relatively so small and they occur at such different times that the net result of their addition, so far as the production of a larger wave is concerned, must be practically nil. Notwithstanding these con- siderations, it is possible that under some conditions, such as in cases of high arterial tension, certain of the predicrotic or postdicrotic waves may be due to the above causes. 204 THE CIRCULATION OF THE BLOOD That the dicrotic is not a reflected wave is clearly established by the fact that if the distance between the dicrotic wave and the main pulse wave is measured at different points of the arterial stream, it will al- ways be found to be the same, which obviously would not be the case were the dicrotic wave reflected. If, for example, we were to examine the contour of the wave produced by throwing a stone into a tub of water, we should find that near the edge the secondary wave was very close to the main wave, whereas near the center the secondary wave would occur much later. Our problem therefore narrows itself down to an investigation of the cause for the dicrotic wave at the central end of the circulation. It occurs, as we have seen, immediately after the beginning of diastole. That it can not be due to anything taking place in the ventricle itself is evidenced by the fact that such a wave is absent from an intracardiac pressure curve (see page 151), although it is present in the very begin- ning of the aorta. Now, the only structures existing between those two points which could be held responsible for this wave are the semilunar valves a conclusion which is sustained by the fact that, if the aortic valves are rendered incompetent by hooking them back, or if the pulse beat is examined in patients suffering from an aortic insufficiency, it will be found that the dicrotic wave is not nearly so evident as usual. To understand how the valves are responsible for the production of the wave, the mechanical changes occurring at the root of the aorta must be clearly understood (see page 155). The stretching of the elastic walls of the aorta which occurs with each systolic outrush of blood is fol- lowed by a powerful and sudden contraction of the stretched walls, and the pressure thus brought to bear on the column of blood in the aorta tends to impel it both forward and backward. The forward movement adds itself to the wave of increased pressure already produced by the ventricular contraction. The backward component travels as far as the semilunar valve, from which it is reflected, and now proceeds peripher- ally along the blood stream during the time at which the original pres- sure pulse is declining. It therefore imprints itself on the pulse trac- ing as a separate wave, and does so all the more markedly when the decline in the main pulse wave is rapid, as in cases in which the periph- eral resistance is low, but fails to be prominent when, on account of a high peripheral resistance, the decline in the main pulse wave is tardy. This explanation coincides exactly with the well-known clinical fact that the dicrotic wave is conspicuous in pulses of low tension, but ill marked or absent in pulses of high tension. One point remains to be considered, and that is the cause for the sudden decline in the main wave at the cessation of the ventricular out- THE BLOODFLOW IN THE ARTERIES 205 put, for, it might be said, why should there be such a sudden fall in pressure near the heart, whereas toward the periphery, as we have seen, the pressure between the heartbeats tends to be maintained on account of the elastic recoil of the stretched arterial walls. The explanation usually given is that the sudden cessation of outflow ^f-h^^d from the ventricle at the end of the sphygmic period causes a negative pressure to be produced in the blood aOhe beginning of the aorta, thus tending to cause jjjreflux of blood towards the he^rt^Jthe .effect of which is (1) to , and (2) *.n prnrf"^ tbA rpflpptprl rHp.rot.if>. wave. If, while fluid is flowing under pressure along a tube, the flow is sud- denly arrested by turning a stopcock, it is possible by the use of manom- eters to show that a negative wave is set up immediately beyond the stopcock, and that this negative wave travels along the tube at a rate depending on the elasticity of its walls. Causes for Disappearance of the Pulse in the Veins The disappearance of the pulse in the capillaries and its consequent absence in the veins we have already seen to be owing to the combined influence of the elasticity of the vessel walls and the peripheral resist- ance. On account of these tAVO factors the pressure conveyed to the blood during systole is stored up to be released during diastole by the recoil of the stretched vessels. Sometimes, however, the pulse gets through to the veins, either because the elasticity of the vessels is not so marked, or because the peripheral resistance has been lowered (vaso- dilatation). In patients with hardened arteries, or in normal individu- als after taking nitrite, which dilates the peripheral arterioles, a pulse may come through at the periphery and appear in the veins. This may be called the peripheral venous pulse, and it is to be carefully distin- guished from the central venous pulse observed in the large veins, as at the root of the neck, before any valves have intervened to block the transmission of the auricular pressure wave back into the column of blood in the veins. If a pulse is seen in a large vein and there is doubt as to whether it is peripheral or central in origin, this doubt can be immediately removed by locally constricting the vein; if the pulse is peripheral, it will disappear on the heart side of the constriction; if it is central, on the side away from the heart. CHAPTER XXIV THE KATE OF MOVEMENT OF THE BLOOD IN THE BLOOD VESSELS Since the object of the circulation is to maintain an adequate move- ment of blood in the tissues and capillaries, it is evident that besides measuring the pressure of bloodflow, we should also measure the rate of its movement, or, as it is often called, the mean velocity. This measure- ment may be undertaken either for a given vessel or for a complete vascular area, such, for example, as that of one of the viscera or one of. the extremities the mass movement of the blood. Or instead of measuring the mean velocity we may desire to know how long it takes for a particle of blood to traverse a given vascular area. Such a meas- urement is called the circulation time; it does not at all tell us how long it takes for all the blood to pass through the given area, but only, as stated, the time required for the circulation of a fraction of the blood through a particular field. VELOCITY OF FLOW IN A VESSEL Special methods have been devised for the measurement of each of these three velocities. For the measurement of the velocity of flow through a main artery or vein, methods similar to those employed by hydraulic engineers are employed; that is to say, the volume of blood, in cubic centimeters, . which passes a given point is measured for < given time, and the result divided by the cross section of the vessel at the point of observation. The result gives us the mean lineal velocity. To measure the outflow of blood in a given time, the simplest method would be to cut across the vessel and collect the blood in a graduate, but obviously in this method an error would be introduced, because cutting the vessel would lower the peripheral resistance and remove the natural obstruction to the flow present in the intact animal. Moreover, the hemorrhage would in itself introduce a disturbing factor on account of the loss of circulating fluid. To make such measurements of any value, it is obviously necessary to retain the peripheral resistance. For smaller vessels this can be done by introducing in the course of the artery a long glass tube bent in the 206 RATE OF MOVEMENT OF THE BLOOD 207 shape of the letter U (Fig. 61 No. 1), or by merely allowing the vessel to bleed into a graduated tube and seeing how long the blood column takes to travel from one end to the other. This method is of considerable value in measuring the velocity of flow from small vessels such as the veins coming from glands and muscles. For larger vessels a so-called stromuhr is employed. There are numerous forms of stromuhr; that shown in the diagram (Ludwig's) (Fig. 61 No. 2) consists of two glass bulbs united above, and connected below with tubes that open flush with the surface of a circular platform of brass. This is pivoted at its center with another similar platform also having flush with the surface the open- ings of two tubes which are connected below with the cut ends of the artery or vein. In a certain position of the upper platform, the tubes' from the artery or vein are exactly opposite those of the bulbs, so that the Fig. 61. Forms of apparatus for measurement of blood velocities. /. Volkman-n's hemodromometer. The blood vessel is attached to the two short side tubes, and according to the position of the stopcock, the blood flows either directly between them or through the U-shaped glass tube. 2. Ludwig's stromuhr. The tubes on the lower end of each of the two glass bulbs pierce a circular brass platform and end flush with its surface. This platform pivots at its center on a similar lower platform with two openings connected with the tubes that lead to the blood vessel. blood can flow through the bulbs from the one end of the blood vessel to the other. To use the instrument the proximal bulb is filled with oil and the peripheral one with physiological saline. The clip is then removed from the artery or vein and the blood flows in and displaces the oil, which in turn displaces the saline into the peripheral end of the blood vessel. When the blood has risen to a mark on the tube joining the two bulbs, the instrument is rapidly rotated so that the oil is brought back again into the proximal position, the rotation being effected so quickly that there is no distinct interruption in bloodflow. The operation is repeated in this way for a given period of time. Counting accurately the number of revolu- tions, then multiplying the number of revolutions by the capacity of the 208 THE CIRCULATION OF THE BLOOD bulbs, we get in cubic centimeters the amount of blood that has flowed through the instrument in a definite unit of time. This gives us the vol- ume flow and, if the result is divided by the cross section of the vessel in square centimeters, we obtain what is known as the mean lineal velocity. Many modifications have been made of this instrument, but it is unneces- sary to go into them here. The general result of such measurements has been to show that the lineal velocity is inversely proportional to the cross section of the vessel at the point of observation. It is obvious that the volume of blood flowing out of the heart to the aorta in a given time is exactly equal to that flowing into it by the vena cava, and likewise that the volume flowing into an organ is exactly equal to that which flows out. Conse- quently the lineal velocity will be inversely proportional to the sec- tional area of the vessel. The principle is the same as that which gov- erns the velocity of flow of a stream: when the bed is narrow, the cur- rent is swift, but it becomes sluggish when the bed is wide. If the arteries were of the same caliber as the veins, the mean velocity of the bloodflow through the two would be the same, but actually it is much greater in the arteries because the lumen of these at a given point in the circulation is only from one-third to one-half that of the corresponding vein. It must be understood that we are dealing above with the mean velocity in a unit of time, and that there must be considerable alteration with each systole and diastole, constituting the velocity pulse (page 200). The degree of this alteration with each velocity pulse is much less at the periphery of the circulation than near the heart. As the periphery is reached, the flow becomes more uniform. It must further be re- membered that, although the mean velocity depends essentially upon the area of the vascular bed, yet it is subject to considerable variations as a result of changes either in the force or rate of the heartbeat or in the facility of outflow from the ends of the arterial system that is, changes in peripheral resistance. It is usually stated that the mean lineal velocity in the carotid artery is about 300 millimeters per second; and in the jugular vein, about 150 millimeters; whereas in the capillaries, where the total area of the vascular bed has become enormously increased, being perhaps some 800 times that of the aorta, the velocity of flow is only about half a milli- meter per second. MASS MOVEMENT OF THE BLOOD IN A VASCULAR AREA Methods. In considering the Hood/low or mass movement of the blood in the different regions of the body, it is usually more practical to BATE OF MOVEMENT OF THE BLOOD 209 measure, not the mean lineal velocity of the inflowing and outflowing blood, but rather how many cubic centimeters of blood are traversing the part per 100 grams of organ or tissue per unit of time. Such meas- urements may be made in a variety of ways. If there are but one artery and one vein to the part, the stromuhr may of course be employed, and it may be inserted in either the arterial or the venous circuit. For measuring the mass movement of blood through such large viscera as the liver, this is indeed the only method that can be employed. The stromuhr is inserted either in the course of the portal vein and he- patic arteries, or, better still, in the vena cava just below the openings of the hepatic vein, the vena cava being shut off for a moment between the liver and the heart, and the blood, as it flows from the hepatic vein, allowed to collect in the stromuhr. For other organs and tissues, how- ever, methods which do not involve any interference with the blood vessels may be employed. One of these is the so-called plethysmo graphic method of Brodie. An organ, such as the kidney, is enclosed in a plethys- mograph (see page 235), and while a record of its volume is being inscribed on a quickly revolving drum, the vein is suddenly clamped, with the result that the kidney volume expands in proportion to the mass of blood flowing into it. When the expansion has reached a cer- tain degree, the clamp is removed and the bloodflow allowed to pur- sue its course. It is then an easy matter, by graduating the plethys- mograph, to determine how many cubic centimeters of blood must have flowed into the organ in a given time. To avoid serious local asphyxia in the tissue, the clamp must be applied to the vein for only the briefest period of time. This method may also be employed for measuring the bloodflow through the extremities. Thus, if the arm is enclosed in the plethysmograph (Fig. 62) and a band encircling the arm above the plethysmograph is tightened so as to constrict the veins but not the ar- teries, the rate at which the volume of the arm within the plethysmograph expands will correspond to the rate at which blood is flowing into it (Hewlett). For the purpose of measuring blood flow through the upper or lower extremities, a much more serviceable clinical method is that of G. N. Stewart. This depends on the principle that, provided the blood passing from the thorax to the hands or feet is of constant temperature, the rate at which heat is dissipated from the hands or feet will be directly proportional to the rate of movement of the blood through these parts. Fortunately for the method, the hands particularly, but also the feet, are more or less perfect radiators at least they are to this extent, that if the temperature in their environment is not much lower than the temperature of the blood, then while this is traversing the part, it will 210 THE CIRCULATION OF THE BLOOD lose heat to the environment until the outflowing, or venous blood, is at exactly the same temperature as the environment; for example, if the hand is placed in water that is a little cooler than that of the blood, and the temperature of the blood in one of the large veins of the hand is measured, it will be found to be the same as that of the water in the water-bath. To measure the rate of flow, therefore, we must ascertain: (1) how much heat has been given out by the part to the water surrounding it in a given time, and (2) the difference in temperature of the inflowing (arterial) and outflowing (venous) blood. We measure the amount of Fig. 62. Plethysmograph for recording volume changes in the hand and forearm. By observ- ing the rate with which the volume increases when the arm is compressed, the mass movement of the blood can be determined. (From Jackson.) heat given out to the water in calories, a calorie being the amount of heat required to raise the temperature of 1 c.c. of water from C. to 1 C.. Suppose, for example, a hand were placed in 3,000 c.c. of water at 33 C., and that after ten minutes the temperature had risen to 33.5 C., then the amount of calories given out would be 3,000x0.5= 1500. Since calories equal cubic centimeters multiplied by change in temperature, it follows that if we divide the figure representing them by the actually observed difference in temperature between inflowing and outflowing blood, the result must equal the number of cubic centimeters of blood that has flowed through the part. The temperature of the in- flowing blood has been found to be practically identical with that of the RATE OF MOVEMENT OP THE BLOOD 211 mouth under the tongue; whereas of course the temperature of the venous blood, as already explained, is equal to the mean temperature of the water during the time that the hand was immersed in it. Further de- tails of the technique of this method will be found eJsewhere, but it may be said here that it is extremely simple and accurate, and that it requires nothing more than (1) an accurate thermometer ranging between about 25 C. and 50 C., with a scale so drawn out that readings can be made to %oo of a degree, and (2) a well-constructed vessel of about 3,000 c.c. capacity, with double walls, the space between them being packed with some heat-insulating material such as ground cork. Results. Regarding the results obtained with these methods, it has been found that the blood supply for each 100 grams of tissue per minute in the viscera, as measured by the stromuhr method, is about as follows: stomach, 21 c.c. ; intestine, 71 c.c.; spleen, 58 c.c.; liver, arterial, 25 c.c.; liver, venous, 59 c.c,; liver, total, 84 c.c.; brain, 136 c.c.; kidney, 150 c.c.; thy- roid gland, 560 c.c. The large blood supplies of the thyroid gland and of the kidney are the most striking results of these observations. By the use of the calorimeter method the bloodflow through the hands and feet of a healthy young man has been found to be about 13 grams per 100 c.c. of hand per minute for the right hand, and about half a gram less for the left. The footflow is only about one-third to one-half that of the hand per 100 c.c. of tissue a difference which is largely owing to the greater proportion of skin and the smaller proportion of bone in the hand. The average footflow or handflow for a given indi- vidual under ordinary conditions is remarkably constant from time to time, but it is extraordinarily sensitive to changes in the temperature of the environment in which the subject has been living for some time previous to the measurement. In one individual, when the room temperature was 20 C., the flow in the right hand, expressed in grams of blood per 100 c.c. of hand or foot, was 10.1; when it was 22.8 C., the flow was 12.8; when it was 25 C., 12.1; when it was 30 C., 18.5. On account of the influence of temperature on the flow, it is extremely important that the measurements should be made in a small room the temperature of which is kept constant, or if it must be made in the wards, the bed should be sur- rounded by curtains. The measurements made on the hands of dispensary patients shortly after coming in from outside air are very likely to be fallacious. The importance of making such bloodflow measurements in the clinic will be alluded to later. Of course the measurements made by the above method in man tell us only the rate of flow in the periphery of the body, and furnish us with no in- formation regarding the flow of blood through the viscera. It is, how- 212 THE CIRCULATION OF THE BLOOD ever, a well-established fact that the bloodflow in the central part of the circulation is more or less reciprocal with that at the periphery, an increase in the one place being accompanied by a corresponding de- crease in the other. The Visceral Bloodflow in Man The visceral bloodflow in man can be measured indirectly in the case of the lungs, either, (1) by finding the quantity of oxygen absorbed by the blood during an interval of time that is less than that required for the blood to travel once round the circulation (60 seconds) and comparing this with the oxygen content of samples of arterial and venous blood, or (2) by causing a person to breathe a known quantity of nitrous-oxide gas and then finding how much is taken up by the blood in the lungs. In the for- mer method the difference in oxygen percentage between arterial and venous blood will be less for a given absorption of oxygen from the alveoli the more rapid the circulation of blood through the lungs; in the latter method, the absorption of a given amount of nitrous oxide will be pro- portional to the rapidity of the bloodflow. Obviously these estimations must be made only over periods of time, less than that taken for any of the blood to complete one circuit of the circulation. Since it is likely that such measurements will find some application in the study of cardiovascular disease, it may be well to indicate briefly how they are carried out. In the oxygen absorption method, the amount of this gas that is absorbed from the lungs in one minute is determined by analysis of inspired and expired air. The ar- terial blood is considered as saturated with oxygen, and the percentage of this gas in the venous blood (entering the lungs) is computed by using the dissociation curve. Suppose 1,000 e.c. of O 2 was absorbed in one minute* and that the arterial blood con- tained 10 per cent more O 2 than the venous, then, since each 100 c.c. of blood carried away 10 c.c. of gas it would take 10,000 c.c. to carry away the 1,000 c.c. of O actually absorbed; and suppose the pulse to be 80 per minute then, with each heart beat 10,000 o = 125 c.c. of blood must have flowed through the lungs. In the nitrous oxide method 1 * 1 * the person inspires a deep breath of a mixture of this gas and oxygen from a meter, and after holding the breath for a few seconds (to allow the gas to mix uni- formly with the alveolar air) he expires sufficiently to bring the lungs to their mid position and again holds the breath for about 30 seconds, after which he finally expires forcibly. Samples of the expired air are taken from the last portions of the two expira- tions, and the percentage of nitrous oxide in them determined. From the percentages of nitrous oxide found in the two samples, the amount of the gas in the air of the lungs at the beginning and at the end of the period can be estimated. Suppose that 55 c.c. N 2 O was found to be absorbed in 0.327 minutes, that the percentage of N o O is 11.08 then, since the absorption coefficient of N" 2 O in blood at 37C is 0.405 (i. e., 1 c.c. of blood dissolves 0.405 c.c, N o O at 37C., Krogh and Lindhard the amount of blood re- *The average consumption of an adult at rest is only about 230-250 c.c. of Os, the above value of 1,000 c.c. being however observed during muscular exercise. RATE OF MOVEMENT OP THE BLOOD 213 55 quired to absorb 55 c.c. is 1.230 c.c. (1.23L.) or in one minute 1.230 = 3.75 liters. 0.327 The methods are admittedly only approximate, but the results are of much interest, mainly because of the indication they give us as to the amount of blood pumped out by the ventricle with each heartbeat, or during a given period of time. The results have been found to vary considerably; thus, Krogh and Lindhard 45 give the output of blood per minute as between 2.3 and 8.7 liters, which would correspond, at a pulse rate of 70, to an output per heartbreat of from 40 to 120 c.c. An immediate and very marked increase has been found to occur during muscular work. By comparing the bloodflow through the hand with that through the lungs, an estimate can be formed in a given individual as to the relative magnitude of the peripheral and visceral moieties of blood. Interesting results, which will be referred to later, have been obtained from such measurements. The Work of the Heart Meanwhile it is of interest to note that we may calculate from the ventricular output of the blood the amount of work that the heart is doing in maintaining the circulation. Of course the calculation is again only approximate, since we have to assume certain figures. If we assume that in a 70-kilogram man the quantity of blood is 4,200 c.c. (see page 85), and that it takes about one minute for all the blood to complete a cir- culation, then the work performed by the left ventricle in one minute will be equal to that done in raising the above quantity of blood to a height corresponding to the mean pressure in the aorta. If we take this pressure as 130 millimeters of mercury, which would correspond to a column of blood 1,755 meters high (13.5x130=1755 mm. blood, or 1.755 meter), the work done by the left ventricle would be 1.755x4.2=7.37 kilogram-meters in one minute, or in twenty-four hours roughly about 10600 kilogram-meters. The work done by the right ventricle is probably about one-third that of the left, this being about the ratio of the pres- sures in the two chambers. The total work of the two ventricles is there- fore about 14000 kilogram-meters. This represents an enormous amount of work; indeed it has been computed that it is sufficient to raise a man of 70 kilograms to about twice the height of the highest skyscraper in New York. The work thus expended in forcing the blood through the capillaries becomes converted by friction in the small blood vessels into heat, the heat equivalent of the above amount of work being roughly about 350 calories (see page 571). 214 THE CIRCULATION OF THE BLOOD THE CIRCULATION TIME The circulation time, or the time taken by a drop of blood to travel between two points in the circulation, can be determined in laboratory animals by a variety of methods, all depending on the principle of seeing how long it takes for a drop of some substance injected into one vessel to appear in the blood of another. For example, to determine the time taken for a drop of blood to pass from the jugular vein into the carotid artery in a rabbit, a solution of methylene blue in isotonic saline is in jected into the former vessel and the moment of its appearance through the walls of the artery determined by a stop-watch. If the walls are too thick to admit of the employment of this method, a strong solution of sodium chloride may be substituted for the methylene blue, and the mo- ment of its appearance at another point of the circulation determined b\ observing the electrical conductivity of the vessel. Since the con- ductivity of a blood vessel depends partly on the concentration of elec- trolytes in the blood flowing through it, the moment at which the salt solution appears will be indicated by a change in electrical resistance (G. N. Stewart). By such methods, it has been found that the time for the pulmonary circulation is very short compared with that of the systemic circulation. In a rabbit it is usually a little less than four seconds; in an average- sized dog of about 12 kilograms, it is about eight seconds; and in man it is computed to be about fifteen seconds. On the other hand, the cir- culation time in such viscera as the spleen and kidney is relatively long, and more susceptible than that of the lungs to different conditions of temperature. In a dog in which the pulmonary circulation time was about 8.5 seconds, that of the spleen was about 11 seconds, and of the kidney about 17.5 seconds. The shortest circulation time of all is of course that in the coronary artery, but that through the retina can not fall far behind it. To determine the total circulation time, we must know: (1) the average amount of blood passing by each part in a given time, and (2) the average circulation time of each part. From such computations, which however are obviously subject to considerable error, it has been reckoned that the total circulation time in man must lie somewhere between 1 and 1.25 minutes. MOVEMENT OF BLOOD IN VEINS Before leaving this part of our subject, a few words may be said con- cerning the forces concerned in the movement of Wood in the veins from RATE OF MOVEMENT OF THE BLOOD 215 the capillaries to the heart. By the time that the venules are reached, owing to friction in the capillaries the blood will have lost most of the force imparted to it by the heart action. Nevertheless, this remaining vis a tergo must be considered as the basic cause for the movement of the venous blood near the periphery. As the venules get larger, two other factors come into play: the massaging influence of the muscles, and the valves of the veins. By the movements of the muscles the veins which lie between will be rhythmically compressed, and this will tend to cause the blood to be moved forward and backward in them, the back- ward movement being however prevented by the operation of the valves. When the tonicity of the muscles is subnormal, as in conditions of ill health, the absence of this massaging action permits the blood to stag- nate in the veins, especially in those of the lower extremities in upright animals, with the consequence that the veins become dilated, particularly just above the valves, thus causing the condition known as varicose veins. As the thorax is approached, two other factors become operative: the aspirating influence of the thorax during inspiration, and the negative intraventricular pressure (see page 152). There is no doubt that the former of these is of considerable importance in maintaining the venous return near the heart, for although the change of pressure induced by in- spiration amounts to only 5 millimeters of mercury, yet it acts so slowly that it produces a considerable influence. The aspirating effect of the ventricle at the beginning of diastole is, however, of no sig- nificance in attracting blood to the heart, for although, as we have seen, it may be considerable, yet it lasts for so short a time that it could not overcome the inertia of the column of blood in the vena cava. Even if the negative pressure did last for a longer period, it could not attract more than a small amount of blood, because it would cause the thin collapsible walls of the veins to come together and thus block the pas- sage towards the heart. CHAPTER XXV THE OUTPUT OF THE HEART IN RELATION TO THE VENOUS INFLOW, CHANGE OF RATE, ETC. The Output of the Heart per Beat In the heart-lung preparation described on page 163 it is possible to make comparison between the output of the heart and such conditions as its rate of filling during diastole, the frequency of its beat and the resistance offered to its systolic discharge. Starling and his pupils have in this way thrown much light on the methods by which the cardiac out- put is adjusted so as to meet the ever-varying demands of the body for blood. According to these workers the fundamental principle which determines cardiac output may be stated thus: the force with which the heart contracts during systole varies directly with its volume at the end of diastole. When the venous inflow is rapid, the heart becomes dilated and it contracts to its full force; when the inflow is slow, it is imper- fectly dilated and when contraction supervenes the beat is feeble. This is the law of the heart, and in the case of the cold-blooded heart, it is rigidly obeyed as is demonstrated by the fact that when the perfusion fluid flow- ing into the venous end is suddenly increased by a certain amount, the ventricular output with the next beat is correspondingly augmented, and the volume of the heart remains unchanged at the end of systole, though it has of course become greater during diastole because of the increased filling. In the warm-blooded heart, at least under the conditions of experi- mentation (heart-lung preparation), there is some lag in the adaptation of the strength of systole to diastolic filling, although the law of the heart is ultimately obeyed. This is well shown in the tracings in Fig. 63, in which C is a tracing of the volume of the isolated heart (obtained by using a cardiac plethysmograph), B.P. the arterial blood pressure and V.P., the venous blood pressure. 50 According to these views the factor that primarily determines the sys- tolic discharge is the extent to which the cardiac muscle fiber becomes stretched by the venous filling of the heart, i.e., the initial length of the fibers. If the muscle be depressed on account of malnutrition, or fatigue, or disease, its response for an equal degree of stretching will be lessened so that to bring about an equal discharge during systole the heart will 216 OUTPUT OF HEART AND VENOUS INFLOW 217 have to become more dilated (i.e., more stretched) during diastole than would a normal heart. If this condition is maintained, the muscle hyper- trophies with the result that less diastolic dilatation becomes necessary because of the cumulative effect of the larger number of fibers. Increase in arterial pressure within certain limits does not affect the amount of the systolic discharge so long as the inflow is kept constant. It will be observed in the preceding paragraph that nothing is said about the tension of the musculature at the beginning of systole; only its length is considered to be important. More recent work by Straub and Wingers 61 shows however that in this particular Starling has erred and BP VP Fig. 63. It will be observed that when V.P. is suddenly increased, the cardiac volume im- mediately becomes greater (indicated by a general fall in the level of C), and that, although the contractions of the ventricle also become more vigorous, they do not do so with sufficient prompti- tude to maintain the systolic volume constant. In other words the output of the heart does not at first keep pace with the inflow, so that the mean volume of the heart becomes progressively greater, and it is onjy after some moments that the contractile power -increases sufficiently so that the output equals the inflow and the mean volume becomes steady. the latter investigator has pointed out that this is probably due to the fact that a heart-lung preparation is too artificial (e.g., the coronary blood flow is certainly abnormal and the pericardium is usually opened) to represent the conditions actually obtaining in the intact animal. The advantage claimed for the preparation was that the rate of heart could 218 THE CIRCULATION OF THE BLOOD be kept constant or altered at will while venous filling, or arterial pressure, was independently altered. But this is no advantage because the same conditions can be fulfilled after vagotomy in otherwise intact animals ; for example, by clamping the aorta in order to raise the arterial resistance, or by injection of saline or clamping of the vena cava in order to vary the venous inflow. Each of these experimental procedures can be maintained for several beats without causing changes that spread suf- ficiently to cause alteration in the other factor and during these few beats, intracardiac pressure curves can be taken by the optical method and the response of the heart precisely analyzed. The important pressures to measure are that just before systole (initi;il pressure) and that at the height of systole (maximal pressure). By clamping the vena cava, for example, the initial and maximal pressures in both ventricles were immediately de- creased whereas by injecting saline into the jugular vein both of these were increased. By raising the arterial resistance (clamping the aorta or by causing reflex general vasoconstriction), the initial pressures for a beat or two were not affected at all in the right ventricle and only moderately so in the left, but the maximal pressures were decidedly increased. By measuring the volume changes along with those of pressure it was found that there was always an increase in initial pressure (in the right ventricle) accompanying an increase in initial volume a result which shows that Starling is wrong in concluding that it is only the initial length of the fibers that determines their contractile power. These relationships, moreover, break down entirely both when the initial pressure is excessively elevated (in which case there is a diminished pressure maximum and a lessened discharge) and when the myocardium is depressed, as by poisoning with chloral. While admitting that the initial length of the cardiac muscle is a most important factor in determining the strength of the succeeding contraction, as it is also in skeletal muscle, Wiggers concludes that changes in volume cannot occur without accompanying alteration in initial tension. He there- fore considers the tone of the heart in diastole as an important factor in determining its systolic force. The Effect of Alteration in Rate of Heart Beat on Output of Blood At this stage it is important to analyze the effect on the output of the heart per minute (the minute volume) brought about ~by inert a.^r in (lie rate of beat. When the venous inflow is slow, the ventricle does not dilate to its full capacity in diastole and acceleration of the beat does not improve the output in a unit of time. The same is true when the inflow is just sufficient to dilate the ventricle to the maximal extent at the moment that systole supervenes, for although more blood is discharged than in the first case, acceleration cannot improve the output. When, on the other hand, the inflow is sufficiently rapid so that the ventricle is practically filled to its limit some time before diastole ends, the output is increased by acceleration. In other words, when there is no so-called period of diastasis following active diastole, acceleration of the beat will not increase the output in a unit of time. The condition in which increased heart rate occurs with greatest certainty is mus- cular exercise. The initial quickening is due to impulses travelling to the cardiac centers in the medulla from centers in the cerebrum (see page 229), and it is clear that the acceleration will be of value in increasing the cardiac output in proportion OUTPUT OP HEART AND VENOUS INFLOW 219 to the relation between active and passive diastole prior to the exercise. The venous inflow also increases because of the muscular activity, so that the diastolic filling is more complete, the initial tension higher, and the systolic discharge greater. As the muscular activity continues, the heart rate continues to accelerate, partly because of increase in the temperature and in C H of the blood (see pages 162 and 168) and partly because of increase in venous pressure (Hooker an d Bainbridge). This in- creased pressure, probably by the tension produced on the walls of the ventricle (right) during diastole, sets up afferent impulses which act on the vagus and sympathetic cardiac centers. Dilatation and Tonus We have seen that in the healthy heart the extent of dilatation and tension during diastole determines the discharge during systole. When the dilation is not accompanied by a large discharge (i. e., the volume does not diminish properly during systole), "pathological dilatation" is said to exist. The essential difference between physiological and pathological dilatation depends on the presence or absence of tonus, by which is meant a sustained partial contraction ' ' by virtue of which the muscle fibers resist stretching more than they would by virtue of inherent elasticity." In other words, for a given volume of venous inflow, a greater pressure will exist in the ventricle when the tonus is high than when it is low. Being, therefore, already partly contracted at the end of diastole the tonic heart will develop greater force than the atonic, during systole; it has less slack to take up, as it were. The Dynamics of the Circulation in Heart Disease When the myocardium is weakened, as after debilitating diseases or in poisoning by chloral, chloroform, etc., the systolic force is diminished so that all the blood is not discharged with each beat, and the ventricle consequently becomes overdistended by the inflow of the venous blood. Although the elongation of the fibers and the greater tension to which they are subjected does not at first succeed in calling forth a sufficiently greater systolic contraction to maintain an adequate discharge, as would be the case in health, a compensation may ultimately set in and the heart contract more powerfully so that the intraventricular pressure increases sufficiently to maintain the normal discharge. If this compensation does not occur the pressures rise in the pulmonary circuit without, however, causing as a rule serious congestion of the lungs because the systolic discharge of the right heart is lessened to a corresponding degree to the left. The congestion therefore usually occurs further back in the venous sys- tem and in the liver. Both first and second sounds are weak until compensation oc- curs. Cardiac stimulants such ;is strophanthin and digitalis assist in the development of compensation. When the aortic pressure is decidedly increased, the pressure maximum of the suc- ceeding systoles is insufficient to expel all the blood that has collected in the ventricle during diastole. The resulting greater stretching and initial tension within the ven- tricle immediately stimulate more powerful contractions and if the pressure be not too great the discharge returns to the normal in a few beats. If the pressure be excessive however, or if it be maintained for too long this compensation begins to fail, the iso- metric phase of systole becomes more gradual, the first sound becomes feebler (while the second sound is still intense) and the venous bloods dams back in the left auricle and pulmonary veins in which the pressure becomes raised so that, with the right ventricle meanwhile discharging its full load of blood, extreme congestion of the lungs occurs. Only later does the right ventricle fail to maintain its output and when this occurs venous congestion a IK! fall of pulmonary pressure become evident. 220 THE CIRCULATION OF THE BLOOD When the aortic valve leaks the ventricle becomes overfilled with blood during diastole so that the initial tension is elevated and in the subsequent systole the pressure at- tained is sufficient to compensate for the regurgitation in the sense that a normal mass movement of the blood is maintained (page 208). If the tonus of the myo- cardium be depressed however, the initial pressure does not properly increase and consequently the maximum pressure attained is inadequate, i.e., decompensation occurs. Decompensation may also be the result of exhaustion of the reserve power of the heart. 61 To understand what this means we must remember that the systolic pressure and discharge are proportional to the initial pressure until this reaches a certain limit beyond which with further increase the discharge, etc., decline. This optimal initial pressure varies with the condition of the myocardium ; it is the index of the reserve power. When it is low, decompensation soon occurs so that "back pressure effects" become evident; when it is normal, the response of the ventricle is adequate to compensate for the leak and the circulation may be maintained for a long time without any pulmonary stasis or venous congestion. When the auriculoventricular valve leaks (mitral incompetence) the back pressure in the auricle occurs mainly, not during the presphygmic period of ventricular systole as might be expected but during the first part of the sphygmic period (Wiggers and Feil). The Reserve Power of the Heart When the heart is well developed, as in a trained athlete, the increase in venous inflow which occurs during exercise (see page 219) will stimulate the ventricle so that the output per beat exactly corresponds to the inflow, and the rate is only moderately increased. But when the musculature is feeble, as in a person of sedentary habits, the dilatation must become considerably greater in order to call out sufficient contractile power; the output per beat, therefore, only increases moderately and con- siderable quickening has to occur so that the minute volume may be adequate to meet the increased demands of the muscles for blood. This means that there must be a certain optimal initial pressure in the ventricle and that when this is exceeded the systolic discharge becomes less. The exact pressure which is optimal will vary with the condition of the myocardium, being;, low when this is subnormal. When the reserve power of the heart is very low, even extreme dilatation during diastole may be insufficient to stimulate contractions that are powerful enough to empty the heart; blood is therefore left over in it at the end of systole, and when the venous blood becomes superadded during diastole, extreme dilatation occurs, the beat becomes very rapid in the attempt to maintain an adequate output and "back pressure effects" become evident. The maximal pulse rate that can ultimately be attained during exercise is about 160 beats per minute. The Testing of Cardiac Efficiency The most practical method for testing the reserve power of the heart is to observe the pulse rate and the systolic blood pressure at frequent stated intervals after a prescribed amount of muscular work. Dumb-bell exercises are useful for this purpose. The pulse rate should be back to normal in at least 4 minutes after the exercise. The blood pressure behaves peculiarly; immediately after the exercise it falls below its height during it, but soon rises again and finally returns to normal within at least four minutes. If the pulse rate and blood pressure are not restored to normal in four minutes after moderate exercise, the cardiac reserve is presumed to be low. CHAPTER XXVI THE CONTROL OF THE CIRCULATION The available blood in the body is parceled out to the various organs and tissues according to their relative activities, and, since these vary from time to time, the question arises as to the nature of the mechanism or mechanisms involved in bringing about this adjustment. Two possible methods of increasing the supply are: an increase in the mass movement of all the blood in circulation, and a reciprocal adjustment of the resistance to the flow in different vascular areas brought about by vasodilatation in one and vasoconstriction in others. Both of these methods might operate together. Two agencies can be thought of as responsible for bringing about the above changes: (1) chemical substances or hormones, present in the blood, and (2) the nervous system. The influence of chemical substances, or hormones (page 766), in the control of the circulation is undoubtedly an important one, and of those known at the present time two groups may be mentioned: (1) sub- stances which alter the hydrogen-ion conp.ftnt.rfl.timi of the blood, and (2) so-called pressor and depressor substances, produced either by duct- less glands, such as the adrenal, or by the activity of tissues. An in- crease in hydrogen-ion concentration of the blood not only affects the heartbeat (see page 168), but causes a marked dilatation of the blood vessels, so that both the central and the peripheral changes will be such as to encourage an increased flow of blood through the active organ or viscus. Thus, during muscular activity of the leg muscles there will be a tendency to an increase in the hydrogen-ion concentration of the blood as a whole, resulting in a greater cardiac activity and a greater outrush of blood through the aorta, and at the same time the vessels of the acting muscle will have become especially dilated because of the production by the active muscles either of lactic acid or of carbonic acid. The active muscle also produces such substances as imidazole, which have a powerful vasodilating action. Such substances are sometimes called depressor. Though the hormone control of the circulation is undoubtedly of great importance, it is probably much less so than that exercised through the nervous system, and here again the control is centered partly in the 221 222 THE CIRCULATION OF THE BLOOD heart and partly in the peripheral resistance. The nerve control of the heart is effected through the vagus and sympathetic nerves, and that exercised on the blood vessels, through the so-called vasoconstrictor and vasodilator nerves. The activity of the nerve centers from which the cardiac and vaso- motor impulses are discharged is controlled by afferent impulses com- ing from the various regions of the body. When a gland becomes more active, we must suppose that stimulation of the sensory fibers has caused afferent impulses to be transmitted to the cardiac and vasomotor centers, upon which they act in such a way as to produce increased heart ac- tion and a local dilatation of the blood vessels of the active gland, with perhaps a constriction of the blood vessels of the rest of the body. THE NERVE CONTROL OF THE HEARTBEAT The Vagus Control With regard to the control exercised through the vagus nerve, we have already seen that the cutting of the two nerves in the neck causes the heart to quicken and the arterial blood pressure to rise, whereas a stimulation of the peripheral end of the nerve causes the heart to be- come slowed, if not stopped altogether, and the blood pressure to fall. For the more detailed investigation of the nature of the vagus control of the heart, it is necessary to observe the exposed heart itself an ex- periment which, for obvious reasons, can be most simply performed in a cold-blooded animal, such as the frog or turtle, but which can also be performed in mammals provided artificial respiration is maintained. The general effect of the vagus in both groups of animals is the same, although apparent differences may exist on account of the relative im- portance of the different parts of the heart in the origination and propa- gation of the heartbeat. The Cold-Blooded Heart. If the vagus nerve on the right side in the turtle (the left nerve is usually more or less inactive in this animal) is stimulated with a very feeble electric current, while simultaneous records are being taken of the contractions of the auricles and ventricles in the manner shown in the accompanying tracing (Fig. 64), it will often be found that there is a weakening of the auricular beats without any change in those of the ventricle. If the strength of stimulus is somewhat in- creased, the auricular beat, besides becoming weaker, will also become slower, but meanwhile the ventricular, although also slower, may become distinctly stronger. At first sight this result may be a little confusing, because it would seem to indicate that the vagus nerve weakens the auricu- THE CONTROL OF THE CIRCULATION 223 lar, but strengthens the ventricular beat. It is clear, however, that the strengthening of the ventricular beat is merely due to the fact that the cavity has become better filled with blood during diastole as a result of the slowing of the auricle. These results indicate, then, that with weak stimulation the vagus exerts its direct influence only on the auricle. If Fig. 64. Simultaneous tracings from auricle and ventricle of turtle's heart. Between the crosses the vagus was stimulated, with the effect that the auricular beat diminished in force but not in frequency, while the ventricular beats were practically unaffected. (From Howell's Physiology.) the stimulation is strong enough both auricles and ventricles cease to beat altogether, and if the stimulus is maintained, the inhibition may go on for a very long time (Fig. 65). Usually, even though the stimulus is maintained the heart begins to Fig. 65. Effect of vagus stimulation on heart of turtle. Note the after effect of augmentation. beat again after a time, at first only occasionally but gradually more rapidly. This is known as escapement, and it indicates that the energy pent up in the heart during the vagus inhibition has at last overcome the inhibiting influence of the nerve, which is meanwhile becoming fatigued. All of these results could be quite satisfactorily explained on the assumption that the action of the vagus is confined to the sinus, 224 THE CIRCULATION OF THE BLOOD which, it will be remembered, dominates the beat in the rest of the heart. There is evidence, however, that the vagus also directly affects the rhythm of the ventricle. It may be stated as a general conclusion from these results that the influence of the vagus upon the heartbeat is chiefly centered upon those parts of the organ in which the rhythmic power is most highly developed. Besides affecting the rate and strength of the heartbeat, the vagus also exercises a control on the conductivity of the cardiac muscle. Thus, if a partial block is instituted in the turtle heart by applying a clamp be- tween the auricles and ventricles, stimulation of the vagus enfeebles the auricular beat and may also cause a complete heart-block as shown in the tracing reproduced in Fig. 66. It is important to point out here, however, that under certain conditions the vagus may appear to increase rather than decrease the conductivity of the tissue in the auriculoven- Fig. 66. Tracing to show that vagus stimulation may diminish transmission from auricles to ventricles. It shows the effect of stimulating the left vagus on partial (2/1) block produced on heart of turtle by application of clamp at auriculoventricular junction. Stimulation at 4- depressed the conductivity and weakened the auricular contractions (lower tracing) without slowing their rate. The result was an increase in the degree of block with cessation of ventricular contractions (upper tracing). Initial auricular rate =. 35 per minute. (From Carrey.) tricular junction; for example, it has been observed in the turtle heart that when a clamp is so tight as to produce complete block that is to say, to render the ventricle inactive while the auricle is still beating at the usual rate stimulation of the vagus, besides causing the auricles to become distinctly slowed, may at the same time cause the ventricles to respond to the auricular beats. This result is probably due to the better chances of slow beats getting through the junction than those which are so frequent as to crowd one another on the narrow bridge which the con- stricted tissue offers to their passage (Fig. 67). Very important work was contributed in this field by G. R. Mines 13 shortly before his lamentable death. He found that the local applica- tion of atropine to the sinus eliminates the effect of stimulation of the (intracranial) vagus on the rate of the heartbeat, while the effect .on the THE CONTROL OF THE CIRCULATION 225 auriculoventricular junction and on the ventricle remains. After the atropinization, vagus stimulation delays the transmission of beat from auricle to ventricle and shortens the time of each beat in the ventricle. It was further found that by the local application of atropine various parts of the ventricle can be rendered irresponsive to the influence of the vagus and the effects of this nerve on the form of the cardiogram modified at will. These results have an important bearing in the in- terpretation of the cause of the T-wave of the electro-cardiogram which will be referred to later. Mines' results show that the proba- ble explanation is that the T-wave is due to the greater duration of the excitatory state at the base than at the apex, for by altering the relative duration of this state at base and apex by the above methods, he could cause the T-wave to appear or disappear. The direct excitability of the heart muscle to external stimuli is also depressed during vagus stimulation. This effect is, however, not evi- Fig. 67. Tracing to show that vagus stimulation may facilitate transmission from auricles to ventricles. It shows the effect of right vagus stimulation on heart-block produced in the turtle by a clamp. Upper tracing records ventricle; lower tracing, auricles. Weak faradization of the right vagus nerve beginning at A affected the degree of block only at -f , when a lengthened period between auricular contractions caused a single ventricular contraction. At B stronger faradiza- tion of the same nerve produced marked slowing of the auricles, in consequence of which the block disappeared and the ventricles contracted after each auricular contraction. Block reappeared when the rate again became rapid. Initial auricular rate = 36 per minute. (From Carrey.) dent in the case of all hearts, but is seen in those of certain fishes (e. g., the eel). The Mammalian Heart, The action of the vagus on the mammalian heart may be investigated either by exposing the heart and connecting the auricles and ventricles with specially designed recording levers (myocardiograph), or if we desire to study the influence on the heart as a whole, by taking a blood-pressure tracing from one of the large arteries by means of a spring manometer. The results are in general similar to those observed on the frog or turtle heart, the main effects being de- veloped on the auricle. Considerable differences are found in the effect on the heart as a whole in different animals, particularly with regard to the facility with which escapement occurs. In the dog when the vagus 226 THE CIRCULATION OF THE BLOOD is continuously stimulated, the heart is likely to remain inhibited for a long time, whereas in the cat the inhibition is very quickly broken into by escapement. If the tracing is taken directly from the heart, it will frequently be observed in the dog that, when the escapement occurs dur- ing vagus stimulation it is only the ventricle that is beating, the auricles still remaining inhibited. If the stimulation of the vagus is discontinued after some time in an animal whose blood pressure is being recorded, the pressure will not only quickly recover, but will usually overshoot the normal level, mainly because of the asphyxia which has been produced during the period of inhibition. The asphyxia raises the hydrogen-ion concentration of the blood and this stimulates both the vasoconstrictor center and the heart action (page 168). The increased heart action is, also partly owing to the fact that during vagus inhibition the beating pOAver of the heart becomes improved (page 230). As an outcome of recent work, 14 it has been show nerve acts mainly on the sinoauricular node, and the left vagus on the auriculoventricular bundle. This is in agreement with the observations described above on the cold-blooded heart (page 222). Stimulation of the right vagus always causes slowing and weakening of both the auricular and the ventricular beats, but stimulation of the left vagus is sometimes observed to have but little influence on the auricular beat, although it may produce a condition of partial heart-block; or, if a clamp is ap- plied to the auriculoventricular bundle so as to produce a partial heart- block, then during stimulation of the left vagus, the block may become complete. There is, however, a considerable overlapping of these in- fluences, at least in the case of the left vagus, for this nerve also acts considerably on the ventricle, influencing perhaps not so much the rate as the force of the contraction. It has been found experimentally that, in order to demonstrate the specific action of the left vagus on the bun- dle, it is most suitable to study the relationship between auricular and ventricular beats when the auricle is beating rapidly as during the application of artificial (electrical) stimuli to it. Ordinarily the con- traction produced by each stimulus passes into the ventricle, but during stimulation of the left vagus it is found that every contraction does not pass. These experiments raise the question as to what the influence of either nerve may be in blocking impulses from the auricles to the ven- tricles when auricular fibrillation is present. It might be expected that the left vagus would prove more effectual in this regard, but actually it has been found that both vagi have the same effect. Tonic Vagus Action. Impulses are constantly passing along the vagi to the heart. On account of this so-called tonic action, the heart rate THE CONTROL OF THE CIRCULATION 227 increases_jyhpri the continuity of the vagus nerve is broken either by cutting or by freezing a portion of nerve (Fig. 27). The effect is usually inconspicuous when one nerve only is cut, but in most mammals it be- comes quite marked when both are cut. Change in the heart rate pro- duced by muscular effort is much more gradual in animals with marked vagus tone, such as hunting dogs, than in those with little vagus tone, as in domestic rabbits. The degree of vagus tone therefore bears a relation- ship to the staying power of the animal for prolonged muscular effort. It is usually ill developed in cold-blooded animals. It is quite marked in the case of man, as is evident on observing the heartbeat before and after giving a sufficient dose of atropine to paralyze the termination of the vagus in the heart. The exact location of the nerve cells that form the center of discharg- ing impulses along the vagus fibers to the heart can not be made out with certainty, but they are no doubt part of the great motor nucleus (ambiguus), from which arise the fibers not only of the vagus but of the glossopharyngeal nerve. The tone of this vagus center is almost without doubt dependent upon the constant transmission to it along the sensory or afferent fibers of impulses coming from various portions of the body. According to the strength or number of these impulses, the tone may be increased or diminished, thus altering the rate of the heart. It is possible of course that the tone can be maintained, independently of the afferent impulses, by the action on the center of chemical meta- bolic products or hormones produced in the cells or carried to them in the blood. We know at least that, like the respiratory center, that of the vagus is excitable by such hormones as the hydrogen-ion concen- tration of the blood. The tonicity of the vagus center is, however, mainly dependent upon the passage to it of afferent impulses, and as evidence for this conclusion may be cited the observation that, after section of most of the afferent nerves to the medulla (as by cutting the spinal cord high up in the cervical region), subsequent section of the two vagi does not produce anything like the usual degree of change in the heart rate. The Afferent Vagus Impulses. The afferent vagus impulses may come from practically any part of the body, having been first discovered by the simple experiment of tapping the abdomen of the frog with the han- dle of a scalpel, when slowing of the heart rate is observed. Cutting the vagi abolishes the reflex. Similar cardiac inhibition is produced by me- chanical stimulation of the tail or gills of an eel. In mammals stimula- tion^ of the central end of any sensory nerve usually slows the heart, though sometimes the opposite effect occurs. The pulmonary branches of the vagus are particularly sensitive in producing reflex inhibition, and distinct results are usually obtained: by stimulation of the termina- 228 THE CIRCULATION OP THE BLOOD tions of the fifth nerve in the mucosa of the upper respiratory passages, as by inhaling ammonia vapor; by stimulation of the sensory nerve end- ings in the pharynx, as by swallowing ; and of the mucosa of the larynx, as when a substance is "swallowed the wrong way." The sensory nerves of the abdominal viscera seem to be particularly active on the vagus center, as is seen in irritation of the sensory nerves of the stom- ach such as occurs in gastritis. Profound inhibition may also be caused by violent stimulation of the mesentery, as from a blow on the abdo- men, or by irritation of the sensory nerves of the intestine, either me- chanical or because of disease. Another interesting illustration of affer- ent vagus stimulation is obtained by pressure on the outer canthus of the eye. This oculomotor vagus reflex, as it is called, is very marked in some individuals. Through which of these afferent paths it may be that the constant stimuli are transmitted to the vagus center to enable it to maintain its tone, can not be said, although it is very likely to be through the vis- ceral nerves. In considering the cause for an observed change in heart rate, we must of course bear in mind the possibility that the action may have occurred, not through the vagus center, but through the sympathetic center. Thus, when the heart becomes quicker, it may be owing either to diminution in the vagus tone or to an increase in the discharges along the sympathetic nerve from the augmentor center. That such reflex action through the augmentor center does occur under experi- mental conditions has been clearly shown; for example, if both vagus nerves are cut and the peripheral end of one of them stimulated mod- erately, so as to hold the heart at about its normal rate, the stimulation of certain sensory nerves may cause increase in the heart rate. Reflex sympathetic control of the heartbeat is however no doubt much less important than control through the vagus center. When it does exist it means that the actual rate of the heartbeat at any given moment must represent the algebraic sum of two opposing influences, with that of the vagus preponderating. The advantage of such a double inner- vation is that it insures prompter adjustment of the beat. If, for ex- ample, for any reason quickening of the heart rate is necessary, it is brought about most promptly if the vagus tone is diminished at the same moment that the sympathetic tone is increased. Such reciprocal action of antagonistic influences is the usual rule in the animal economy. Thus, when the knee joint flexes, it does so not merely because stimulating impulses are transmitted to the hamstring muscles, but also because at the same moment inhibiting impulses are transmitted to the extensor muscles (see page 915). Left Ant Caval vein .Right Ant Caval vein Preganqf ionic rrettrdn ^ l ionic neuron 'Auricular sfeptum Position vonBezold's Ganqlion in Auricular septum In f Vena Hook from 'Heart lever didders Gdnqlion in auriculo-ventricular junction Simulating electrodes c . . . , . in sino-auricu/ar junction [Crescent] Sympathetic fibres- dotted lines Fig. 68. Diagram to show the innervation of the heart in the frog or turtle. The electrodes are represented as applied to the white creseentic line where they will stimulate some postganglionic libers. (From Jackson.) THE CONTROL OF THE CIRCULATION 229 Several possibilities have to be kept in mind when we attempt to determine the exciting cause for an observed change in the heart rate in man. Thus, a slowing of the rate may be due to mechanical stimulation of the vagus trunk, as in pressure on the nerves by a tumor or aneurism in the neck. That such mechanical irritation may stimulate the vagus is easily demonstrated in many individuals by applying pressure to the vagus where it lies in the neck in front of the sixth cervical vertebra. Such pressure sometimes produces so profound an inhibition of the heart that temporary loss of consciousness occurs. It is often an unsafe ex- periment to perform. Change in the heart rate in man may be caused by direct stimulation of the vagus center, as by the presence of a tumor or a blood clot in the medulla, or by the action on the center of some unusual hormone in the blood. A general increase in intracranial pressure also stimulates the vagus center. The slowing of the heart which occurs in asphyxia might be due either to the action of hormones (hydrogen-ion concentration) in the blood as the result of the asphyxia, or to the increased intra- cranial pressure. That the latter is the more important cause is shown by the fact that, if the rise in blood pressure is prevented by connecting an artery with a mercury valve, that is, with a tube dipping into a cylinder of mercury to a depth corresponding to the normal blood pressure, so that when the pressure tends to rise the blood escapes, the slowing of the heart is not observed. The excitability of the afferent vagus fibers in the lungs is greatly increased during the earlier stage of chloroform administration. Finally it should be pointed out that, although we have no voluntary control of the activity of the vagus center, its activities are subject to great variation as a result of impulses transmitted from centers higher up in the cerebrospinal axis. It is by the operation on the vagus center of such impulses that changes in heart rate occur during emotional ex- citement, fright, etc. The increased heart rate in muscular exercise is probably dependent upon a number of causes, such as the irradiation of the motor impulses on to the cardiac centers (see page 430), the rise in temperature and changes in the hydrogen-ion concentration of the blood, etc. Mechanism of Action of Vagus on the Heart. Physiologists have nat- urally been curious as to the exact manner in which the vagus nerve brings about inhibition of heart action. Similar inhibition as a result of stimulation of efferent nerves exists in the case of the dilator fibers to the blood vessels (page 239) and the sympathetic nerve to the intes- tine (page 501). Inhibition of voluntary muscles can be produced only through the central nervous system by stimulation of afferent nerves 230 THE CIRCULATION OF THE BLOOD (page 915). It is not the nerve fibers themselves that are responsible for the inhibitory effect, for it has been found that if the peripheral end of a cut vagus nerve is connected with the central end of one of the anterior roots of the cervical portion of the spinal cord, the axons of the latter when they grow down into the vagus trunk during the regeneration which follows, stimulation of the regenerated fibers will still produce inhibition of the heart. The nature of the fibers can not therefore be the factor upon which the inhibiting influence of the vagus is dependent. This leaves the terminal apparatus of the vagus fibers in the heart as the structures in which the stimulus conveyed to them is rendered inhibitory in nature. There has been considerable speculation as to what kind of change must be occurring in the heart in order to cause the inhibition, but practically nothing that is definite is known. One significant fact, how- ever, is that the electrical current led off through nonpolarizable elec- trodes from two portions of the auricle one of which is injured, does not take the same direction when the vagus nerve is stimulated as that which it takes when the motor nerve of a similarly observed muscle is stimu- lated. A positive, instead of a negative variation is observed. Now, since a negative variation is always accompanied by active chemical breakdown changes occurring in the muscle to supply its energy of contraction, it is assumed that the positive variation accompanying stim- ulation of the vagus must indicate that, instead of a katabolic process, a building up, or anabolic process, is being excited. This conclusion would fit in perfectly with the well-known fact that, after the heart has been held in standstill for some time by vagus stimulation, the beats are stronger after the inhibition has passed off than they were before. The vagus seems to have a conserving influence on the heart. During the inhibition produced by it energy material is apparently stored up in the heart, so that when the beat is reestablished it is stronger than before. The Manner of Termination of the Vagus Fibers in the Heart. This subject is of considerable pharmacological and therefore therapeutic in- terest. In approaching the problem it must be remembered that the vagus fibers belong to the so-called bulbar autonomic system of nerves (see page 894). They are therefore fibers which have cell stations situ- ated near their peripheral termination cell stations, that is to say, in which ganglionic medullated fibers, by forming synapses around nerve cells, become connected with postganglionic nonmedullated fibers. The existence of ganglia in the heart, particularly of the frog, has been known for a long time. These ganglia are located at the sinoauricular junction, at the interauricular septum, and in the ventricle near the THE CONTROL OF THE CIRCULATION 231 auriculoventricular junction. The function of the ganglia is to serve as cell stations on the course of the vagus nerves. (Fig. 68.)* Nicotine is a drug which in certain concentrations, if applied locally to sympathetic ganglia, specifically paralyzes the synapses between the ends of the preganglionic fibefs and the cells from which the prost- ganglionic fibers arise. If this drug is applied in a 1 per cent solution to the heart, stimulation of the vagus trunk no longer produces inhibi- Fig. 69. Frog heart tracing showing the action of nicotine. The vagus trunk was stimulated as indicated. In the normal (lower) tracing inhibition occurs hut after nicotine (second tracing) no inhibition follows. Stimulation of the crescent in the next two lines still is followed by inhibi- tion. The final effects of the drug are shown in the last two (upper) tracings. (From Jackson.) tion, but if the stimulus is applied to a portion of the heart known as the white crescentic line, inhibition still occurs, because at this point the postganglionic nerve fibers come near to the surface and therefore are stimulated (Fig. 69). On the other hand, atropine is a drug which paralyzes the postganglionic fibers, so that after its application to the heart inhibition cannot be produced by stimulating either the vagus 'The reader is referred to (lie chapter dealing with the autonomic nervous system for a de- scription of the relationships of the fibers and ganglia. 232 THE CIRCULATION OF THE BLOOD trunk or the white crescentic line. Pilocarpine and muscarine are drugs which have an action exactly opposite or antagonistic to that of atro- pine; that is, they stimulate the postganglionic fibers and produce a slowing and possibly an enfeebling of the beat. In the mammalian heart a large number of the fibers in the right vagus nerve proceed directly to the sinoauricular node, where it can be shown histologically that considerable masses of nervous tissue exist. On the other hand, the great majority of the fibers in the left vagus proceed to the auriculoventricular bundle, in which also nervous struc- tures are abundant (page 183). As already indicated, the experimental results which follow stimulation of either nerve can be explained by the influence which the nerve exerts on the particular structure to which the majority of its fibers proceed. In brief, stimulation of the right vagus is likely to produce slowing and weakening of the beat, whereas stimulation of the left vagus is more likely to institute a condition of partial heart-block. On account of the different results which may be obtained by stimu- lating the vagus, some authorities have assumed that the heart must contain four kinds of fiber, more strictly, of vagus nerve endings, one for each kind of influence which the vagus can develop. These four influ- ences are, it will be remembered, on the strength, the rate and the propagation of the heartbeat, and the excitability of the cardiac muscle. It is, however, almost certainly unnecessary to make such an assump- tion, for the results can be explained as merely dependent upon dif- ferent degrees of stimulation of the same kind of fiber and upon the exact part of the heart to which the fiber runs. Sometimes, for ex- ample, when the right vagus nerve is stimulated very feebty, there may be only a diminution in the force of the beats without any change in their rate, indicating that the effect has been upon the musculature of the auricular walls and not on the sinoauricular node. If the stimulus is increased a little, then both an enfeebling and a slowing of beat occur, indicating that the stimulus has now passed both to the auricular mus- culature directly and to the sinoauricular node. The Sympathetic Control The effect of the sympathetic nerve on the heart may be described as being exactly opposite to that of the vagus. The pathway along which the fibers of this nerve travel to the heart is more or less a devious one. They arise in the mammal from nerve cells in the gray matter in the upper thoracic portion of the spinal cord. The fibers leave by the cor- responding spinal roots and pass by the white rami communicantes into the sympathetic chain, up which they travel to the stellate and inferior Medulla oblongata Cervical 1- Accessory n.~ +o trapezi Spinal medulla- (cord) Kami communican- tes going to Symp. gang, (preganglionic) Ansa subclavia- (Annulus of Vieussens)r "Thoracic 3 nerves ^ N.I Postgsnglionic fibers are dotted thus N.IX ^j-Jugular ganglion (dang, of the root) A iX- Depressor (Fall in pressure or slowing of heart.) (Sensory) Separate nerve in rabbit and opossum. Nodosum ganglion (Gang, of the trunk) ( Hunf * ^Inhibitory cranial autonomic fibers Harrington) -Superior cervical ganglion -Descending sympathetic fibers in cord ^Electrodes (slowing or stoppage of Subclavian heart. Augmentation in some animals.) Aortic arch -Cervical va Fig. 79. Capillaries from abdominal wall of guinea pigs after injection of india ink. The black particles are absent from the corpuscles which will be seen to be distorted in shape. Note that in a transverse section of a capillary the corpuscles may become folded. (Krogh.) come altered to meet the varying demands. Indeed Krogh has shown by mathematical calculations based on: (1) the depth of actual muscle tissue which each capillary supplies, (2) the rate of oxygen consumption and (3) the diffusion rate of oxygen through tissues that the oxygen pressure necessary to supply the muscle fibers is remarkably small, even during the heaviest muscular work. This means that the oxygen pressure in the muscular tissue must at all times be practically the same as that of the venous blood, and that the call for oxygen by the tissues is readily met by diffusion from the capillary blood. That a measurable pressure of oxygen in the tissues is difficult to demonstrate does not contradict this conclusion THE CONTROL OP THE CIRCULATION 253 because the oxygen is so rapidly used up. In lirine, where this is not the case, the pressure of 2 is about the same as in the venous blood, and when a neutral gas is placed in the pleural or peritoneal cavities, it ultimately becomes mixed with oxygen up to 3-4 per cent. (Tobiesen cf. Krogh.) Still more definite proofs of the ability of capillaries to dilate inde- pendently of changes in the corresponding arterioles have been obtained by Krogh and by Hooker. co Krogh found that irritation of the ventral aspect of the frog's tongue, as by punctate stimulation with a hair, caused, after a brief latent period, a marked vasodilatation and increased blood- flow. This dilatation was seen by the microscope sometimes to affect both the capillaries and small arterioles over a considerable area. That the former had been actively dilated, and not merely passively distended by greater inflow of blood from the arterioles, was shown by the fact that sometimes it was possible to restrict the irritation so that only a capillary, or even a short piece of one, dilated. In such cases the dilatation often started at the venous end of the capillary and spread towards the arterial end. This made it appear as if the blood were flowing towards the heart. After clamping the lingual artery so as to slow down the circulation, irri- tation caused the capillaries to dilate without any increased flow of blood through them, this occurring, however, immediately the clamp was re- moved. Urethane or epinephrine, applied locally, dilated the capillaries, but not the arterioles. After cocainization or degeneration of the nerves, the dilatation in- stead of being diffuse was then more localized; merely cutting the nerves, however, had no effect. This indicates that a local nerve axon reflex, similar to that described on page 898, is concerned in the spread. There must be sensory nerve fibers on the walls of the smallest vessels and the impulses which are set up by their stimulation must travel up the nerve to a collateral, down which they then pass to neighboring parts of the vessel as dilator impulses. Hooker has also shown that stimulation of the cervical sympathetic in the cat causes the capillaries around the hair follicles of the ear to exhibit movements which are inde- pendent of changes in the arterioles. These discoveries explain the association of local hyperemia with cyanosis (the blood is flow- ing so slowly in dilated capillaries that its oxygen is used up), the pathogenesis of shock (page 307), the initial stages of inflamma- tion, and it is altogether likely that they will before long lead us to an explanation of the causes for clinical hypertension. It is possible that examination of the capillary circulation in the skin at the base of the finger nails by Lombard's method 62 * would be of considerable value in the differentiation of cases of hypertension. *A drop of glycerine is placed on the skin at the base of the nail, with the hand firmly supported on a rest, and a strong light thrown on it. An objective magnifying up to 35 times is then focussed until the capillary loops are seen. CHAPTER XXIX PECULIARITIES OF BLOOD SUPPLY IN CERTAIN VISCERA Up to the present we have been considering the circulation of the blood from a general point of view. There are certain organs and tissues, how- ever, in which the general mechanism is altered in order to meet pecu- liar requirements of blood supply. Thus, it is evident that the brain, incased as it is in the rigid cranium, will be unable to contract and expand as a result of vasoconstriction or vasodilation. On the other hand, we know that the blood supply to this organ does vary con- siderably from time to time. What is the nature of the mechanism by which such changes are brought about? In the case of the liver the cir- culation is peculiar on account of the fact that blood is carried to the organ by two vessels, in one of which it is supplied under high pressure and in the other, under low pressure. We must investigate the rela- tionship of these two sources of blood supply. The circulation through the coronary and pulmonary vessels must likewise receive special atten- tion on account of the highly specialized functions of these organs. THE CIRCULATION IN THE BRAIN Anatomical Peculiarities Serious curtailment of the blood supply to the brain is guarded against by the existence of the circle of Willis. Besides the four main arteries the vertebrals and the two carotids the spinal arteries contribute to the blood supply of the circle, and consequently in certain animals, such as the dog, the four main arteries may be ligated without causing death. In man, however, ligation of both carotids is usually fatal. The free anastomosis displayed in the circle of Willis is not maintained in the case of the arteries which run from it to supply the brain structure. On the contrary, these vessels are more or less terminal in character; that is to say, the capillary systems of the different vessels do not freely anasto- mose, so that obstruction of one vessel, or an important branch, is followed by death of the supplied area. The vessels which go to the pia mater, how- ever, break up into numerous smaller branches, which freely anastomose before entering the brain tissue. 254 PECULIARITIES OF BLOOD SUPPLY IN CERTAIN VISCERA 255 The venous blood is collected by the small, very thin-walled and valve- less cerebral veins. These run together to form larger veins dis- charging into the sinuses, the openings into which are kept patent by the arrangement of dura mater around the orifices. The sinuses exist between the dura and skull and are so constructed that they can not be compressed, particularly those at the base of the brain. From them the blood is conveyed mainly to the internal jugular vein, some of it however escaping by the anastomoses existing between the cavernous sinus and the opththalmic veins, and by the venous plexus of the spinal cord. The most striking peculiarities of the veins are their patulous con- dition and the absence of valves, so that any change in the blood pres- sure in the internal jugular vein must be immediately reflected in that of the venous sinuses. This explains why compression of the abdomen causes venous blood to flow from an opening made in the longitudinal sinus. In considering the cerebral circulation, another factor that must be borne in mind is the presence of cerebrospinal fluid. This is contained in the subarachnoid spaces of the brain and spinal cord, these spaces, in the case of the brain, being often considerably enlarged to form the cisternae. The cerebrospinal fluid is also present in the ventricles of the brain, which it will be remembered communicate with the subarachnoid spaces through the foramen of Magendie, etc. There is free communication between the cerebral and spinal portions of the fluid, as is evidenced by the fact that blood clots appear in the spinal fluid (collected by spinal puncture) when there is hemorrhage into the ventricles of the brain. It is unlikely that the cerebrospinal fluid is of much importance in connection with the control of the blood supply to the brain tissue. It may be merely a lubricating fluid; at least it is so small in amount (60 to 80 c.c. in man) as to be apparently of little value in bringing about an alteration in brain volume. Although normally so scanty, its secretion can become re- markably stimulated under certain conditions as in fractures of the base of the skull. Under these conditions in man, it may drain away at the rate of about J3QO c.c. a day or more. In cerebrospinal rhinorrhea as much as 700 c.c. of cerebrospinal fluid may run away in a day. The fluid is apparently produced in the choroid plexus, for when the pathways by which the ventricles communicate with the subarachnoid space are obstructed it collects in the ventricles, producing internal hy- drocephalus. Under certain conditions absorption is also very rapid, as shown experimentally by the rate with which physiological saline is absorbed when it is injected into the subarachnoid space. This absorp- tion is believed to occur through various pathways, the most important of which is, directly into the blood stream, through the capillaries lining 256 THE CIRCULATION OF THE BLOOD the subarachnoid spaces of the brain and cord. There is no evidence to support the view that absorption occurs through the Pacchionian bodies. A smaller degree of absorption occurs by way of the lymphatics. The cerebrospinal fluid is believed to undergo a slow circulation from the ventricles through the foramen of Magendie into the subarachnoid spaces of the spinal cord down which it travels on the posterior aspect, and then ascends on the anterior aspect where the greater part of its ab- sorption occurs. The biochemical properties of this important fluid will be found described elsewhere (page 121). Important papers on its pro- duction are those of Becht 63 who believes that this depends solely on alterations in venous and arterial pressure in the skull and that none is formed by secretion. Physical Conditions of the Intracranial Circulation Considered from a physical standpoint the circulation through the brain has been recognized for long to be unique in comparison with that of any other organ or tissue in the body with the exception of the bone marrow. Encased in the rigid cranium, the volume of the brain can not, like that of other vascular areas, expand and contract in proportion to changes in the blood supply; neither can the caliber of its blood vessels become altered, unless some special mechanism may exist whereby a part of the crainal contents is quickly expelled from and aspirated into the rigid case. In a general way, the physical conditions of the intracra- nial circulation are similar to those existing in a flask full of water and pro- vided with a thin-walled rubber tube suspended in the water with its free ends connected with glass tubes passing through the stopper of the flask. If fluid be made to circulate through the tubing, no change in the caliber can be produced by altering the pressure of inflow; but the rate of dis- charge from the other end of the tube will be proportionate to the pres- sure. Although the tubing itself is readily distensible and elastic, these properties are entirely annulled by the incompressible fluid in which the tube is suspended. If any expansion or contraction of the tubing as a whole is to occur, provision must be made for changes in the volume of fluid in the flask, by inserting in the stopper a third tube connected with an overflow flask, and in applying this second model to represent the circulatory conditions as they exist in the brain, the question arises as to whether the cerebrospinal fluid which lies in the large subarachnoid spaces at the base of the brain and in the ventricles, by communicating through the foramen of Magendie with the spaces surrounding the spinal cord, may not be capable of functioning as the overflow fluid. This is at least conceivable, especially when one bears in mind that some outflow PECULIARITIES OF BLOOD SUPPLY IN CERTAIN VISCERA 257 is also possible along the sheaths of certain of the cranial nerves. He- cent investigation has, however, clearly demonstrated that under nor- mal conditions the amount of cerebrospinal fluid is too limited to make it of any significance in this connection. As explained on page 255 the main function of the fluid is to equalize the pressure between the brain and the cord. Although it is therefore improbable that the vessels as a whole could expand or contract, it is still possible that some provision might exist by which extra room could be made to allow of localized dilatation of certain parts of the vessels. The veins, for example, might contract in proportion as the arteries dilated and the possibility becomes all the more likely when we consider that because of the great capacity of the cerebral veins, their lumina might be considerably constricted without any serious obstruction being offered to the bloodflow through them. Such a reciprocal dilatation and constriction of the proximal and dis- tal halves of a thin-walled rubber tube suspended in water in a closed flask can be demonstrated provided some resistance be inserted between the two halves. This resistance would be represented in the intracra- nial vessels by the capillary area. It is impossible to say to what extent this reciprocal mechanism between arteries and veins may prevail, but in any case it can not well extend beyond the cerebral veins to the sinuses, since these are partly embedded in the cranium itself and are protected by relatively thick membranes on their free sides. The mech- anism may be employed for permitting the arteries of a local area to expand, but it can not obtain over any large area, since otherwise the total outflow of blood from the sinuses through the jugular foramen would be curtailed, which we know to be contrary to what actually oc- curs when the arterial pressure is raised, and which moreover would be highly detrimental, since it would cause self-strangulation of the intra- cranial bloodflow. These physical considerations lead us to expect that there can not be any dilatation or constriction in the intracranial vessels which is com- parable with that which occurs in other vascular areas, although it may take place to a degree which is limited by the extent to which the cere- bral veins can be passively contracted or expanded without curtailment of the bloodflow. Acting to this extent, the dilatation produced in the arteries by each cardiac systole accounts for the rise in pressure which occurs simultaneously in the venous sinuses (as measured in the torcu- lar Herophili), but it is unlikely that the amount of blood supplying the brain will be determined by local dilatation or constriction of the blood vessels, as is the case, for example, in a gland or muscle. Of this we are certain, that the total volume of blood within the brain case at any 258 THE CIRCULATION OF THE BLOOD given moment can undergo no considerable change. Provision for more or less blood must therefore be afforded by changes in the velocity of now. Physiological Conditions of the Intracranial Circulation We must now proceed to test these hypotheses by physiological ex- periment, for, if they are found to apply to the intracranial circulation, the conclusion becomes inevitable that changes in the total blood supply to this, the most important organ in the body, are dependent not on any local adjusting mechanism in that organ itself, but upon conditions pre- vailing in other parts of the body, with the possibility that a local vaso- dilatation may be provided for by a secondary compression of neigh- boring venules, or perhaps even by an active constriction of the arteri- oles of neighboring inactive centers. The questions of greatest practical importance are, therefore, as fol- lows: (1) What determines the intracranial pressure, and how does this vary during each heart beat? (2) If there can be no change in the actual volume of blood in the vessels as a whole, what provision is made to cause changes in blood supply with varying degrees of activity of the brain, and how are these changes brought about? (3) Is it possible with- out change in the total volume of blood in the brain for certain vascular areas to expand at the expense of others that correspondingly constrict? The Pulsations of the Brain and the Cause of Intracranial Pressure. Examination either of the fontanelles in an infant or of the surface of the brain exposed by trephining shows distinct pulsations, but this does not prove that similar pulsations occur in the intact brain, for the ab- se.nce of a part of the cranial wall might be responsible for the pulsa- tion. The presence or absence of pulsation must be sought for in the still rigid brain case. This has been done by closing a trephine hole by a glass window through which the cranial contents can be seen when strong illumination is used : pulsations of the vessels are clearly visible. To determine the exact relationships of the pulsations, the trephine hole is connected with a delicate recording tambour by screwing into it a brass tube closed at its inner end by a thin rubber membrane. It has been found that the arteries expand somewhat with each cardiac sys- tole, and that there are further expansions with each expiration, but not with inspiration, as is the case in other vascular areas. The room for the expansions is no doubt provided mainly by compression of the cerebral veins, thus causing the blood within them to exhibit corres- ponding waves of pressure. The reason why expiration and not in- spiration causes the increase in volume is that there are no efficient valves between the right side of the heart and the cerebral veins. This allows PECULIARITIES OF BLOOD SUPPLY IN CERTAIN VISCERA 259 the expiratory rise in venous pressure which is well known to occur in the former to be directly transmitted to the brain. This brings us to the second part of our first question : What determines the intracranial pressure ? To answer it we must know something of the method by which the pressure is measured. This has been most successfully done by Leonard Hill, 19 who devised an instrument called the cerebral pressure gauge, consisting of a brass tube closed at one end by rubber membrane and screwed into a trephine hole. The outer end of the tube is joined to a narrow glass tube connected with a pressure bottle. The whole system is filled with fluid except for a minute bubble of air in the narrow glass tube. Any changes in pressure in the brain cause correspond- ing movements of the bubble, and the magnitude of the change is meas- ured by readjusting the pressure bottle so as to bring the bubble back to its original level. It has been found that the pressure may vary from zero to 50 mm. Hg. (as in strychnine convulsions), and that these variations depend entirely on circulatory conditions, there being no compensatory mechanism by which the pressure is kept constant. The average pressure under physiological conditions is 100-130 mm. H 2 0. The intracranial pressure varies directly with the venous pressure within the skull, and it passively follows changes in the pressures in the arteries and veins of the systemic circulation. This implies that the efficiency of the cerebral circulation will be dependent very largely upon alterations in the capacity of the splanchnic area, the greatest reservoir of blood in the body. By actual measurement it has also been found that: 1. The pressure within the lateral sinuses of the brain (measured by connecting a tube and manometer with the torcular Herophili) varies absolutely with the intracranial pressure. It therefore exhibits pulsa- tions which mirror precisely those observed in the cerebral pressure gauge. 2. Both these pressures passively follow changes in the pressure in the right auricle. They also run more or less parallel with changes in arterial pressure, and there is never any change in either of them which can not be traced to some general circulatory condition. A few of the many experiments performed by Leonard Hill and others will serve to prove these far-reaching conclusions: 1. In asphyxia caused by cessation of the respiratory movements in a curarized animal, the cerebral venous pressure at first falls with the fall in systemic pressure and then rises as the arterial hypertension sets in. In the last stage, however, al- though the arterial pressure is quickly falling, the venous pressure rises and with it the cerebral venous pressure. During vagus inhibition the fall in arterial pressure is so marked that the intracranial pressures fall at first and only rise later, correspond- ing to the rise in venous pressure (Fig 80). 2. During administration of ether, alterations in cerebral pressure become marked only when there is extensive muscular movement or hyperpnea. Chloroform, on the 260 THE CIRCULATION OF THE BLOOD other hand, by acting more directly on the heart so as to produce a fall in arterial and a rise in venous pressure, causes at first a decided rise in cerebral pressure and later a fall following the development of decided arterial hypotension. 3. Amyl nitrite, injected into the jugular vein, causes at first a rise in venous pressure and therefore in cerebral pressure. Later, however, marked arterial hypo- tension develops, and the intracranial pressure declines. Fig. 80. To show simultaneous records of the arterial blood pressure (A), the venous pres- sure (B), the intracranial pressure (C), the pressure in the venous sinuses (>). The fall in ar- terial pressure produced by stimulation of the peripheral end of the vagus will be found to cause a fall of intracranial and cerebral venous pressure, accompanying that in the arteries, but a rise in that of the venous system. (From Leonard Hill.) 4. During epileptic fits induced experimentally by excitation of the cortex, there is a rise in venous pressure and correspondingly in intracranial pressure. In the more violent convulsions produced by absinthe, there is very little change in systemic venous pressure while the arterial pressure shows extreme variations, with which the intra- cranial pressure runs parallel. With adrenalin, where both arterial and venous sys- temic pressures rise enormously, there is of course a great rise in intracranial pressure PECULIARITIES OF BLOOD SUPPLY IN CERTAIN VISCERA 261 and there is never any local change in the latter which would indicate that this potent drug had locally caused these vessels to constrict. 5. The alterations in systemic pressure induced by the operation of the force of gravity and coming into play when the position of the body is changed, if not per- fectly compensated for by constriction of the splanchnic area, will cause correspond 5 ing changes in the intracranial tensions. Under the influence of gravity, for example, the intracranial and the intracranial-venous pressures may fall below zero. The comparatively slight amount of extra room which can be provided in the cranial cavity by compression of the venules and capillaries has suggested to some writers that a self -strangulation of bloodflow might occur when the pressure suddenly rises in the basal and cerebral arteries. The increased pressure would be transmitted undiminished through the incompressible brain substance to the thin-walled vessels and compress them because of the lower pressure within. This is, however, impossible for any curtailment in the bloodflow through the venules and capillaries could only be transitory, since the compression will be overcome by the arrival of the pressure wave through the blood stream itself. For it is obvious that the arterial pressure trans- mitted directly must be greater than that pressure after it has overcome the tension of the arterial wall and is transmitted to the venules through the brain substance. Whenever this readjustment has occurred, the cerebral vessels become expanded to the greatest extent possible and they become virtually rigid tubes comparable with the rubber tube suspended in water in a closed flask, as in the schema referred to above. The only variation in intracranial blood supply which can occur is one affecting the velocity of flow or if you prefer the term the mass movement of the blood; the volume can not change. After all, however, that is what is necessary to meet the demands for more blood, and the conceptions which have been formed by studies on expansible vascular areas, such as the kidney and spleen, that increased blood supply runs parallel with increased volume, do not apply. That the mass movement of the blood in the cranium increases when the arterial pressure rises has been shown by direct experiment. Hill and Nabarro found it increased from two to six times during the con- vulsions produced by absinthe. Local Readjustments of Blood Supply in Different Parts of the Brain. Limited though any change in caliber of the cerebral arteries can be, it is nevertheless sufficient to make it possible that local variations in blood supply might occur as a result of active constriction or dilatation of the vessels. Just as the blood supply of a muscle or gland may be varied independently of any change in general blood pressure, by local changes in the caliber of its blood vessels, so might that of the brain be varied, and this might occur to a limited extent for the supply as a whole, as by constriction of the circle of Willis, or to a greater extent in one or other of the arteries which spring from the circle. By the latter adjust- ment a greater blood supply might be directed into an area which had become especially active, the flow to other relatively quiescent areas being meanwhile somewhat curtailed. 262 THE CIRCULATION OF THE BLOOD Vasomotor Nerves, These possibilities raise the question as to whether there are functionally active vasomotor nerves to the cerebral vessels. Histologists have definitely demonstrated nerve fibers running on to the cerebral vessels, especially by the use of the intra vitam methylene blue method of staining (Huber, Hunter, etc.), but this does not of course necessarily indicate that the fibers normally cause the arterial walls to expand and contract. The only basis upon which such a claim could be put forth is an actual demonstration of changes in intracranial blood- flow occurring independently of changes in systemic arterial or venous pressures. Leonard Hill and Bayliss, and later Leonard Hill and Macleod, 20 have most diligently sought for such evidence, but with entirely negative results. Ee cords were taken of the intracranial pressure, the cerebral venous pressure and the pressure in the circle of Willis (by a cannula inserted in the peripheral end of the internal carotid artery), as well as the arterial and venous pressures in the systemic vessels carotid and jugular). Since any vasomotor fibers must presumably be derived from the vasomotor centers, and since these fibers must gain the cerebral vessels through the stellate ganglion and ultimately travel into the cranial cavity along the outer coats of the arteries, the above pressures were simultaneously observed before and during electrical stimulation at these places. It was found that any change that did occur could in- variably be attributed to changes in the circulation as a whole ; there was never any alteration in pressure locally in the brain for which the occurrence of local constriction or dilatation of the vessels had to be assumed. Other observers have attempted to investigate the problem by meas- urement of the volume of blood leaving the brain, but with similarly negative results. But an objection can be raised to these experiments on the ground that there might be feebly acting vasomotor influences, the effect of which would become entirely masked by the much more potent influence exerted on the bloodflow by changes in the circulation as a whole. As pointed out by Wiggers, the only way by which local changes in the bloodflow through the intracranial vessels can be expected to reveal themselves is by measuring the entire outflow, a measurement which, however, it is im- possible to make in an intact animal on account of the many pathways through which the venous blood can leave the skull. Measurement of the outflow by one of them does not by any means indicate the magnitude of total outflow. To overcome these difficulties, Wiggers proceeded to measure the outflow from all the cranial vessels of oxygenated Locke's solution perfused into the cerebral artries under constant pressure. It was found that the otherwise constant rate of outflow became decidedly curtailed when adrenalin was added to the Locke's solution. If we assume that this drug acts only on arterial muscle having functionally active vasoconstrictor nerves, then the result would prove the presence of such fibers to the cerebral vessels, but even granted this, PECULIARITIES OF BLOOD SUPPLY IN CERTAIN VISCERA 263 the result does not warrant the conclusion that, under normal conditions in the intact snimal, such fibers display any activity. Wiggers does not claim that .his results prove that a local vasomotor mechanism is important, but thinks that "they are favor- able to the view that cerebral vasoconstrictor nerves are present.*' Intracranial Pressure One word more with regard to what is known as intracranial pressure, that is, the pressure in the space between the skull and the brain. Under ordinary conditions it must be equal to that in the cerebral capil- laries, and may be measured by connecting a sensitive manometer with a tube screwed into the cranium as described above. It has been found to vary from mm. Hg in a man standing erect to 50-60 mm. Hg in a dog poisoned by strychnine. It becomes increased, not only by com- pression of the veins of the neck and by an increase in general arterial pressure, but also in pathological conditions, such as hydrocephalus. A new growth in the brain, if it occupies more space than the tissue which is destroyed, exerts pressure on all parts of that region of the cranial cavity, but this pressure may not be transmitted equally throughout the cranial contents, for the falciform ligaments and the tentorium sup- port a part of it, thus directing the spread of pressure along certain pathways. The structures at the base of the brain, the optic nerves, the veins of Galen and the Sylvian aqueduct are most affected in this way. If the pressure is rapidly applied, however, it may rise through- out the cranial contents. In such cases the pressure is, of course, cir- culatory in origin, since immediately after death from cerebral tumor the intracranial pressure is not found to be raised. The major symptoms of cerebral compression are no doubt due to anejnia of the medulla oblongata, which may be the result either of pressure applied locally in the bulbar region, where the presence of a very small foreign body or only trivial tumor formation is sufficient to destroy life, or of pressure transmitted from the cerebral cavity, in which case, on account of the support offered by the tentorium, a much larger growth is required to affect the medulla. Internal hydrocephalus produced by blocking of the aqueduct of Sylvius and the veins of Galen causes the greatest rise in intracranial tension, and may affect the me- dulla, because the brain is driven downwards so as to pinch the bulb against the occipital bone. It must be emphasized that it is not the pressure per se that causes the symptoms, but the attendant anemia, the symptoms of acute cerebral anemia and of compression being iden- tical (Leonard Hill 19 ). To relieve the compression, trephining is the common practice. The trephine hole should be as large and as near to the source of compression (tumor, etc.) as possible. 264 THE CIRCULATION OF THE BLOOD CIRCULATION THROUGH THE LUNGS The pulmonary or lesser circulation, as it is called, is quite different from the systemic circulation. In the first place, the pressure in the pul- monary arteries does not amount to more than about 20 mm. Hg, or about one-sixth of that of the systemic arteries, so that the peripHeTat resistance in the blood vessels of the lungs is much less than that of the body in general. This lower resistance is owing partly to the large diameter of the arterioles and the small amount of muscular fibers in their walls, and partly to the fact that the capillaries are held con- stantly in a somewhat dilated condition on account of the subatmos- pheric pressure in the thorax (see page 323). Another peculiarity of the pulmonary circulation is that the caliber of the vessels is to a very large extent dependent upon the changes that occur in the iTit.rai.Tim^^jft_jrrfissLUJ!fi with eagli inspiration and ex- piration. They become Dilated on inspiration and contracted on ex- piration. The extent to which these respiratory changes affect the amount of blood contained in the lungs, is very considerable. At the height of inspiration it is computed that a little more than eight per cent of the whole blood in the body is contained in the lungs, whereas on expiration it diminishes to between five and seven per cent. A third peculiarity is that although nerve fibers have been seen run- ning to the w T alls of the pulmonic blood vessels no changes in their caliber can be demonstrated, in mammals, by stimulation of the vagus or sympa- thetic. In the frog, however, constriction of the pulmonary artery occurs when the vagus is stimulated (Carlson and Luckhardt). When the pul- monary vessels are perfused and the outflow measured, a diminution in the latter is said to occur when epinephrine is added to the injection fluid. In short, the conclusion which we must draw is much the same as that for the blood vessels of the brain namely, that although we must admit that a vasomotor supply may be present, yet it is one which is so feeble that it is " readily overpowered by changes in the * * * dis- charge of the right heart or by back pressure effects from the systemic side of the circulation" (Wiggers 57 ). When the venous inflow to the right heart is increased, the discharge into the pulmonary artery correspondingly increases, in obedience to the law of the heart (page 216) and a certain degree of congestion of the fpulmonary vessels results. But when this becomes sufficient to cause an [effect on the blood flow in the pulmonary veins, the left ventricle im- I mediately also responds by augmented output so that the engorgement I in the pulmonary circuit does not become excessive. It has indeed been I thought by some that the left ventricle is so much more sensitive to changes in venous inflow than is the right that it succeeds in preventing PECULIARITIES IN BLOOD SUPPLY IN CERTAIN VISCERA 265 any pulmonary congestion under the above conditions. When moderate or slowly increasing resistance is offered to the systolic discharge from the left ventricle, no back pressure effects develop in the pulmonary veins (again in obedience to "the law of the heart"), but if the aortic resistance becomes so high that the ventricle dilates beyond its physiological capac- ity and fails to discharge its normal volume, then pulmonary venous con- gestion develops. But it may not be till much later that the "back pressure" shows itself in the pulmonary artery and this has been ingeni- ously explained as being due to a diminution of the capacity of the right ventricle, and therefore of the discharge of blood from it, because of bulging of the intraventricular septum. When the above pressure differ- ences become persistent, and especially when the myocardium begins to break down, various pathological disturbances of the circulation (cardiac decompensation) supervene as described in the various textbooks on clinical medicine. CIRCULATION THROUGH THE LIVER The liver is the only gland in the body receiving both venous and arterial blood, the former being supplied to it at a very low pressure by way of the capacious portal vein, and the latter at very high pressure by the strikingly narrow hepatic artery. Except for the relatively small amount of blood which is supplied to the walls of the blood vessels and the biliary ducts, none of the hepatic artery blood mixes with that of the portal vein until the vessels enter the hepatic lobules. Beyond this point the two blood streams mix and the combined stream is drained away by the sublobular and hepatic veins. Methods of Investigation To study the relative importance of these tAvo sources of blood supply, and also to .nvestigate th2 in alveolar air. The person expires forcibly through the tube with the stopcocks as in I. A is closed and the tube placed in water to cool the air, after which B is turned as in II. The entrapped column of air equals 100 c.c. A solution of caustic alkali is now sucked into C with stopcocks as in II. B is then turned as in I but with A still closed, and the alkali solution allowed to enter b, after which B is turned off, the excess of alkali solution in C allowed to run. out and the burette shaken. The burette is then submersed up to a in a cylinder of water, with B as in III. After allowing for cooling, the level at which the water stands gives the per cent of CO^. 20 $9 in, inspired air Co* 130 to 50 30 Fig. 126. Curves to show the relationship between the O 2 and CO 2 tensions in alveolar air (dotted lines) and arterial blood (continuous lines). It will be observed that the tension of CO 2 in blood is slightly above that in alveolar air, but that the reverse relationship obtains for O 2 . In the upper part of the curve the Q-> tension in the alveolar air was experimentally altered, causing a corresponding alteration in the 6 2 tension of the blood. ' This result is of practical significance in connection with O 2 alterations in gas poisoning, pneumonia, etc. (From A. and M. Krogh.) THE CONTROL OF THE RESPIRATION 359 oratory and in clinical work, a special chapter has been devoted to the subject, giving in detail the more recent methods devised by K. G. Pearce. Lastly, it should be noted that several observers believe that a more reliable estimate of the alveolar tension of C0 2 (and of 2 ) can be made by analyzing a sample of ordinary expired air and calculating the per- centages of C0 2 and 2 in the alveolar air by allowing a constant dead- space capacity of 140 c.c. (Krogh, etc.). If we compare the C0 2 tension of arterial blood, as measured by the Krogh method, with that of alveolar air, we shall find that there is a remarkable correspondence, indicating, therefore, that, when the arterial 44 % Cox ia it sptred 220 Fig. 127. Same as Fig. 126, except that in this case the tension of CO 2 in the alveolar air was experimentally altered. (From A. and M. Krogh.) blood leaves the alveoli, its partial pressure or tension of C0 2 is exactly equal to that in the alveolar air. This is shown in the accompanying curves of experiments performed by Krogh. The dotted line in these curves represents the tension of C0 2 or 2 in alveolar air, and the con- tinuous line, these tensions in arterial blood. Close correspondence will be observed between the C0 2 curves even when sudden changes in alveolar C0 2 were induced by artificial means. In the case of the 2 tensions, however, that of the blood is always lower than that of the alveolar air, the differences being especially marked when the 2 ten- sion in the alveoli is raised (Figs. 126 and 127). Tension of C0 2 and 2 in Venous Blood. If we examine the C0 2 tension of the venous blood coming to the lungs, we shall find that it is distinctly 360 THE RESPIRATION higher than that in the alveolar air. The earliest method for measuring it consisted in passing a lung catheter into the right bronchus and then blocking the passage above the open end of the catheter by inflating a rubber collar or ampulla. The renewal of air in the right lung is thereby prevented, and a sample of the stagnant air can be removed and analyzed. In such a case, however, the blood will have circulated several times round the body, and with only one lung operating the risk is incurred that more C0 2 is being discharged into the blocked lung than cor- responds to the tension of C0 2 of venous blood under normal conditions. Much more practical methods are those of Haldane, Yandell Hender- son and R. G. Pearce, which are much the same in principle. In Pearce's method, the person first of all inspires from a gas meter containing a gaseous mixture with about 10 per cent of C0 2 . Immediately after fill- ing the lungs, he makes a rapid forced expiration into a tube provided with a valve having four openings. This valve is turned through a complete circuit during the expiration, so that four fractions of the ex- pired air can be collected in rubber bags connected with side tubes opening opposite the four openings in the valve. The first fraction will contain a little less than 10 per cent C0 2 , the second distinctly less, while the fourth will contain the same as the third, indicating that equi- librium between the C0 2 of the alveolar air and the blood must have been attained. This figure therefore gives us the tension of C0 2 in the venous blood of the lungs. In Henderson's method the rebreathing is per- formed into gas receivers containing 6 per cent C0 2 . These results then indicate that the whole process by which C0 2 is exchanged in the lungs is dependent on the law of gas diffusion ; the gas diffuses from a place of higher to a place of lower pressure, and does so until equilibrium is attained. The tension of 2 in venous blood may be determined by the applica- tion of similar principles. After a deep expiration nitrogen is inspired from a rubber bag for 3-5 breaths (10-20 sees.) and a sample of alveolar air is immediately taken. The percentage of 2 in this alveolar air will be found to be the same whether it be taken after 3 or 5 breaths, show- ing that an equilibrium with the tension of this gas in the blood must have been reached. If taken earlier, the 2 will be too high, and if taken later it will be too low (because the blood will have circulated twice round the body). This 2 percentage corresponds with the partial pres- sure (tension) of 2 in the mixed venous blood but it is somewhat lower than the latter because under the conditions of the experiment the C0 2 in the alveolar air is only that of arterial blood (40 mm.) (Bar- croft, etc. 84 ). THE CONTROL OF THE RESPIRATION 361 FALLACIES IN THE ESTIMATION OF THE ALVEOLAR GASES Methods such as that of Haldane and Priestley, which calculate the mean percentage composition of the alveolar air by analysis of a sample taken from the end of a prolonged forced expiration, give values which are too high for C0 2 and too low for 2 . There are several reasons for this: (1) In the time taken for the prolonged deep expiration an appreciable amount of C0 2 will be given off by the blood to the alveolar air, and oxygen will be absorbed that is, the sample will not contain the same percentages of C0 2 and 2 at different stages of expiration. (2) The portion of the tidal air which reaches the alveoli dilutes the alveolar air and thus causes the amount of C0 2 given off by the blood to vary during the different phases of respiration. If we bear in mind that the tensions of C0 2 in the alveolar air and in the blood leaving the lungs are always the same (page 360), and that the entire fall in C0 2 tension in the alveolar air occurs during inspiration, then it is clear that the blood in the pulmonary capillaries must have a maximum tension and load of C0 2 at the end of expiration, and a minimum tension and load of C0 2 at the end of inspiration. Accordingly, the average of the per- centage of C0 2 and 2 at the end of inspiration and expiration, as de- termined by the Haldane-Priestley method or by any of its modifications, must fail to give the correct mean tension of these gases in the alveolar air during expiration. The error which makes the C0 2 higher than it should be, makes the percentage of 2 less than it should be. These in- fluences taken along with the fact, which will be shown later, that the evolution of C0 2 from the blood is relatively more rapid at low than at high tension of C0 2 , indicates that the blood in the pulmonary capil- laries during inspiration must contribute a greater part of the C0 2 excreted during a respiratory cycle than that in the pulmonary capil- laries during expiration, and moreover that a greater part of the C0 2 excreted must be evolved at a tension which is below the mean tension of the C0 2 present in the entire time of the expiration. We conclude, therefore, that the average tension of C0 2 in the alveolar air, determined by the actual tension under which the gas is evolved from the blood, is less than the average tension of C0 2 in the alveolar air during the time of a respiratory cycle. In the case of 2 the conditions are different. While the diluting effect of the alveolar tidal air is marked in altering the amount of C0 2 given off during the different phases of a respiration, it can have little influence on the amount of 2 taken up by the blood under normal con- ditions. This is evident from a study of the dissociation curve of hemo- globin (page 396), which shows that at tensions above 65 mm. Hg the 362 THE RESPIRATION hemoglobin is practically saturated with 2 . Since the tension of 2 in the alveolar air under normal conditions is greater than 65 mm. (95-100 mm.), the rate of absorption of 2 must be practically maximal during the respiratory cycle that is, it will not change at different phases of it. While the relationship of the alveolar gases is continually changing at different stages of the respiratory cycle, their mean relationship for periods including several respirations or for complete respirations is more or less constant, being controlled by the type of the metabolism, and mathematically expressed by the respiratory quotient (page 582). The average relative percentages of the two gases in the alveolar air must therefore be the same as in the tidal air. In the alveolar air col- lected by the Haldane method, however, the above factors cause the respiratory quotient to be less than that in the tidal air. These points have been insisted upon because much of the knowledge of the gaseous exchange between the blood and the air in the lungs, as well as the control of respiration, has been built upon data obtained by the Haldane-Priestly method, and in considering this work, which we shall do in subsequent pages, it is advisable that we be aware of the limitations of the method employed. The method has been an invaluable one for opening up a hitherto entirely unexplored field of research, but now, the pioneer work having been done, we must employ methods which will enable us to explore more exactly. (R. G. Pearce.) CHAPTER XL THE CONTROL OF RESPIRATION (Cont'd) VARIATIONS IN THE ALVEOLAR C0 2 AND THE ACID BASE BALANCE IN HEALTH AND DISEASE At this stage and before we proceed to examine the respiratory dis- turbances due to blood changes, it will be advantageous to review the general nature of the adjustments which may occur in the body to com- pensate for disturbances in the acid-base equilibrium, and to indicate the type of symptoms which develop if these compensations are not promptly effected. To understand the relationships it is most satisfactory to study a C0 2 absorption curve such as is shown in Fig. 128. As has already been explained on page 50 this curve is obtained by finding the percentages of C0 2 (ordinates) absorbed by blood exposed at 38 C. in a tonometer to varying tensions of C0 2 (abscissae). Two such curves are shown in the chart to represent the extreme limits which are found in normal blood. The percentage of C0 2 going into simple solution in the blood is readily calculated for each tension, by multiplying this by 0.0672, the coefficient of solubility of C0 2 in blood (slanting straight line near the abscissa). It is then an easy matter to calculate P H for the different parts of the C0 2 curve (see equation on page 50). If this be done for curves of vary- ing heights (e.g., blood of different individuals or blood of the same individual under varying conditions) it has been found by Y. Henderson and Haggard that lines joining the same P H 's all meet at the zero point. By drawing such lines on the chart we can at a glance tell P H if we know the total C0 2 absorbed and the partial pressure of C0 2 . Taking the normal range of P H as 7.3-7.5 and of total C0 2 at 40 mm. C0 2 pressure, 43 and 55 vols. per cent, we can see from the chart the relationship of these values to P H in various abnormal conditions. These may be classi- fied as follows: Decrease in P H (increased H-ion concentration). 1. Addition of C0 2 (I in diagram). The simplest form of this is where atmospheres containing excess of C0 2 are respired. A true acidosis is established and it is compensated for by greatly increased breathing (to get rid of the C0 2 ), by excretion of excess of acid by the kidneys and by an increase in the bases of the blood. The condition may occur in 363 364 THE RESPIRATION asphyxia and in emphysema where it is due to the curtailment of the respiratory epithelium. In the latter case cyanosis is also common be- cause 2 diffusion is even more interfered with (see page 407). When it is compensated the blood condition shifts to area II on the chart, i.e., P H is normal but the blood takes up an excess of C0 2 . This variety of acidosis is more fully discussed in Chapter XLI. 2. Addition or accumulation of nonvolatile acids, either acids absorbed from the intestine or injected intravenously or when metabolic acids fail to be adequately excreted such as in severe diabetic ketosis and nephritis. This causes an alkali deficit. Under these conditions (area III in chart) P H is low and the C0 2 absorbing power is much reduced because 20 30 40 50 60 70 80 MILLIMETERS CO, TENSION 90 Fig. 128. Curves showing relationship between total COs in solution and PH at varying CO 2 pressure. (Redrawn from D. D. Van Slyke.) the foreign acid has appropriated some of the base (BHC0 3 + HA = BA + H 2 C0 3 ). Compensation is effected by deepened breathing (air hunger) to get rid of C0 2 , excretion of acid urine and increased excretion of ammonia. If compensation fails, coma is the result. If it is adequate the blood condition shifts to the left on the diagram to occupy area IV, i.e., P H falls within normal limits although the blood cannot take up its proper amount of C0 2 . During muscular exercise both of the above types of acid may come into play, the nonvolatile acid in this case being lactic (page 438). THE CONTROL OF THE RESPIRATION 365 Increase in P n (diminished H-ion concentration). 3. Addition of excess of alkali to the Hood, either by intravenous in- jection of carbonate solutions, or by overdosing with bicarbonates or phosphates. There is a great excess of total C0 2 as shown in area V of chart, - When the alkalosis is extreme, symptoms of tetany supervene (page 805). The compensation in this case involves the following: (a) Diminished excretion of C0 2 brought about by lessened breathing (-the" percentage of-GG 2 in the alveolar air is however increased because of the curtailment in the amount of air breathed), (b) Liberation of lactic acid into the blood, (Macleod and Knapp). (c) Increased excretion of alkali (as bicarbonate) and diminished excretion of ammonia in the urine. When compensation is adequate the blood picture shifts to area II (the same position as for compensated C0 2 excess). 4. Removal of excess of H 2 CO & (free C0 2 ) from the body. This results from increased breathing, being readily induced either voluntarily or as a result of stimulation of the respiratory center by anoxemia (page 378) or by excitation of afferent respiratory nerves. H 2 C0 3 being re- duced, there is a relative excess of BHC0 3 making the condition one akin to that following alkali administration (i.e., a true alkalosis, although the absolute amount of alkali is much less) and being accompanied by the same symptoms (tetany) and compensated byv the same mechanisms (increased excretion of alkali by the urine, disappearance of ammonia and probably production of lactic acid) as when excess of alkali is admin- istered. The blood condition immediately after the removal of the C0 2 corresponds to that of area VI on the chart, it then shifts to that of area IV (the same as in compensated alkali deficit). This form of al- kalosis is particularly important from the clinical standpoint since it must tend to be produced in all forms of hyperpnea not due to actual addition or accumulation of acids in the organism. It is the condition established in mountain sickness' (page 415). In all the above cases if compensation proceeds satisfactorily the blood condition ultimately returns to area VII on the chart, i.e., P H lies between 7.30 and 7.50 with the vol. of combined C0 2 between 43 and 55 when the blood is exposed to 40 mm. C0 2 pressure. Finally it should be pointed out that analysis of the percentage of C0 2 in the alveolar air and of the percentage of C0 2 in arterial blood (simultaneously collected), or of P H and the total C0 2 of blood, will give us the necessary data to find the actual blood condition from the chart, provided only that the breathing is normal. If the conditions be uncompensated, the alveolar C0 2 will be either too high or too low ac- cording to whether there is hypnea or hyperpnea. CHAPTER XLI THE CONTROL OF RESPIRATION (Cont'd) THE NATURE OF THE RESPIRATORY HORMONE The practical importance of the observations described in the foregoing chapters in the investigation of the relationship between C H of the blood and respiratory activity will now be plain, and it remains for us to consider the physiological evidence that such a relationship exists. In the first place, let us consider the behavior of the acid-base equilibrium during conditions of abnormal breathing hyperpnea and dyspnea.* As C0 2 accumulates and O 2 becomes used up in a confined space, the breathing becomes intensified. In searching for the exact cause of this effect, we must first of all ascertain whether the hyperpuea is due to the deficiency of 2 or to the accumulation of C0 2 or to both acting to- gether. Many of the experiments bearing on these problems can be more satisfactorily performed on man than on laboratory animals, be- cause anesthesia is not necessary and the subjective symptoms experi- enced are of great value in the interpretation of the results. If an in- dividual is placed in a large air-tight chamber (2000 liters' capacity), and the depth and rate of breathing observed as the C0 2 accumulates and the 2 becomes used up in the air of the chamber, no distinct change in respiration will be observed by the person himself until the C0 2 per- centage of the air has risen to almost 3. Above this point, however, the hyperpnea becomes more and more pronounced, until finally, when the C0 2 percentage has risen to about 6 and the 2 percentage has fallen to 13.5, it becomes unbearable (dyspnea). From the results of the fore- going observation alone we could not, however, decide whether the excitation of the respiratory center is due to the deficiency of 2 or to the increase of C0 2 . If the experiment is repeated with the difference that the C0 2 as it accumulates is absorbed by soda lime, no perceptible hyperpnea will develop even when the 2 is as low as in the previous experiment. We may conclude, therefore, that in the first experiment C0 2 accumulation must have acted as the main respiratory stimulus, and that oxygen deficiency, if it stimulates at all, must do so to a less degree than increase in C0 2 . *Hyperpnea means slightly increased breathing; dyspnea, labored breathing, but yet with suffi- cient ventilation to maintain life; asphyxia, the results of insufficient breathing. 366 THE CONTROL OF THE RESPIRATION 367 There is an obvious reason why the adjustment of pulmonic ventilation should not depend primarily upon changes in 2 supply to the respira- tory center. If it were so, many other tissue activities and other nerve cen- ters would suffer from the 2 deficiency before there was time for the breathing to become stimulated sufficiently to make good the loss of 2 . As a matter of fact, headache, dizziness, nausea and even fainting are almost certain to be caused whenever any muscular exercise is attempted in an atmosphere containing a deficiency of 2 but no excess of C0 2 (cf. moun- tain sickness) . An adequate 2 supply of the body is, therefore, insured by changes in C0 2 tension of the blood. Quantitative Relationship between C0 2 of Inspired Air and Pulmonary Ventilation. These results suggest, as the next step in the investigation of our problem, the determination of the quantitative relationship be- tween the C0 2 percentage of the respired air and the amount of air breathed (pulmonic ventilation).* That there is such a relationship has been most successfully demonstrated by R. W. Scott, 20 who used for his purpose decerebrate cats.f The trachea was connected, through a T-tube provided with valves, with tubing leading to a large bottle and a Gad-Krogh spirometer, so that the animal breathed out of the bottle into the spirometer, these two being also connected together. The spirom- eter was made to record its movements on a drum, so that an accurate record of the depth and frequency of the respirations was secured. Sam- ples of air were removed from the bottle by ground-glass plunger syringes at frequent intervals during the time that the animal was respiring into the tubing. The results are given in the accompanying curve (Fig. 129), which shows that there is a perfect correspondence between the C0 2 percentage in the air of the bottle and the pulmonary ventilation. Moreover, when the bottle was filled with 2 instead of air to start with, the same results were obtained, showing that the C0 2 accumulation alone was responsible for the hyperpnea. In these cases the percentage of 2 remaining in the system after hyperpnea had become extreme, was far above that at which excitation of the center as a result of 2 deficiency is possible. Experiments of a similar type had previously been performed by Por- ter and his pupils, 21 but their object was not so much to show the close parallelism between the C0 2 content of the respired air and the pulmonic *A distinction is somewhere drawn between pulmonic ventilation and alveolar ventilation, the former being the total amount of air that enters and leaves the lungs, and the latter, that which en- ters and leaves the alveoli. This distinction is based on the assumption that the capacity of the dead space may vary considerably from time to time, which, as pointed out elsewhere, is erroneous. For practical purposes pulmonic ventilation is the safer value to give. fDecerebrate animals must be used in these experiments, since anesthetics markedly depress the activity of the respiratory center. 368 THE RESPIRATION ventilation as to demonstrate the changes produced in the sensitivity of the respiratory center in pneumonia. Possibility that C0 2 Specifically Stimulates Center. After showing that C0 2 acts as an excitant of the respiratory center, the question arises as to whether the action depends on the raising of the C H of the blood, or whether it may be a specific action of the C0 2 . Many attempts have SOQ 400 300 ZOO /OO L Fig. 129. Composite curve obtained from the data on sixteen experiments, showing the re- spiratory response to CO 2 in the decerebrate cat. Abscissae = percentage of CO 2 in the inspired air. Ordinates = the percentile increase the tidal air per minute. (From R. W. Scott.) been made to decide this question experimentally, the general principle of the experiments being to determine whether C H of the blood runs parallel with the C0 2 content of the respired air and with the hyperpnea. Using the gas-chain method (page 31), Hasselbalch and Lundsgaard 22 found that the hyperpnea produced in rabbits by breathing in C0 2 -rich THE CONTROL OF THE RESPIRATION 369 air runs approximately parallel with the increase in the C H of the blood, but on account of the experimental difficulties encountered they could not decide whether changes in CH are alone responsible for the effect. These authors had previously demonstrated that changes in C H can be induced in blood removed from the body by experimental alteration of the C0 2 tension within the physiological limits. An increase of one millimeter in C0 2 tension was found to cause an increase in C H of 0.0065 x 10- 7 (see page 27). R. W. Scott's experiments, above referred to, have, however, yielded more definite results. By using the colorimetric method for determining CH of the blood (see page 32), it could be readily shown, as is evident THE EFFECT OF REBREATHING CARBON DIOXIDE ON THE MINUTE VOLUME AND ON THE H-ION CONCENTRATION AND TOTAL CARBONATE CONTENT OF THE ARTERIAL BLOOD IN THE DECEREBRATE CAT FIFTEEN MINUTES PRELIMINARY PERIOD* REBREATHING PERIOD AFTER RE- BREATHING PERIOD W g S3 1 c o. C a a c c > VH Jl s h o Sna 1 cfl'g o JU-0 O * bi a II 0. *H 3 0^ 0. Ui O ^ e i-i _0 5 gj & .n gj IH * Time S C & jQ c O, .h a | "a 0^3 H JO IH O*rt " o b '3 675 | 0-3" s fa +J 2 rt rt CJ'^ o rt a] -M c u-S u-3 M 14 fc c be 2 '&" 3 a o rt rH |S 1 | id "0 i to o rt *~ l 9 o rt *~ l U U ^ P4 H PH OH fn cu & f-H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 k K . c.c. c.c. c.c per per c.c. c.c. cent cent 27 36 2.2 10.30 a.m. 38.5 48 864 7.45 30.3 64 3,968 540 6.45 7.25 53.4 7.4 34.2 28 33 2.2 1.50 p.m. 138.8 22 616 7.40 40.2 40 2,880 367 5.02 7.20 58.0 7.35 42.0 29 35 2.0 10.30 p.m. |38.6 28 784 7.40 38.1 40 3,840 389 5.35 7.25 51.1 7.4 40.0 30 31 32 31 2.3 2.0 11.00 p.m. 11.15 a.m. 38.7 38.8 32 32 768 896 7.45 7.40 30.2 32.0 54 60 4,536 4,800 489 |436 6.40 5.95 7.20 7.20 51.1 50.0 7.4 7.4 35.0 35.2 Averaj ye 32.4 785 7.42 34.1 *PH is the actual value given in the table. This is inversely proportional to CH. from the table (cols. 8 and 14), that a marked rise in C H became evident when the inspired air contained 5 per cent or more of C0 2 . That this rise was due to increase in the C0 2 tension was shown not only by finding a greater percentage of C0 2 (cols. 9 and 15) in the blood, but also by being able to demonstrate that when C0 2 -free air was bubbled through the blood removed during the dyspnea, C H immediately returned to the normal which it also did in the -blood removed after the animal had breathed for a few minutes in outside air (col. 16) when the C0 2 content likewise returned (col. 17). Had the increase in acidity been caused by nonvolatile acids lactic, for example these results, particularly the latter, could not have been obtained. 370 THE RESPIRATION Although there is therefore no doubt that the C H of the blood may be raised because of an increase in C0 2 in solution in the blood plasma a C0 2 acidosis, as we may call it (see page 371) this does not prove that the stimulation of the respiratory center is brought about solely by C H - The increase in C0 2 might in itself also serve as a stimulus. That such is actually the case was demonstrated by finding that, if C H of the blood was first of all lowered by injecting alkali intravenously, hyperpnea still developed in proportion as the C0 2 accumulated in the inspired air; and that CH of the blood, when the hyperpnea was at its highest, was below that of normal blood. Some other factor than C H must obviously be responsible for this result. This is undoubtedly dependent on the C0 2 . Further corroboration of the claim that C0 2 has a specific stimu- lating effect on the respiratory center that is independent of CH, has been furnished by Hooker, Wilson and Connett. 23 These authors suc- ceeded in keeping the centers of the medulla alive by perfusion with defibrinated blood through the blood vessels of the brain, and found that, although the respiratory movements of the diaphragm became de- pressed with a decrease and excited with an increase in C H of the per- fusion fluid, a greater activity of the center was produced when the fluid contained a high tension of C0 2 than with another fluid of the same C H but with a low tension of C0 2 . We conclude that, although the C H is the important respiratory hormone, carbon dioxide per se also has a stimu- lating influence. A similar conclusion had previously been arrived at by Lacquer and Verzar 54 who studied the activity of the respiratory center in young rab- bits perfused through the aorta with isotonic solutions in which the C H was caused to vary by the addition of different acids. It was found that when C H was about the neutral point (even slightly on the alkaline side), but the solution contained C0 2 , much more marked stimulation occurred than when CH was raised by adding some other acid. Collip 77 has also ob- served that the respirations may be stimulated by intravenous injections of sodium bicarbonate. These conclusions are confirmed by observations on the influence of C0 2 on other living cells than those of the respiratory center. Rona and Neukirch 55 found that the isolated intestine when made to beat in oxy- genated saline solution (page 497) is very sensitive to C0 2 , and Jacobs 56 has more recently made some very interesting observations on the toxic effects of C0 2 as compared with that of other acids, on the tadpoles of the toad, and on several species of protozoa. It was found that a saturated solution of C0 2 is incomparably more toxic than are solutions of various inorganic and organic acids of the same C H as the C0 2 solution. On the other hand neutral solutions of NaHC0 3 are nontoxic, which indicates THE CONTROL OF THE RESPIRATION 371 that it cannot be the HC0 3 -anion as some have supposed, that is the toxic agent. The order of resistance of various protozoa to CO 2 was found to bear no relation to that which they bear to other acids. It may be, however, that the more toxic effect of the C0 2 is dependent upon the greater rate at which it penetrates cell membranes than other acids. It would there- fore enter the cells of the respiratory center and by dissociation cause alteration of C H in the protoplasm. In the light of this possibility it is of interest that whereas other acids cause cessation of the movements of flag- ella in protozoa with no visible changes in the interior of the cells, C0 2 has little effect on the flagella but causes marked alterations in the intra- cellular activities. Jacobs has also shown that an acid reaction may develop within the petals of certain flowers when these are suspended in a bicarbonate solution that is itself faintly alkaline. He has also succeeded in arranging a laboratory experiment in which a similar migration of undissociated carbonic acid through a layer of xylol caused a neutral solution in contact with it to become acid. 78 Relationship Between Alveolar CO, and Respiratory Activity. Vari- ations in the respiratory hormone, whatever this may be, are associated with changes in the C0 2 content of the alveolar air. Increase in the alveolar C0 2 immediately stimulates respiration unless under certain conditions which will be discussed later. Indeed the respiratory center is so very sensitive towards this stimulus that whenever the percentage of C0 2 in the inspired air tends to rise, pulmonary ventilation is excited to a degree which is just sufficient to maintain the tension of* C0 2 in the alveolar air at the normal level. There is therefore, no better method for testing the excitability of the center than to measure the increase of pulmonary ventilation produced by adding known percentages of CO., to the inspired air. If the amount of C0 2 in the inspired air is sufficient to raise the C0 2 in the alveoli, in spite of the greater breathing, hyperp- nea becomes very marked ; thus, it has been found that if enough C0 2 is inspired to cause an increase of from 0.2-0.3 per cent in alveolar C0 2 in man the alveolar ventilation is doubled, or, more precisely stated, an increase of ten liters in the air entering and leaving the alveoli per minute results from raising the alveolar C0 2 tension by 2.2 to 3.1 mm. Hg (Douglas 24 ). The relationship between breathing and alveolar C0 2 is by no means al- ways so simple as in the instances just described. In these hyperpnea is sec- ondary to an increase in alveolar C0 2 but there are many cases where the reverse relationship obtains namely, where decreased alveolar C0 2 is sec- ondary to hyperpnea caused by stimulation of the respiratory center by some other agency than increase in C0 2 tension of the blood. These agen- *The tension is found by the equation given on page 374. 372 THE RESPIRATION cies include afferent nerve stimulation, lowering of the 2 -tension, or in- crease in C H of the blood brought about by other acids than C0 2 such as occurs in clinical cases of acidosis (see page 654). The whole question is very closely linked with that of the control of the reaction of the body fluids and with the etiological factors in acidosis. When it is fully answered, many obscure clinical conditions in which respi- ratory disturbances occur will be much better understood than they are at present. On account of the great importance of the subject, considerable attention will be devoted in the next few pages to some of the researches which have been made bearing on the relationship between the C0 2 of the alveolar air and the various modified types of breathing that can be produced experimentally or which become developed under altered physi- ological conditions. Even at the risk of repetition let us consider what will happen when excess of C0 2 is added to the blood in the body, either as a result of respiring C0 2 -rich air or because of increased production of this gas in the tissues, as in muscular exercise. The tension of C0 2 in the blood will first of all increase and tend to raise CH since, as we have seen, this TT QQ depends on the molecular ratio - (where B HC0 3 stands for B HC0 3 NaHC0 3 and other similar compounds). The excess of C0 2 will be re- moved, partly by increased breathing and partly by basic substances, including Na, being set free to combine with it. In these ways the TT p(") ratio ~ will tend to come back to its normal value but now with both free C0 2 and total bicarbonates absolutely greater than before, so that the C0 2 combining power of the blood will be raised (page 42). On the other hand, when fixed acids are added to the blood they will appropriate some of the base (B) and liberate H 2 C0 3 so that both the tension of C0 2 and C H will rise (P H become less), equilibrium being then reestablished by increased breathing and blowing off of C0 2 . If the increased breathing does not suffice in both these cases to restore the TT c*r\ normal J* J* ratio, other compensating mechanisms come into play, B namely, increased excretion of acid by the urine and combination with ammonia (page 381). When the H 2 C0 3 is lowered as by increased breathing, changes of an opposite character occur and these will be discussed in the following pages. CHAPTER XLII THE CONTROL OF RESPIRATION (Cont'd) THE ALVEOLAR C0 2 -TENSION. ANOXEMIA. The Constancy of the Alveolar C0 2 -Tension Under Normal Conditions Since a close relationship exists between the alveolar C0 2 -tension and the respiratory activity, it is to be expected that the two would bear a strict proportionality to each other, and since the breathing under normal conditions does not vary much, the C0 2 -tension should also be constant. Many observations show this to be the case. The tension is remarkably constant from day to day and even from month to month in the same individual, provided the physiological conditions are the same. A slight seasonal variation is said to exist, a rise in the temperature of the en- vironment of the individual causing a slight depression in the C0 2 -ten- sion, while a fall in temperature causes a slight rise (Haldane). These changes are independent of any demonstrable change in rectal temper- ature and, therefore, are probably due to the influence of the temperature on the skin. Since it is the number of molecules of C0 2 in a given volume of alve- olar air (i. e., the partial pressure or tension) that is of importance, it is only when the barometric pressure is the same that the percentage of C0 2 in the sample of alveolar air can be constant. To allow for this, all results are reduced to standard barometric pressure (760 mm. Hg). If the barometric pressure is lowered, there will have to be a higher percentage of C0 2 in the sample in order that there may be the same tension of this gas in the air of the alveoli ; and vice versa when the bar- ometric pressure is raised. The equation by which this tension, ex- pressed in millimeters of mercury, is determined is : 100 :760 ( aq) : :a:p, where a is the percentage actually found in the air of the sampling tube and p the tension. A correction ( aq) is introduced in this equation to allow for the vapor tension of the air in the alveoli, for of course H 2 molecules will behave like C0 2 molecules in causing a partial pressure. When reduced to this standard, it has been found that the tension of C0 2 in the alveolar air tends to remain constant under the varying baro- metric conditions that obtain at sea level or at moderate departures from this. This is shown in the following table : 373 374 THE RESPIRATION (1) BAROMETRIC PRESSURE (MM. HG) (2) CO, ACTUALLY FOUND IN DRY ALVEOLAR AIR (PER CENT) (3) PARTIAL PRESSURE OF C0 2 IN MOIST ALVEOLAR AIR AFTER CALCULATING FOR BAROMETRIC PRESSURE Top of Ben Nevis Oxford Foot of Dolcoath Mine Compressed air cabinet 646.5 755 832 1260 6.62 5.95 5.29 3.52 5.23* 5.53 5.48 5.64 (B'-A) P' - "P \irViprp P ficmrfx; in last column; B' = figures in first column; A = aqueous tension of alveolar air;_ P' = figures of second column; B barometric pressure at sea level. A is obtained from tables giving the aqueous tension at different temperatures. The Alveolar C0 2 -Tension in Conditions of Anoxemia The foregoing observations have shown that the respiratory hormone is related to the tension of C0 2 in the blood supplying the respiratory center, and that this tension acts partly by causing alteration in C H of the blood, and partly because C0 2 has direct effect on the center. These conclusions do not imply that other changes in the composition of the blood may not act on the respiratory center, indeed there is plenty of evidence to show that deficiency of oxygrw fffl fhe ^^-^ 1 blood or anoxe- mia also acts. This influenceTs, howeverTless evidentjliaji .that of changes in^^HjDr^XlJD^-and it varies with the degree of deficiency, being stimulatory when the deficiency is moderate, and inhibitory when it is extreme. It is not surprising, therefore, that some considerable confusion should have existed as to the precise role of 2 deficiency in its effect on the respira- tions and it is only within the last year or two that the problem has been satisfactorily elucidated. The most important indication that alterations in the C0 2 -tension of the blood cannot alone be responsible for changes in respiratory activity is afforded by the observation that the alveolar C0 2 does not always, as in the cases described in the previous chapter, run parallel with alveolar ven- tilation. The opposite relationship often obtains, namely, decreased al- veolar C0 2 and hyperpnea (see page 379), and it is our purpose in the present chapter to show how this is often associated with a condition of oxygen deficiency. It is most important that we consider this phase of the subject in some detail because of the application which it has in the eluci- dation of many problems of respiratory disturbance, as met with in the clinic. The disturbances in respiratory function which can be brought about experimentally in normal animals are in many cases exactly like those which are met with in various diseases, particularly those which depend on inadequate absorption of oxygen by the blood. The General Effects of Deficiency of Oxygen, or Anoxemia. Various THE CONTROL OF THE RESPIRATION 375 methods have been employed in the investigation of this subject. The most important of these are as follows: (1) Breathing from a tank con- taining varying mixtures of oxygen and nitrogen (Dreyer apparatus). This apparatus was used extensively in the British Army in testing the ability of candidates for the aviation service to withstand low oxygen. Its greatest value is that the alteration in oxygen content of the inspired air can be made either gradually or quickly. (2) Breathing from a tank through tubes provided with valves which direct the expired air so as to make it pass through an apparatus for absorption of the C0 2 , before it re-enters the tank. The subject, therefore, re-breathes the air of the tank from which he gradually absorbs the oxygen. In this method the 2 -content of the inspired air falls gradually, and effects are pro- duced which must be similar to those which would be caused by slow ascent to higher altitudes. The rate at which the 2 falls can be varied by altering the size of the tank. When a very rapid fall is desired rubber bags can be used in place of tanks. An apparatus on this principle was employed for testing aviators particularly in the United States Army. (3) Breathing in an air-tight cabi.net containing properly arranged soda lime absorbers to take up the C0 2 ; the oxygen decreases at a rate which is inversely proportional to the size of the cabinet. (4) Breathing in a strongly built steel chamber connected with a powerful pump by means of which the chamber can be partially evacuated and the pressure main- tained at any desired level. Such a chamber has been used by Haldane, Kellas and Kennaway in important experiments, the results of which we shall consider immediately. (5) Adding a sufficient percentage of carbon monoxide gas to the inspired air to combine with a considerable propor- tion of the hemoglobin of the blood and so render it incapable of carrying the oxygen. The observations made by the use of these methods have been com- pared with those made during life at high altitudes, particularly in con- nection with mountaineering. The latter observations are of particular importance in' the study of the adaptive processes which come into play to render persons who have become accustomed to high altitudes immune to the distressing symptoms from which others suffer. There is considerable variability in the reactions of different persons to decreased oxygen and these cause symptoms which are partly subjective and partly objective in nature. Slight differences are observed according to whether the anoxemia is produced by a lowering of barometric pressure (decompression), or by simply reducing the percentage of oxygen in the inspired air. In the former case there is also a slight difference between the symptoms following decompression in an experimental cham- ber, and those observed on a high mountain. In a general way the symp- 376 THE RESPIRATION toms are less marked when the barometric pressure is reduced than when the oxygen percentage is simply diminished. The Symptoms During Gradual Reduction of the Percentage of Oxy- gen. Measurements have been made of a number of physiological func- tions in a large number of healthy young men who were condidates for the flying corps of the various armies participating in the recent war. * -*'- 7?5H /N UEC/L. P7? M/N. 180 160 140 TOO 80 60 40 _. 4 6 8 10 11 M- 76 18 W 14 26 3L8 30 " JB.H Fig. 130. The behavior of the respiratory volume, the blood pressure, and the pulse during progressive anoxemia. The results are of value in showing to what extent the candidates can be expected to withstand the rarefied air met with at great altitudes. The accompanying chart (Fig. 130) taken from the "Air Service Manual" of the United States Army depicts the results obtained on a perfectly normal individual. The percentage of 2 in the air breathed at vari- ous stages of the test is read on the right edge of the chart by find- THE CONTROL OF THE RESPIRATION 377 ing the ordinate which is crossed by the thick continuous line. In this observation the subject withstood oxygen deficiency without alarm- ing symptoms until a percentage of nearly six was reached. These symptoms are giddiness and then fainting along with various psy- chological effects. The respiratory volume, the pulse rate and the systolic and diastolic blood pressures are also shown (see legend of figure for details) and it will be observed that the respiratory vol- ume did not change greatly until the oxygen percentage had fallen to below twelve. Usually, however, the volume is slightly increased even with a slight reduction in the oxygen, the depth rather than the rate of the breathing being responsible for the change. Ellis 57 has more particularly investigated the precise percentage of oxygen at which the respiratory volume becomes just perceptibly increased and found it to be somewhat above eighteen. By the time 8 per cent 2 is reached the majority of men show about a doubling in the amount of air breathed. If they do not respond in this way, they are almost certain to break down by fainting, because the increased respiration is the mechanism by which the deficiency of oxygen is compensated for. There is usually little change in the pulse rate until the oxygen has fallen below fifteen per cent. A total acceleration of 15-40 beats per minute is normal when the 2 is lowered to seven per cent. The systolic blood pressure usually remains unchanged down to a partial pressure corresponding to 14 or even 9 per cent of oxygen. Below these levels the systolic pressure rises 15-20 mm. Hg. above normal. If it rises more than this it is considered unfavora- ble, since it indicates that vasodilatation has not occurred as it ought to as a result of the low oxygen. A sudden fall in systolic pressure precedes fainting. The diastolic pressure remains practically unchanged throughout the test, but it ought to show a slight decline as the systolic pressure rises. The decline shows that vasodilatation is occurring. When the anoxemia is continued for a time, the pulse usually returns towards the normal rate and the systolic and diastolic pressures, if they were affected, also tend to return to the normal levels (Lutz and Schneider 58 ). These authors found the same results whether the 2 was reduced by lowering of the barometric pressure or by reduction of the percentage of the gas. Cyanosis is a normal reaction but it should be delayed in its onset. In some cases a pale and death-like color develops in place of cyanosis. This is an unfavorable sign. The actual time taken to reach the limit of endurance in the test de- pends of course on the capacity of the spirometer. This is usually chosen so that a test occupies from 20 to 30 minutes. The Symptoms During Lowering of the Barometric Pressure in a Pneu- 378 THE RESPIRATION matic Chamber. These have been carefully described by Haldane, Kellas and Kennaway, 59 who exposed themselves and others to 380 mm. baro- metric pressure (corresponding to about 19,000 feet above sea level) for at least an hour on several successive days, the pressure being lowered to this level very quickly (5 minutes). The symptoms varied somewhat in the different subjects. The respirations increased in depth and frequency at first but then returned more or less to normal in four out of six of the observed persons. In one they remained very rapid, and in another they showed a great tendency to become periodic. The pulse al- ways increased at first and then returned almost to normal. Cyanosis was always present but did not diminish with continued exposure. Al- though mental and sensory symptoms were not perceived, the sudden raising of the oxygen percentage caused the light to become brighter and sounds to become louder. Vigorous muscular work caused marked hyperpnea, quick pulse, increased cyanosis and mental confusion. There were none of the symptoms of mountain sickness (page 415) and no ill-effects were experienced on leaving the chamber. At still lower pressures the above mentioned changes in pulse and respira- tions became much more pronounced and decided mental symptoms ap- peared, for example it became very difficult to count the pulse, or to record the other observations. Loss of memory was common, but there was no loss of consciousness. A most interesting mental symptom sometimes observed was fixedness of ideas. This has also been noted in coal gas poison- ing, and in high balloon ascensions. Thus, in one experiment the sub- ject persisted in having the pressure held at a certain (very low) level for no evident reason, just as those exposed to carbon monoxide can not be diverted from remaining exposed to the gas. These symptoms have been described in some detail since it is im- portant that they be compared with those in clinical cases in which oxygen deficiency exists. They constitute the reactions of healthy in- dividuals to conditions which may become established as a result of disease, and it is obviously most important in such cases that they be distinguished from the more definitely pathological symptoms. On comparing the above symptoms to those of mountain sickness it will be noted that there is a marked difference (see page 415). One pe- culiarity of the latter condition is that prolonged stay in the rarefied air brings about an acclimatization, so that after some days the person is practically normal. The attempt was made in the above experiments to bring about a similar acclimatization with partial success. The Nature of the Changes Produced in the Body in Anoxemia. A careful study of this aspect of the problem is most important, not only because it throws much light on the manner of control of the respiratory THE CONTROL OF THE RESPIRATION 379 center, but also because it helps us to explain the nature of the altera- tions which occur in the acid-base equilibrium of the body in all condi- tions which tend to bring about anoxemia. The immediate increase in breathing is the first of the symptoms which demands attention. Ob- viously it cannot be due to the excitation of the respiratory center by an increase in the free C0 2 of the arterial blood (see page 366). Two possi- bilities remain either the reduction of free oxygen in the blood per se excites the center, or this reduction in oxygen causes incompletely oxi- dized acid substances such as lactic acid to appear in the blood so as to raise C H . A clue as to which of these causes is responsible is fur- nished by observation of the C0 2 tension of the alveolar air. It will be recalled that this does not change when the barometric pressure is slightly altered (page 373), but it is otherwise when the reduction is extreme; a progressive decrease occurs. Thus, in one of the experiments, the alveolar C0 2 tension to start with was 40.7 mm. Hg. After lowering the barometric pressure to about 500 mm., the alveolar tensions were : after 25 minutes, 36 mm. ; after 90 minutes, 36.8 mm. ; after 175 minutes, 25.7 ; after 465 minutes, 34.9 mm. This shows clearly that increase of free C(X in the blood cannot be the cause for the hyperpnea. Further evidence for this conclusion is afforded by the fact that the breathing after some time returned towards the normal although the alveolar C0 2 tension remained low. Increase in C H of the arterial blood on account of the appearance of unoxidized acids, has been considered as the possible cause for the symp- toms of anoxemia. This hypothesis demands close attention because it has been very widely accepted and has seemed to be supported by nu- merous observations, both physiological and clinical. Physiologists, for example, have known for long that lactic acid accumulates in the blood and appears in the urine in all conditions in which there is de- ficient oxidation in the tissues. Thus, Araki found it in the urine after asphyxia produced either by obstruction to breathing or by inspiring coal gas. Hopkins and Fletcher showed that it appears in muscle when- ever this is made to contract in deficiency of oxygen, and Ryffle found it increased in the blood and was present in the urine excreted after vigor- ous muscular exercise. From the clinical side the evidence has been fur- nished by observing the behavior of patients suffering from acidosis due to the appearance of oxybutyric acid (page 737), as in diabetes, or of other acids, as in certain cases of nephritis. In these cases hyperp- nea is accompanied by a lowered tension of alveolar C0 2 and by a lowered capacity of the blood to combine with C0 2 , that is a lowered alkaline reserve (page 38). If the alkaline reserve of the blood be determined after exposure of an animal (man) to low oxygen, it has also been found to be decreased. 380 THE RESPIRATION Taking all these facts together, a strong case appeared to be made for the acidosis hypothesis and this seemed to be almost established since Haldane and his collaborators were apparently able to apply it in ex- plaining the results of their numerous investigations of the respiratory function. It will be observed, however, that all of the evidence is cir- cumstantial in nature, and that the changes observed in anoxemia may be due to entirely different causes. Lowering of the alkaline reserve of the blood, lowering of the tension of C0 2 in the alveolar air, and hyperp- nea can undoubtedly all result from deficiency of oxygen-supply to the tissues, but the sequence in which the changes occur may be exactly the reverse of that which is assumed in the acidosis hypothesis; it is pos- sible, namely, that the deficiency in oxygen first excites the respiratory center, the increased breathing then causes a blowing oif of the free C0 2 from the blood, and the alkali that is thus set free is then excreted from the blood in the endeavor to hold C H at a constant level. If some method were available for precise measurements of C H of the arterial blood at frequent intervals it would be possible to settle this question once and for all, but such is not the case, and we must seek for proof for the new hypothesis by indirect means.* Assuming then that the respiratory center is excited by a slight degree of anoxemia, let us see how the known facts fit in. The diminution in oxygen in the blood excites the respiratory center and causes increased breathing. This results in a blowing off of C0 2 from the blood into the alveolar air, so that there comes to be relatively more C0 2 excreted than 2 absorbed, and the respiratory quotient becomes raised. We have shown this very clearly in experi- ments on decerebrated cats breathing in oxygen-poor atmospheres ; even when the oxygen percentage was only slightly reduced, R.Q. had already risen to over unity. As a result of this blowing-off of C0 2 , C H of the blood must tend to fall and a condition of alkalosis, rather than of acidosis results. This may explain the tendency for the breathing to return to- wards the normal. It is important for practical reasons to realize that when this condition becomes established increased breathing will not compensate for the anoxemia, for the advantage gained by higher satura- tion of the blood with oxygen is counteracted by the alkalosis in which the hemoglobin holds on to the oxygen with greater avidity. When such is the case, as already pointed out elsewhere (p. 401), the oxy- gen is not so readily given up to the tissues. Under these con- ditions neither increased breathing nor increased bloodflow can force more oxygen into the tissues, so that both the respirations and the pulse become less rapid after the initial acceleration. It is this prolonged *It may be stated, however, that CH of the blood of man after he has been for some time in rarefied air is still normal as judged by determination of the dissociation curve of his blood (page 402) in a partial pressure of CO 2 equal to that of his alveolar air (Barcroft). This result shows at least that the acid-base equilibrium is ultimately restored under the altered conditions. THE CONTROL OF THE RESPIRATION 381 anoxemia that causes the symptoms of mountain sickness and many of these of pathological conditions such as pneumonia or CO-poisoning. The only possible treatment is to raise the- tension of oxygen in the alveolar air sufficiently to force some into solution in the plasma (see page 445). After a time acclimatization to anoxemia occurs because of adjustments which bring C H back to its normal value. These depend on excretion of the excess of alkali by the kidneys and the conversion of a greater pro- portion of ammonia into urea (page 650). Hasselbach and Lindhard, 60 Collip and Backus, 79 and Grant and Goldman 80 were the first to show that CH of the urine becomes reduced early in anoxemia, but returns to the normal after acclimatization is established, and that the excretion of ammonia is also relatively decreased but remains low even after ac- climatization. Haldane, Kellas and Kennaway 59 have confirmed these observations by showing that the titrable acid of the urine is reduced by one-half, or more. Diminution in the NH 3 excretion is always ob- served when the excretion of fixed alkali is increased in proportion to the acid,* and it indicates that the function of the organs which are respon- sible for conversion of NH 3 to neutral urea, must become stimulated as a protection against the alkalosis. The question therefore arises whether the relative decrease in the acid excretion by the kidneys (rendering the urine more alkaline) runs parallel with the diminished production of am- monia. It was found that the ratio of ammonia to acid rose markedly, showing that the conversion of NH 3 into urea did not occur as promptly as the increased excretion of alkali by the kidney. Part of this delay is no doubt dependent upon the time required for the ammonia already present in the blood and tissue fluids to be excreted before the increased urea formation could become evident in the urine. The results as a whole show that the kidneys (and liver) on the one hand, and the respiratory center on the other, respond to changes in C H of the blood which are far below those that can be detected by exist- ing physical or chemical methods of measurement (Haldane). The respiratory center constitutes the first line of defence against any change in C H by increasing or decreasing the rate at which C0 2 is blown off from the blood. The kidneys constitute a second defence by altering the ratio of acid base whicfcthey allow to pass into the urine, andjhe liver and other organs form a third line of defence by altering the amount of free "NH 3 which they permit to enter the blood. The readjustment of the alveolar C0 2 and of the acid and ammonia excretions do not return to the normal until some time after the subject has been breathing at normal barometric pressure. This will be made evident by consulting Fig. 142-A. *Benedict and Nash have recently shown that it is solely in the kidneys that the ammonia for these purposes is formed (page 563). CHAPTER XLIII THE CONTROL OF RESPIRATION (Cont'd) APNEA PERIODIC BREATHING Apnea If a man breathes forcibly and quickly for about two minutes, tie will experience no desire to breathe for a further period of about the same duration he becomes apneic. When the desire to breathe returns, the breathing is at first very shallow, and frequently periodic in type, but gradually it becomes more marked, until at last normal respiration is reestablished. How may the results be explained? The cause for the absence of breathing is lowering of the C0 2 -tension in blood. This re- moves the natural stimulus to the respiratory center because a temporary condition of alkalosis with low C0 2 -tension is induced. Evidence of the establishment of alkalosis by forced breathing has been obtained by ex- amination of the acid excretion in the urine, (see page 381). The C0 2 - tension is lowered because this gas has been " washed out" or "blown off" from the blood into the overventilated alveoli. Although this over- ventilation also raises the pressure (tension) of oxygen in the alveoli, little, if any, more of this gas can be absorbed into the blood because even at the normal alveolar tension, the blood takes up at least 95 per cent of its possible load, in consequence of the dissociation curve. These differences in the rate of C0 2 -loss and 2 -gain cause the respiratory quo- tient to become very high during the forced breathing ; it may indeed rise nearly to 2. During the apneic period the person by an effort can expire some alveolar air, and if this be analyzed it will be found that very little C0 2 is being expelled from the blood though the 2 is being absorbed as usual. Consequently R.Q. becomes very low (0.2 has been observed). Gradually, however, the C0 2 rises again in the blood and the alkalosis disappears so that the respiratory center becomes excited, although at first only feebly and irregularly. If alveolar air be analyzed when the first indication of breathing returns it is said by Haldane that the C0 2 tension is not yet back to the normal level (see Fig. 131). This indi- cates that some other influence is helping to excite the respiratory center and this no doubt is anoxemia for the percentage of 2 in the alveolar air has now fallen to a very low level. 382 THE CONTROL OF THE RESPIRATION 383 Fig. 131. Curves showing variations in alveolar gas tensions after forced breathing for two minutes. Thin line = Oo tension; thick line CC*2 tension. Double line normal alveolar CO 2 tension. Dotted line shows the alveolar CO 2 tension at which breathing would recommence at the end of apnea with the alveolar O 2 pressures shown by the thin line. The actual breathing is indicated at the lower part of the figure. It is periodic to start with. (From Douglas and Haldane.) 384 THE RESPIRATION In agreement with this explanation it has been found that, if the last two or three forced respirations preceding the apnea are made in an atmosphere of 2 instead of air, so as to fill the alveoli with 2 , the apnea can be maintained for a very much longer period; and when the natural desire to breathe returns, the C0 2 tension of the alveolar air, instead of being below the normal, is above it. The effect of 2 in pro- longing apnea, must, therefore, be dependent on the fact that it prevents anoxemia. By this means the period during which the breath can be held after breathing 2 is sometimes phenomenal; in one individual, for example, after breathing forcibly for a few minutes and then filling the lungs with 2 , apnea lasted for eight minutes and seventeen seconds. The excess of 2 also serves to drive out considerably more C0 2 from the blood in the alveolar capillaries (cf. page 399). Collip, 79 and Grant and Goldman 80 have called attention to the peculiar "tetany"-like symp- toms that develop in many individuals after the forced breathing. The condition of alkalosis induced by the forced breathing is believed to be responsible for the tetany (cf. page 381). THE SUPPOSED NERVOUS ELEMENT IN APNEA The apnea following forced breathing has been attributed to a sort of inhibition of the respiratory center brought about by its repeated stimulation by afferent impulses transmitted to it along the vagus nerves, as a result of the frequent collapse and distention of the alveoli. In justification of this nervous interpretation, it was claimed that apnea could not readily be produced in animals after severing both vagus nerves. More recent work has shown that this is not an accurate obser- vation, for if the severing of the vagi is accomplished not by cutting but by freezing, then apnea is as readily produced as in an intact animal (Milroy 28 ). That chemical and not nervous factors cause the apnea is further demonstrated by the well-known experiment of Fredericq, who, after ligating the vertebral and one of the carotid arteries in two dogs, anas- tomosed the central end of the remaining carotid of the one to the peripheral end of the carotid of the other animal, thus establishing a crossed circulation. He then found by applying forced artificial respira- tion to the one animal, that the apnea which supervened affected the other animal and not that to which the artificial respiration had actually been applied. Another proof of the chemical theory of apnea is furnished by the observation that if forced breathing is per- formed in an atmosphere containing C0 2 in about the same partial pres- sure as in the alveolar air, no apnea supervenes, and if the experiment is repeated several times with progressively declining percentages of THE CONTROL OF THE RESPIRATION 385 C0 2 in the air each time, the length of the apneic pause proportionally increases as the C0 2 pressure in the inspired air diminishes. Periodic Breathing TYPES OF PERIODIC BREATHING In the best known of these, called Cheyne-Stokes respiration, a period of hyperpnea supervenes upon one of apnea, each period following in regular sequence. After an apneic period, the breathing begins at first AAA-/ I 132. Various types of periodic breathing. (From Mosso's "Life of Man in the High Alps.") faintly, gradually becomes more pronounced until it is markedly exag- gerated, and then fades off again to the apneic pause. Sometimes the apneic period is immediately followed by one of intense hyperpnea, there being no gradual increase in the respiratory movements. Between these two types all varieties of the condition are met (Fig. 132). The conditions in which periodic breathing occurs may be divided into 386 THE RESPIRATION physiological and pathological groups. Of the physiological conditions the following may be taken as examples: (1) Breathing in an atmosphere containing a deficiency of 2 ; thus, periodic breathing is very readily pro- duced in persons living in rarefied air. It is often more particularly after returning to air at normal barometric pressure, that the periodic breathing sets in. (2) Breathing through a long tube having a small vessel contain- ing soda lime inserted between the tube and the mouth, the whole capacity of this vessel and tubing being about a liter. This will cause periodic breathing in persons that are susceptible to oxygen deficiency. Even breathing through the tube without soda lime will sometimes cause a periodic type of breathing in such individuals. (3) The initial breathing follow- ing an apnea induced by forced ventilation of the lungs. In this post- apneic periodicity, the apneic periods may at first be quite marked, but as breathing returns they become gradually shorter and the breathing in- tervals gradually longer, until normal respiration is restored (Fig. 131). (4) Restricted breathing, brought about either by limiting the quantity of inspired air or by restricting the respiratory movements by corsets. The pathological conditions in which periodic breathing becomes devel- oped are particularly those associated with renaj^^disej^^njLiie^ebral hemorrhage. In many of these cases, the periodic breathing does not appear to depend on the same factors as are concerned in the experi- mental types. The symptoms would rather appear to depend on some influence of the higher cerebral (supranuclear) centers on the respiratory center. At least some other evidence of disturbance of the cerebral func- tions is always forthcoming, such as a slight paralytic stroke, and the periodic breathing is nearly always aggravated during sleep. Many of these cases are greatly benefited by administration of caffeine. In both the physiological and the pathological groups, the breathing may develop a periodic character only when the person is asleep. Infants or very old people may indeed exhibit a certain degree of periodic breathing apart from any of the above mentioned causative factors. CAUSES OF PERIODIC BREATHING Great interest attaches to an investigation of the causes of periodic breathing, but it can not be claimed that any perfectly satisfactory ex- planation has as yet been offered. Pembrey 31 attributes it to a diminished excitability (a raised threshold) of the respiratory center due to faulty blood supply, the supposition being that, when thus depressed, the normal C H of the blood is unable to excite the center, so that breathing stops. During the resulting apnea, C0 2 again accumulates until it has raised the C H sufficiently to excite the depressed center. Hyperpnea follows, causing a washing out of the C0 2 and a resulting diminution of THE CONTROL OF THE RESPIRATION 387 the effective stimulus, so that again the center fails to be stimulated and apnea supervenes, and so on. Support for this explanation would appear to be furnished by the fact that, when patients exhibiting periodic breath- ing are made to breathe an atmosphere containing a high percentage of C0 2 , the periodicity of the breathing may give place to regular breath- ing; a result which may also be obtained by making such patients breathe in atmospheres rich in oxygen. In the former case, the stimulus is raised to meet the depressed excitability of the center; in the latter, the excitability of the center is increased because of better oxygen supply so that it is enabled to react to the diminished stimulus. But even granted that the excitability of the center is depressed, it is difficult to see why this should occasion a periodic type of breathing unless we as- sume that it is only when stimulus (i. e., C H of blood) and threshold of excitability of the center are adjusted at a certain physiological level that smooth and continuous action can go on. The fact that alterations in the excitability of the respiratory center in clinical conditions are very commonly associated with periodic breath- ing, suggests that similar alterations must be responsible for the experi- mental forms. Support for this view is found in the fact that most of these latter are produced under conditions where there must be a cer- tain degree of anoxemia. Apparently when the oxygen tension of the blood falls to a certain degree the excitability of the center becomes altered and when it is so, the respiratory hormone, afforded by the ten- sion of C0 2 , causes an irregular stimulation, but why it should do so is impossible to explain. A further factor that may come into play is dependent on the time taken for blood to travel between the pulmo- nary alveoli and the medulla. This may explain the gradual rather than sudden development of the apneic and hyperpnejc phases. At present all we can do in attempting to explain this mysterious phenom- enon is to examine the exact conditions under which it occurs. The most simple to consider first is the periodic breathing that is produced in a person susceptible to 2 'want, by breathing through a tube and bottle (of a total capacity of 1 liter), containing soda lime. In such a case no outside air can enter the lungs, so long as the breathing is normal on account of the dead space having been too greatly prolonged. The oxygen tension of the rebreathed air, therefore quickly falls (while at the same time the C0 2 is being ab- sorbed), until at last a point is reached at which the respiratory center is directly stimulated by anoxemia (see page 374). The deep breaths (hyperpnea) which follow, being of greater volume, cause some out- side air to be inspired so that the 2 want is made good and the hy- perpnea again disappears, possibly to the extent of apnea, since now, in 388 THE RESPIRATION consequence of a coincident ''washing out" of C0 2 , there has been a lowering of the C0 2 -tension of the blood below the threshold value. During the apnea the 2 is rapidly used up, till a point is reached at which the center again becomes excited. In such an experiment the effects of anoxemia such as cyanosis may show themselves. That breath- ing under these conditions should be periodic and not merely show a steadily increasing hyperpnea is probably due, as we have seen, to the un- equal rates at which the 2 and C0 2 tensions change in the blood. Be- cause of a " buffer action" the latter fluctuates much less than the for- mer. Another cause for the periodicity is no doubt the delay between the gas exchange in the lungs and the arrival of the arterialized blood in the brain. "When the 2 tension of the blood supplying the respiratory cen- ter falls to so low a level that excitation of the center occurs, the resulting increased breathing aspirates outside 2 into the alveoli. After a moment or so, the 2 is carried by the blood to the center, so that its stimula- Fig. 133. Quantitative record of breathing air through a tube 260 cm. long and 2 cm. in diameter. (From Douglas and Haldane.) tion by 2 deficiency is removed, and it is left in a condition in which it fails to discharge any impulses, since there is a subnormal C0 2 tension of the blood as a consequence of the hyperpnea. A little time must now elapse before the C0 2 again rises or the 2 falls sufficiently to excite the center. Periodic breathing can be brought about by this method in de- cerebrate cats, the animals being caused to respire through a tube and small flask containing soda lime, the total capacity being about 80-100 c.c. That 2 deficiency is responsible for the result is indicated by the fact that administration of 2 through a catheter inserted in the trachea brings about normal breathing. A similar although less marked degree of periodic breathing can sometimes be induced by merely respiring through a long tube without any provision for the absorption of C0 2 . In this case it is more difficult to explain the cause of the periodic breathing, but that the main factor concerned is one of 2 deprivation is evidenced by the fact that in this, THE CONTROL OF THE RESPIRATION 389 as in the previous experiment, the periodic nature of the respiration is immediately changed to the regular breathing if 2 is introduced into the tube. The interest of the experiment lies in the fact that a similar relative elongation of the dead space is probably accountable for the periodic breathing seen in the winter sleep of hibernating animals. Dur- ing this condition, on account of the depression of metabolism less 2 is required and less C0 2 is produced, so that the exchange of gases through the pulmonary endothelium is greatly diminished. The dead space, however, remains of the same capacity, which amounts to the same thing as if the latter had been prolonged under unchanged con- ditions of pulmonary gas exchange. Important evidence that changes occurring in the tensions of 2 and C0 2 in the alveolar air, and therefore in the arterial blood of the respiratory center, are largely responsible for periodic breathing has been secured by studying the condition that develops after a period of apnea produced by voluntary forced breathing. The results of such observations are given in the curve shown in Fig. 131, in which the thin line represents the 2 tension of the alveolar air, the thick line the C0 2 tension. The double line running across the chart repre- sents the average tension of C0 2 during quiet normal breathing. The respiratory movements are represented by the tracing at the foot of the curve, along the abscissa. It will be observed that the oxygen ten- sion falls very rapidly during the apneic period, until just before breath- ing recommences it may be as low as 30-35 mm. Hg instead of the nor- mal of about 95. Meanwhile the C0 2 tension rises from the very low level of 12 mm., at first very rapidly, then more gradually, although, when breathing recommences, it has not yet gained the normal level. As a result of the first periods of breathing, the 2 tension suddenly increases, but the C0 2 falls only slightly. During the next apneic stage the 2 quickly comes down again, and the C0 2 rises so as almost to at- tain normal tension before breathing again supervenes. As the apneic periods subsequently become less pronounced, the C0 2 tension comes to stand almost at its normal level, whereas considerable variations in the 2 tension continue to occur. If the lungs are filled with oxygen by in- haling the gas with the last few deep breaths the return of breathing is delayed and it is not periodic in character. Besides affording substantial support to the hypothesis which was stated above, there are several other interesting features of these results which demand attention. In the first place, it is plain that the body is possessed of some mechanism by which it can prevent great fluctua- tions in the C0 2 tension of the blood, whereas towards 2 no such "buffer action" is displayed. It will further be observed that the CO, 390 THE RESPIRATION tension of the alveolar air rises very rapidly during the first part of the apneic period, and then more gradually, the explanation being that during the forced breathing the C0 2 has been washed out from the blood but not from the body as a whole. As the C0 2 tension becomes lowered in the blood, C0 2 will diffuse out of the tissues to maintain equilibrium between blood and tissues. There must be some considerable lag in this process however, so that when the forced breathing ceases a much lower tension exists in the blood than in the tissues. The rapid rise in alveolar C0 2 early in the apnea is therefore due to diffusion from the tissues up to the equi- librium point. Periodic breathing is produced by forced respiration more readily in rarefied air than at sea level. It was found by Douglas, 26 after breath- ing forcibly for one minute at sea level, that the breathing when it returned showed 8 to 10 different periods of apnea and hyperpnea. On repetition of the experiment at an altitude giving a barometric pres- sure of 600 mm., 25 such periods followed the apnea; at a height cor- responding to 520 mm., 40 periods. Indeed, at high altitudes periodic breathing may be brought about by the slightest alteration in normal respiration ; even taking a deep breath may be sufficient to cause distinct periodicity in the succeeding respirations, and in many persons living at high altitudes periodic breathing is very apt to occur during sleep. As in pathological cases exhibiting Cheyne-Stokes respiration, the peri- odic breathing at high altitudes can be immediately removed by inspir- ing oxygen. Having now considered in detail the conditions which cause altera- tions in the acid base equilibrium of the body, it will repay the reader to refer back to pages 50 and 3'62 where they are discussed from a more general point of view. CHAPTER XLIV RESPIRATION BEYOND THE LUNGS Up to the present our studies in respiration have concerned the various mechanisms involved in bringing about a constant change in the com- position of the alveolar air. We must now consider the nature of the means by which the oxygen is conveyed to the tissues and the carbon di- oxide removed from them. In the first place, it is important to note that it is not for purposes of oxidation in the blood itself that the 2 is required. In its respiratory function this fluid serves as a transporting agency between the lungs and the tissues, in which reside the furnaces of the body that con- sume the 2 and produce the C0 2 . This does not imply that there is no oxidation in the blood itself; indeed, we should expect a certain degree of oxidation because of the fact that the blood contains some living cells the leucocytes. It is scarcely necessary nowadays to offer evi- dence for the foregoing conclusion. One well-known experimental proof consists in replacing the blood in a frog with physiological saline solution and then subjecting the frog with the saline in its blood vessels to an atmosphere of pure 2 , when it will be found that the animal continues to absorb the normal amount of 2 and exhale the normal amount of C0 2 . It respires normally without any blood in the blood vessels. In order that this transportation of gases between the lungs and the tissues may be efficiently performed,, the blood must be provided with means for carrying adequate amounts of gases to supply the requirements of the tissues, both during rest and during their varying degrees of activity. Not only, therefore, must the 2 and C0 2 capacity of the blood be very considerable, but it must be capable of very rapid adjust- ment from time to time. Our problem naturally resolves itself into three parts: (1) the call of the tissues for oxygen (Bar croft) ; or, as it is styled, tissue or internal respiration; (2) the mechanism by which the blood transports the proper amounts of gases to meet the requirements of the tissues; and (3) the mechanism by which the blood gases are exchanged in the lungs ex- ternal respiration. For convenience, however, we shall change this nat- ural order and consider the transportation of the gases first. 391 392 THE RESPIRATION THE TRANSPORTATION OF GASES BY THE BLOOD The Transportation of Oxygen It is plainly not by mere solution in the plasma of the blood that the transportation of 2 occurs, for at the partial pressure of this gas ex- isting in the alveolar air at the temperature of the body the amount that could be dissolved in the blood would be only one-fortieth of that which is actually found to be present. If there were only plasma in the blood vessels, it would require a volume of fluid amounting to 150 kilograms or more in order to convey the necessary amount of 2 from the lungs to the tissues; that is, the contents of the vascular system would weigh twice as much as the average weight of a man. The substance that carries the 2 in the blood is the hemoglobin, which may be described as a highly complex iron compound of protein espe- cially evolved for the purpose of transporting 2 . In some of the lower animals other compounds exist in the blood for this purpose, but none of them is to be compared in its efficiency with hemoglobin. They are merely poor imitations of it. Regarding the conditions under which hemoglobin combines with or delivers up 2 , the first question that presents itself is whether or not the reaction is a strictly chemical one. If so, a definite amount of 2 must be capable of combining with a definite amount of hemoglobin. It is impossible to secure hemoglobin of sufficient purity to test this rela- tionship directly on hemoglobin itself, so that we must test it indirectly by examining the combining equivalent between 2 and that portion of the hemoglobin molecule upon which the combining power depends. This is the part of the molecule containing iron. Now, if we compare the amount of 2 w r hich hemoglobin can take up with the amount of iron present in the hemoglobin, we shall find that one atom of iron becomes combined with two atoms of 2 . Evidently, then, we are here dealing with a definite chemical reaction occurring between the 2 and the iron of the hematin portion of the hemoglobin. This relationship is known as "the specific oxygen capacity of hemoglobin. " In showing that the union of 2 and hemoglobin occurs according to chemical laws, we throw into prominence consideration of the mechanism by which the 2 , combined with hemoglobin in the blood, is rapidly de- livered up in the capillaries so as to supply the tissues with their require- ment, and is then as rapidly recombined again in the lungs. Moreover, we must reconcile the idea of a specific 2 capacity with the well- known observation that the hemoglobin in the circulation may be united with considerably less 2 than the total amount possible. In other words, we must recognize that, although it is essentially a chemical reaction, RESPIRATION BEYOND THE LUNGS 393 the combination of 2 with hemoglobin is greatly influenced by other factors, and that it is these that are likely to be of physiological impor- tance. In order to understand the conditions under which hemoglobin will take up and give off 2 in the animal body, we must study the combining power of hemoglobin when it is exposed to different partial pressures of 2 (for laws governing this, see page 353). In the blood, the ex- tremes of the partial pressure of 2 are represented, at the one end, by that in the alveolar air, which we have seen to be about 100 mm. Hg, and at the other, by that existing in the tissues, such as muscle, which has been shown to be not more than 19 or 20 mm. Hg. We must further bear in mind that the 2 in its passage from the alveolar air to the hemo- globin and from the hemoglobin to the tissues, is transmitted in solution through the plasma; that is, so far as the supply of 2 to the tissue cells is concerned, the plasma serves as the immediate source. Since the tis- sues are using up 2 at a very great speed, especially when active, and are thus tending to lower the tension of 2 in the plasma, favorable con- ditions have to be created whereby the hemoglobin liberates 2 at the same rate as that at which it is leaving the plasma. In brief, it is the 2 tension of the plasma in the tissue capillaries that is the important' factor, the hemoglobin merely serving as a storehouse, which delivers its 2 at just such a rate as to maintain the plasma-oxygen tension at a constant level. It is obviously of the greatest importance that we should understand how this mechanism of an adequate plasma-oxygen tension is maintained. Methods of Investigation. We must remember that the combination of 2 and hemoglobin, being a definite chemical reaction, will be re- versible, and must, therefore, obey the laws of mass action (see page 23) according to the equation: Hb -f- 2 ^Hb0 2 . In order to ascertain the position of the balance of this equation at different partial pressures of 2 , that is, the relative quantities of oxy- and reduced hemoglobin formed in a solution of hemoglobin when this is shaken with 2 at differ- ent pressures, we may proceed as follows: A few c.c. of the hemoglobin solution are placed in each of a series of vessels called tonometers, like those shown in Fig. 134. In addition to the hemoglobin solution, each tonometer contains a mixture of nitrogen and 2 in different propor- tions. Suppose we use six vessels and in No. 1 have pure nitrogen; in No. 2, nitrogen containing 5 mm. partial pressure of 2 ; in No. 3, 10 mm. ; in No. 4, 20 ; in No. 5, 50 ; and in No. 6, 100. We now rotate the tonometers in a water-bath at body temperature for about twenty min- utes, so that, by the formation of a thin film of hemoglobin solution over the walls of the vessel, perfect equilibrium between the atmosphere and 394 THE RESPIRATION the fluid may be attained (see page 355). A measured quantity of hemo- globin solution (0.1 or 1.0 c.c.) is then removed from each tonometer and placed, together with some very dilute ammonia to lake the blood, in one of the small bottles of the differential manometer, shown in Fig. Fig. 134. Barcroft's tonometer for determining the curve of absorption of oxygen by hemoglobin or blood. (From Starling's Physiology.) 135.* This manometer consists in principle of a graduated U-shaped tube of narrow bore, containing clove oil, the free end of the U-tube being connected with small bottles provided with some device so that Fig. 135. Barcroft's differential blood gas manometer. The capillary U-tube contains clove oil. The pockets on the sides of the blood bottles should be deeper. For manipulation see context. two fluids can be placed in each of them but kept unmixed until the bottle is violently shaken. The three-way stopcock between the small bottles and the manometer serves to permit communication of the manometer with the outside air. *The blood-gas manometers are made in two sizes for use with 1 c.c. and 0.1 c.c. quantities of blood, respectively. The results with these small quantities are as accurate as with larger amounts. RESPIRATION BEYOND THE LUNGS 395 An equal quantity of hemoglobin solution that has been saturated with oxygen i. e., oxyhemoglobin is placed in the bottle on the other end of the manometer tube from that containing the bottle with the un- saturated hemoglobin solution. The bottles having been attached to the manometer with the stopcocks open to the outside, the apparatus is placed in a water-bath until the temperature conditions are constant. The manometers are then closed to the outside air and the bottles are shaken in order that the hemoglobin solution that is unsaturated with 2 may take up 2 from the atmosphere in the bottle until it becomes saturated. The resulting shrinkage in the volume of the atmosphere on the side of the unknown hemoglobin solution causes the clove oil Fig. 136. Barcroft blood gas manometer. This form can be used either as a differential manometer (page 403) or for direct measurement of pressure. For the latter purpose one bottle is removed and the pressure of gas generated in the other bottle is measured by the height to which it raises the clove oil in the distal tube of the manometer, the meniscus in the proximal limb being readjusted to its original level by compression with the brass screw of the rubber tube shown in the center. meniscus to move towards that side, the degree of movement being pro- portional to the initial unsaturation of the hemoglobin. The manometer tubes are then again brought into communication with the atmosphere so that the meniscus of clove oil may move back to its old level, and the bottle with saturated hemoglobin is removed from the manometer and a drop or two of a saturated solution of potassium ferricyanide placed in the separate compartment of the bottle without allowing it to mix with the hemoglobin. The bottle is then reattached, the temperature conditions readjusted, the manometer closed off from the ouside air, and the apparatus again shaken so that the ferricyanide mixes with the 396 THE RESPIRATION hemoglobin solution. This drives off all the 2 from the oxyhemoglobin solution, and, therefore, raises the pressure in the atmosphere of that bottle so that the clove oil moves to the opposite side of the manom- eter, the degree of displacement being proportional to the amount of oxyhemoglobin. We have now all the necessary data for estimating the relative amounts of reduced hemoglobin in the hemoglobin solution as removed from the tonometers, for it is plain that the second estimation, as described above, tells us how much oxyhemoglobin might have been formed had all the hemoglobin been saturated and the first one, how much 2 had yet to be taken up by the original hemoglobin solution to produce saturation. The Dissociation Curve. The next step is to plot the results obtained from the various hemoglobin solutions in the form of a curve. This is known as the dissociation curve of "hemoglobin. It is plotted with the relative percentages of reduced and oxyhemoglobin in each of the solu- tions along the ordinates, and the partial pressures of 2 in millimeters of mercury to which they were exposed along the abscissae. The curve thus drawn is exactly of the same shape as that which would be pro- duced if we were to place the tonometers in a row at distances from one another corresponding to the partial pressure of 2 which each con- tained, and then to mark on each tonometer the relative amounts of reduced and oxyhemoglobin found in the solutions after shaking. A line joining these marks on the tonometers would then exactly corre- spond to the curve drawn by the method described above. This will be clear from the accompanying figure from Barcroft's book (Fig. 137). In such a chart the space below the curve can be taken to represent the percentage of oxyhemoglobin (red in chart), and that above it of reduced hemoglobin (blue in chart), at the varying partial pressures of 2 which are indicated along the abscissae as being contained in the at- mosphere of the tonometers, and which must be proportional to the partial pressure of 2 in the solution in which the hemoglobin is dis- solved. Difference between Curves of Blood and Hemoglobin Solutions. The curve obtained from pure hemoglobin solutions is very far, however, from clearing up the problem as to how the blood absorbs and discharges 2 . On the contrary, it makes this problem appear all the more difficult, for, according to the curve (Fig. 138-Curve A) the hemo- globin is already more than half combined with 2 at a partial pressure of this gas of no more than 10 mm. Hg, which means that in the low partial pressure of 2 existing in the capillaries the oxyhemoglobin, in- stead of readily yielding up its load of 2 , would greedily retain prac- tically the whole of it. The curve, in other words, would satisfactorily 20 4 100 mm. pressurt Percentage saturation with oxygen 10 20 30 40 50 QO 70 80 90 100 Oxygen pressure mm. Fig. 137. Upper left hand, percentage saturation of hemoglobin with oxygen at 37 C. cor- responding to oxygen pressures of 0, 10, 20, 40 and 100 mm. of oxygen, respectively. Upper right hand, the same spaced with the oxygen pressure as the abscissae. Lower figure, dissociation curve representing the equilibrium between oxygen, oxyhemoglobin (red) and reduced hemoglobin (purple). (From Joseph Barcroft.) RESPIRATION BEYOND THE LUNGS 397 explain why hemoglobin should readily absorb 2 from the alveolar air, but would fall far short of explaining how this 2 is readily released when it is required in the tissues. Obviously there is some artificial con- dition present in the above experiment which can not obtain in the nat- ural environment of the blood. Since hemoglobin takes up 2 in proportion to its iron, it can not be 10 ZO 30 W 30 60 70 80 nerve. The nmonnt of s ju'u-o is controlled through the produed 1\\ nervous stimulation VTES, bO"W6VeP, never found 10 l>e so ns ihnt produced by secretin, nnd for se\ ernl years nfler the discovery of mivsiouxiY TIII' UI,ANI>S 11 u la lie r hormone, much doubt existed as to the correctness of Pavlov's claim. As ill many oilier fields of physiological science, investigators at- tempted to show that one or the other mechanism obtained, and they were n of inclined to consider the possibility that both mechanisms might exist side by side. That such is the case, however, is clear from the most recent work, in which it has been found that if proper precautions are taken, repeated stiTrmlntinn -nf t.hn irnqnn nevvfl flnna nn.11 fnyfhftafirrftfiny> of pancreatic juice which, besides being less copious thantKaT following ii. in. !''>:. 147. Three preparations of pancreatic acini stained by eosin orange tolni.lm i.iuo. The I'-ini of Fig. I were from a gland after vagus stimulation, and it is noted that besides free ex- trusion of the granules, globules staining with orange (and appearing in deep black in the photo- iraph) have formed and may be present in the ductules. Some in Iheir sl.mnm: properties, !>eeonuii>; li-hl led (d.uk >M.I\ in photo>;r, wen i loin danda excited by secretin. No globules appear; the gram M'l" 11 in the clear protoplasm. (Prom llabkin, Rubaschktn and Ssa I he globules. Innvev I-1O. The .leini in I I secret in injection, differs from it in the important fact that it contains not t.rypsinogen but activejCTisin. Since the normal pancreatic juice contains trypsino^cn, this last mentioned fact would appear to indicate that \ a^'iis cont rol of Ihe normal secretion can not he an important affair. The vjiirns secretion of pancreatic juice is, moreover, paralyzed by atro- pine, which has no action on the secretin mechanism (cf. Bayliss). 464 DIGESTION The copious secretion of pancreatic juice produced by secretin, on the one hand, and the scanty, thick secretion produced by vagus stimula- tion, on the other, calls to mind similar differences observed in the secre- tion of saliva as the result of chorda-tympani or sympathetic stimulation. It will be remembered that from these latter results it was concluded that there must be secretory and trophic fibers concerned in the control of the activities of gland cells. Interesting corroboration of this conclusion has recently been obtained by histological examination of the pancreas fol- lowing secretin or vagus activity. After the repeated injection of secre- tin, it is difficult to observe any signs of fatigue in the cells ; the zymogen granules remain practically as numerous as in a resting gland, but in the clear protoplasm of the outer third of the cell, it is said that fine channels of fluid can be seen. Through these channels water is believed to pass from the blood towards the lumen and in its course to carry with it some of the zymogen granules, without, however, changing them. Thus, when the gland cells are stained with eosin and orange, after secretin activity some of the zymogen granules can occasionally be seen in the lumen of the acini stained with eosin like those in the cell itself. After vagus stimulation the appearances are different ; not only are the granules more decidedly removed from the cells, but they undergo a preliminary change ; they lose the property of staining with eosin and become stained with orange, at the same time increasing in size so as to form vacuoles. These vacuoles may wander into the ductules, and when they are present here they are stained by orange (Figs. 146 and 147) (Babkin, etc. 10 ). Why there should be both a nervous and a hormone control of the pan- creatic secretion is not clear. This gland, unlike the gastric and salivary glands, is not called upon to become active all of a sudden, and it is dif- ficult to see what could serve as the normal stimulus operating through the nervous pathway. Taking it all in all, it is probably safe to con- clude that the nervous mechanism is relatively unimportant, and that under normal conditions it seldom if ever is called into operation. Cor- roboration for this view is afforded by the fact, above mentioned, that the pancreatic juice produced by vagus stimulation contains active tryp- sin, which is not the case with normal pancreatic juice. CHAPTER LII PHYSIOLOGY OF THE DIGESTIVE GLANDS (Cont'd) Up to the present we have been concerned with the physiological activi- ties of digestive glands in general, but now we must study each of them separately in order to find out the conditions under which they become stimulated to activity in the normal process of digestion. The secretion of each gland has a definite role assigned to it in the complex and lengthy process of digestion. It takes up its work where the preceding secre- tion left off; e.g., the pepsin of gastric juice digests protein so far as proteoses and peptone; the trypsin of pancreatic juice then attacks the proteoses and peptone, and the resulting lower degradation products are finally attacked by the erepsin of the intestinal juice. The secre- tions of the various glands are, therefore, required in a certain definite order they are correlated; and we must now give some attention to the precise conditions upon which the activity and correlation depend. THE NORMAL CONDITIONS UNDER WHICH THE GLANDS BECOME STIMULATED TO INCREASED ACTIVITY To make possible such observations on the normal activities of the glands, a preliminary operation has to be performed so as to bring the duct of the gland to the surface of the body and permit of the observa- tion of its secretory activity after the animal has recovered from the immediate effects of the operation. We owe to Pavlov 1 the surgical technic by which these conditions can be fulfilled. The general principle of the operation, in the case of glands provided with ducts, consists in making a circular cut through the mucous membrane surrounding the opening of the duct and then, after dissecting the duct free, stitching the edges of the cut to the skin wound. Healing then takes place without the formation in the duct of any stricture due to cicatricial tissue. After the wound has healed, the secretion can readily be collected in a receiver attached over the duct fistula, the animal being in every other way in a perfectly normal condition. In the case of glands not provided with a duct, other methods must be adopted to collect the secretions. These will be described elsewhere. 465 466 DIGESTION THE NORMAL SECRETION OF SALIVA The duct fistula can in this case be made either for the submaxillary gland, representing a mucous gland, or for the parotid, representing a serous gland. Under ordinary conditions there is very little secretion from either duct. When secretion occurs, it is, of course, caused by influences acting on a nerve center or centers in the medulla oblongata, the exact location of which for the different glands has been worked out in recent years by Miller. 11 The impulses acting on these centers may be transmitted along afferent nerves coming from the mucous membrane of the mouth, nares, etc., or by impulses which we may call psychic, trans- mitted from the higher nerve centers. The reflex secretions caused by impulses traveling by the afferent nerve from the mouth, etc., have been called unconditioned, and those from the higher nerve centers, condi- tioned. With regard to the former, there is considerable discrimination in the type of stimulus that will be effective. Thus, if the dog for most of the experiments have been performed on this animal is given meat, a secretion of thick, mucous saliva will be observed to occur (submaxil- lary gland). On the other hand, if the meat is dried and pulverized, the secretion which it calls forth will be very copious and watery (par- otid gland). There is, then, an obvious association between the nature of the secretion and the function it will be called upon to perform when it becomes mixed with the food. The mucous secretion called forth by meat will serve to lubricate the bolus of food and thus facilitate its swallowing, whereas the thin watery secretion produced by the dry powder will have the effect of washing the powder from the mouth. It is evident that the mechanical condition of the food partly deter- mines its exciting quality. Mechanical stimulation of the mucosa in it- self is, however, not an adequate stimulus, for if pebbles are placed in the mouth, little secretion occurs, whereas with sand, secretion immedi- ately becomes copious. The nerve endings also respond to chemical stimuli. Thus, weak acid causes a copious secretion, while alkali has no effect; disagreeable, nauseous substances also excite secretion. The above dif- ferences in the response of the glands according to the mechanical condi- tion of the food has been observed in the case of the parotid gland, increase in the submaxillary secretion being obtained only when actual foodstuffs are placed in the mouth. The investigations that have been made on the conditions of psychic secretion of saliva are still more interesting and important. Their im- portance depends not so much on the information they give us concern- ing the secretion of saliva as such, as on the methods they afford us for investigating the various conditions that affect the psychic processes PHYSIOLOGY OF THE DIGESTIVE GLANDS 467 associated with, the taking of food. It is from the psychic rather than from the physiologic standpoint, therefore, that these observations are of importance, for they permit us, by objective methods, to study on dumb animals problems that would otherwise be beyond our powers of investigation. Many of the results, with their bearing on the functions of the higher nerve centers, have been discussed elsewhere (Chapt. Oil). Meanwhile, however, even at the risk of repetition it may not be out of place to cite a few of the most interesting experiments. If we tease a hungry animal with food for which he has a great appe- tite, a copious secretion of saliva immediately occurs. If we go on teas- ing him without giving him food, and repeat this procedure on several succeeding days, it will be found that gradually he no longer responds to the teasing by increased salivation. Evidently, therefore, the reflex is conditioned upon the animal's afterward receiving the food. The experiment may be performed in another way. If, for example, we offer the animal some food for which he has no appetite, no secre- tion of saliva will occur; but, if at the end of the process we give him appetizing food, it will be found after repeating this procedure on several successive days that the presentation of the unappetizing food calls forth a secretion. He has learned to associate the presentation of unappetizing food with the subsequent gratification of his appetite. The experiment can even be performed so that a definite interval of time elapses between the application of the stimulus and the salivation: if the animal is teased on successive days with food for which he has an appetite but is not given the food until after ten or twenty minutes, presentation of this food will come to be followed by salivation not immediately, but after the exact interval of time that had been allowed to intervene in the training process. During this interval there must be an inhibition of psychic stimulation of the salivary centers by other nerve centers. It is of great interest that this inhibition may itself be inhib- ited by various forms of stimulation of the nervous system (see page 957). THE SECRETION OF GASTRIC JUICE Methods of Investigation There being no common duct, the secretion of the gastric glands is a much more difficult problem to investigate than is that of glands which, like the salivary, are supplied with ducts. One of the most interesting chapters in the history of physiology concerns the methods which, from time to time have been evolved for the collection of this juice and for studying the digestive processes in the stomach. Prominent among the problems confronting the earlier investigators was the question whether the main function of the stomach is to crush or triturate the food or to act on it chemically. The great French scientist Eeaumur and a little later the Italian Abbe Spallanzani 468 DIGESTION (1729-1799) attacked this problem by methods that anticipated those of Behfuss and Einhorn. Spallanzani ultimately devised the method of swallowing small perforated wooden tubes containing foodstuffs and covered by small linen bags. After the bags were passed per rectum, he found that considerable erosion or digestion of the food had occurred, but that the wooden tubes, however thin-walled they might be, were not crushed. In order to secure samples of the gastric juice free from food, the only method available to the older investigators consisted in swallowing sponges at- tached to threads, which after being for some time in the stomach were withdrawn and squeezed dry of juice. The next great contribution came from this country, where, in 1833, Dr. Beaumont, while a surgeon in the service of the American troops located at Mackinaw, made ob- servations on a Canadian voyageur by the name of Alexis St. Martin, who by the premature discharge of his gun had wounded himself in the stomach, the wound never healing but leaving a permanent gastric fistula. Beaumont arranged to keep Alexis St. Martin in his service for several years, during which time he made numerous observa- Fig. 148. Diagram of stomach showing miniature stomach (5) separated from the main stomach (V) by a double layer of mucous membrane. A.A., is the opening of the pouch on the abdominal wall. (Pavlov.) tions on the process of digestion in the stomach observations many of which are of great value even at the present day. By none of these methods, however, could a sample of pure gastric juice be secured while the digestive process was actually in progress. To make the collection of such a sample possible, Heidenhain devised a method of isolating portions of the stomach wall as pouches opening through iistulse on the abdominal wall. The results of Heidenhain 's experiments are, however, open to the objection that the secretion in the isolated pouches may not really correspond to that occurring in the main stomach, since the connections of the pouches with the central nervous system must have been severed. In order that these connections might remain as nearly intact as possible, the Russian physiologist, Pavlov, 1 devised an ingenious operation in which the pouch, or " minia- ture stomach," remains connected with the main stomach through a considerable width of mucous and submucous tissue and in which the nervous connections are not severed. The essential nature of this operation will be evident from the accompanying diagram. (Fig. 148.) PHYSIOLOGY OF THE DIGESTIVE GLANDS 469 The most recent investigations have been made by Cannons and by Carlson.* The former fed animals food impregnated with bismuth subnitrate, and then exposed the animal to the x-rays. A shadow is produced by the food mass in the stomach, and from the changes in the outline of this shadow facts have been collected, not only concerning the movements of the viscus, but also concerning the rate of discharge of food into the intestine and therefore the duration of the gastric digestive process. Carlson's contribution has been rendered possible by his good fortune in having in his service a second Alexis St. Martin, a man with complete closure of the esophagus and a gastric fistula large enough to permit of direct inspection of the interior of the stomach. Seizing the opportunity thus presented, Carlson during the last four or five years has devoted his attention exclusively to a thorough investigation, not only of the movements of the stomach, but also of the rate of secretion of the gastric juice under different conditions. He has also, with praiseworthy enthusiasm and keen scientific spirit, extended his observations both on laboratory animals and on himself and his coworkers, so as not to incur the error, which is all too frequently made of confining the observations to one species of animal. The Nervous Element in Gastric Secretion The first stimulus to the secretion of gastric juice is nervousjn origin, and is dependent on the gratification of the appetite and the pleasure of taking food. This fact, after having been suggested by observations made in the clinic, was first thoroughly investigated by Pavlov, who for this purpose observed the gastric secretion flowing either from a fistula of the stomach itself, or from a "miniature stomach," in dogs in which also an esophageal fistula had been established. When food was given by mouth to these animals, it was chewed and swallowed in the usual manner, but before reaching the stomach, it escaped through the esopha- geal fistula. This experiment is known as that of "sham feeding." Within a few minutes after giving food the gastric juice was found to be secreted actively, and if the feeding process was kept up, which could be done almost indefinitely since the animal never became satisfied, the secretion continued to flow. Thus, in one instance Pavlov succeeded in collecting about 700 c.c. of gastric juice after sham feeding an animal for five or six hours in the manner above described. After the stomach has emptied itself of the food taken with the pre- vious meal, it is said by Pavlov to contain only a little alkaline mucus. The more recent work of Carlson, however, shows that this is not strictly the case, there being more or less of a continuous secretion of gastric juice An the entire absence of food. The amount varies from a few c.c. up to / 60 c.c. per hour, more secretion being produced when it is collected every | five or ten minutes than if it is collected every thirty or sixty, thus indicating that, ordinarily, some escapes through the pylorus into the duodenum. The secretion contains both pepsin and hydrochloric acid. As to the cause of this continuous secretion, little is known. It may be an example of the periodic activities of the digestive glands described by 470 DIGESTION Boldyreff, or it may in part be due to a psychic stimulation dependent upon the thought of food. That the latter is probably not the cause, is indicated by the fact that, at least in Carlson's patient, the psychic juice could not be made to flow short of giving food. The sham feeding causes stimulation of the gastric secretion through impulses transmitted to the stomach along the vagus nerves; for it has been found, in animals in which the vagus nerve has been cut, that the sham feeding no longer induces a secretion of gastric juice. The ques- tion therefore arises as to how the nerve center is stimulated. Three possible causes may be considered: (1) mechanical^ stimulation of the sensory nerves of the mouth; (2) chemical stimulation of these nerves; (3) the agreeable stimulation of the taste buds and olfactory endings concerned in the tasting of food. In investigating these possibilities, mechanical stimulation was readily ruled out by showing that mere taking of solid matter in the mouth did not excite any secretion, although it might cause a flow of saliva. Mere chemical stimulation could not be the cause, for no secretion was induced by placing substances such as acetic acid or mustard oil in the mouth. By exclusion, then, it would appear that the adequate stimulus must consist in the agreeable stimula- tion of the taste buds, etc. that is to say, in the gratification of appetite. Further justification for this conclusion was readily secured by noting that foodstuffs for which the animal had no particular desire or appe- tite failed to excite the secretion. Most dogs, for example, although they may take it, are not particularly fond of bread, and when fed with it, these animals did not produce any appetite juice. In one animal that showed considerable liking for bread, active secretion occurred when he was fed with this foodstuff. Pavlov further noted that usually it was not necessary actually to allow the animal to take the food into his mouth, but that mere teasing with savory food was sufficient to cause the secretion, and that in highly sensitive animals even the noises and other events usually asso- ciated with feeding time were sufficient to excite the secretion. In the case of a hungry animal, the mere approach of the attendant with food, or some other noise or action definitely associated with feeding time, was a sufficient excitant. The appetite juice when started was found to persist for some time after the stimulus causing it had been removed. Carlson has succeeded in confirming in man most of these observa- tions. He noted, however, that the secretion produced by seeing or smelling or thinking of food is much less than would be expected from Pavlov's observations on dogs. Even when his subject was hungry, Carlson did not observe that the bringing of a tray of savory food into the room caused any secretion of gastric juice. It is, of course, to be PHYSIOLOGY OF THE DIGESTIVE GLANDS 471 expected that the quantity of the psychic secretion will not be the same in different individuals. It has been observed by Pavlov, for example, to vary considerably in the case of dogs, and it is very likely that it will vary still more in man, with his more highly complicated nervous system. In no case could Carlson observe any secretion of gastric juice to be pro- duced by having his patient chew on indifferent substances, or by stim- ulating the nerve endings in the mouth by substances other than those directly related to food. In man the rate of secretion is proportional to the palatability of the food, the smallest amount, during twenty minutes' mastication of pal- atable food, being 30 c.c. and the largest 150 c.c., in a series of 156 obser- vations. A typical curve showing the amount of the secretion is given in Fig. 149. To construct this curve the gastric juice was collected diy- \ 25' 30' 50* 55 Chewing food Fig. 149. Typical curve of secretion of gastric juice collected at 5-minute intervals on mas- tication of palatable food for 20 minutes. The rise in secretion during the last 5 minutes of mastication is due to chewing the dessert (fruit) for which the person had great relish. (From Carlson.) ing five-minute intervals while the man was chewing a meal of average composition and of his own choice. An interesting feature depicted on this curve is that the secretion rate was highest in the last five-minute period, this being the time during which the dessert was being taken, for which this man had a great relish. Quite clearly there was a direct relation between the rate of the secretion of the appetite juice and the palatability of the food. It will further be observed that it took only from fifteen to twenty minutes after discontinuing the chewing before the juice returned to its original level. The practical application of these facts in connection with the hygiene of diet and the feeding of patients during convalescence, is obviously very great. However perfect in other regards a diet may be, it will probably fail to be digested at the proper rate unless it is taken with relish. Frequent feeding with favorite morsels is more likely to be fol- 472 DIGESTION lowed by thorough digestion and assimilation than occasional stuffing with larger amounts. We see too in these experiments an explanation of the well-established practice of starting a meal with something savory. A hors d'oeuvre is nothing more than a physiological stimulant to appetite. It is also interesting from a practical standpoint to observe that with those who have a keen relish for sweetmeats the taking of des- sert has a real physiological significance, for, as in Carlson's patient, it stimulates toward the end of a meal a further secretion of the gastric juice, and thus insures a more rapid digestion of the food. Good cooking, it should be remembered, is really the first stage in digestion, and it is the only stage over which we can exercise voluntary control. The Hormone Element in Gastric Secretion Although gastric digestion is initiated by the appetite juice, it is clear that this alone can not account for all the secretion that occurs during the time the food is in the stomach. After an ordinary meal this occupies usually about four hours, whereas we have seen, particularly from Carlson's observations, that the appetite juice lasts only for some fifteen or twenty minutes after the exciting stimulus has been removed. The appetite juice, in other words, serves only to initiate the process of secretion, and the question arises, What keeps up the secretion during the rest of gastric digestion? The answer was furnished by Pavlov, who observed animals in which not only a miniature stomach had been made, but a fistula into the main stomach as well. The behavior of the secre- tion of gastric juice as a whole could be followed by collecting that which was secreted in the miniature stomach, for it was shown, in con- trol experiments, that this secretion runs strictly parallel with that in the main stomach, being quantitatively a definite fraction of it accord- ing to the relative size of the miniature stomach and qualitatively identical. The miniature stomach, in other words, mirrors the events of secretion in the main stomach. It was observed that when the animal was allowed to take the food into the main stomach by the mouth and esophagus, the secretion from the miniature stomach continued to flow until the process of gastric digestion had been completed, a result which was quite different from that obtained after sham feeding. The only possible explanation for this result is that the food in the stomach sets up secretion as a result of local stimulation. To investigate the nature of this local stimulation, whether mechanical_or_ chemical, food and other substances were placed in the main stomach through the gastric fistula without the animal's knowledge so as to avoid possible psychic stimulation, and the secretion observed from the miniature stomach. When the mucous membrane of PHYSIOLOGY OF THE DIGESTIVE GLANDS 473 the main stomach was stimulated mechanically, as by placing inert objects such as a piece of sponge or sand in the stomach, no secretion occurred. Evidently, therefore, the stimulus_is_dependent upon some chemical quality of the food. By introducing various foods it was found that there is considerable difference in the degree to which they can excite the secretion. Water and egg white had a slight effect; bread and starch in the dry state had no effect. On the other hand, when protein that had been partly digested by means of pepsin and hydrochloric acid was introduced into the stomach, it immediately called forth a secretion. The conclusion is that the partly digested products, even of insipid food, are capable of directly exciting the secretion. These include proteoses and peptones, and it was, therefore, of great interest to find t^at a. solution of commercial peptone is also an fvffee.t.ivp ptinmlns. This is a result of deep significance, for it indicates that the food which has been partially digested by the appetite juice will serve as a stimulus to continued secretion. The psychic juice has been aptly called the "ignition juice," because by producing partial digestion it serves to ignite the process of gastric secretion. Experimental evidence of its great importance in gastric digestion was secured by Pavlov in experiments in which he placed weighed quantities of meat attached to threads in the stomach through a gastric fistula, and after some time removed them and determined by the difference in weights the extent to which they had become digested. It was found that when the appetite juice was excited by sham feeding at the same time that food was placed directly in the stomach, its diges- tion was much more rapid than in cases in which it was placed in the stomach without the animal's knowledge, as when he was asleep. Other foods having a direct stimulating effect on the gastric secre- tion are meat extracts and, to a certain extent, milk. This effect of meat extract is interesting in connection with the practice of taking soup as a first or early stage in dining. It not only excites the appetite juice, but also serves as a direct stimulus to the gastric secretion. As to the nature of the mechanism J)y which this direct secretion takes place, it was shown by Popielski 12 that the secretion still occurs after all the nerves proceeding to the stomach are cut. Evidently, therefore, it is independent of the extrinsic nerve supply of the viscus. As a result of his experiments Popielski concluded that the secretion must depend on a local reflex mediated through the nerve structures present in the walls of the stomach itself. Another explanation of the result has, however, in recent years been given more credence by the experiments of Bayliss and Starling on the influence of hormones on the secretion of pancreatic juice (cf. page 460). Edkins 18 suggested that a similar 474 DIGESTION process in the stomach might account for the continued secretion of gastric juice. To test such a possibility this investigator, after ligating the cardiac sphincter in anesthetized animals, inserted a tube into the pyloric end of the stomach, through which he placed in the stomach about 50 c.c. of physiological saline. After this had been in the stomach for an hour, he found that no water was absorbed, and that it contained neither hydrochloric acid nor pepsin. On the other hand, if during the time the saline was in the stomach a decoction of the mucous membrane of the pyloric end, made either with peptone solution or with a solution of dextrine, was injected intravenously in small quantities every few min- utes, the saline contained distinct quantities of hydrochloric acid and pep- sin. Furthermore, it was found that, if the peptone solution or the dextrine solution alone was injected intravenously, there was no such evidence of gastric secretion. The conclusion which Edkins drew from his experi- ments is to the effect that the half-cjigested products of the earlier stages of gastric digestion act on the mucous membrane of the stomach so as to produce a hormone, which is then carried by the blood to the cells of the gastric glands, upon which, like secretin, it directly develops an exciting effect. This hormone has been called gastrin. These observa- tions of Edkins have been confirmed, and they explain very simply how gastric secretion is maintained after the cessation of the secretion of the appetite juice. ,By such a mechanism gastric juice would continue to be secreted so long as any half -digested food remains in the stomach. The action of gastrin is the first instance of a hormone control of the digestive glands. In the earlier stages of digestion, the secretion of saliva and appetite juice is mediated through the nervous system, because these juices must be produced promptly. In the later stages of gastric diges- tion, such promptitude in response on the part of the gland is no longer necessary, so that the slower, more continuous process of hormone con- trol is sufficient. Quantity of Gastric Juice Secreted According to Carlson, the total amount of gastric juice secreted in man on an average meal composed of meat, bread, vegetables, coffee or milk, and dessert, amounts to about 700 c.c., being divided into 200 c.c. in the first hour, 150 in the second, and 350 c.c. during the third, fourth and fifth hours. These figures were estimated partly on the basis of observations made on the man with the gastric fistula, and partly from the data supplied by Pavlov's observations on dogs. Carlson believes that Pavlov overestimated 'the relative importance of the appetite juice in gastric digestion. He found, for example, that after division of both vagus nerves in dogs normal gastric digestion might be regained a few PHYSIOLOGY OF THE DIGESTIVE GLANDS 475 days after the operation, although, of course, under such circumstances no appetite juice could have been secreted. Moreover, he observed that cats when forcibly fed with unpalatable food may digest that food as rapidly as when they eat voluntarily. In support of his contention, Carlson states that he has frequently removed all of the appetite juice from his patient's stomach before the masticated meal was put into it without any evident interference with the digestive process. Fat has a distinct inhibiting influence on the direct secretion of gas- tric juice; cream takes considerably longer to be be digested than milk, 345678123456789 10 123456 Flesh. 200 gm. Bread, 200 gm. Milk, 600 c.c. Fig. 150. Cubic centimeters of gastric juice secreted after diets of meat, bread, and milk. (From Pavlov.) Hours 1 10.0 c An 2345678234 5678923456 Mm of'Protei Column <=> g { / ^ / \| \ x-"' . / \ t \ ^ \ / \ / \ I \ ^ Flesh, 200 gm. Bread, 200 gm. Milk, 600 c.c. Fig. 151. . Digestive power of the juice, as measured by the length of the protein column digested in Mett's tubes, with diets of flesh, bread, and milk. (From Pavlov.) and the presence of oil in the stomach delays the secretion of juice poured out on a subsequent meal of otherwise readily digestible food. By col- lecting all of the gastric juice from the miniature stomach after feeding by mouth with quantities of different protein-rich foods containing the same quantities of nitrogen, interesting observations have been recorded concerning the amount of juice secreted and its proteolytic power. The results of some of the experiments are shown in the accompanying curves (Figs. 150 and 151). 476 DIGESTION It will be seen that the most abundant secretion occurs with meat, that of milk being not only smaller but also slower in starting. The digestive power is greatest in the case of bread. THE INTESTINAL SECRETIONS Pancreatic Juice Regarding the natural secretion of pancreatic juice, little need be added to what has already been said (see page 460). The secretion begins when the chyme., enters the duodenum, and attains its maximum when the outflow of this is greatest. By collecting the juice from a permanent fistula of the pancreatic duct, it has been found that the amount varies with different foods. When quantities of food containing equivalent amounts of nitro- gen are fed, the greatest secretion is said to occur with bread and the least with milk. Such differences are probably dependent upon the amount of acid secreted in the stomach and passed on into the duodenum. It was thought at one time that, besides variation in quantity, the nature of the enzymes in the pancreatic juice might vary according to the kind of food. This, however, has been shown not to be the case. Bile The secretion of bile runs pracJieallyLjsarallel with that of pancreatic juice. The liver is producing bile more or less continuously, since besides being a digestive fluid it is also an excretory product. The bile produced between the periods of digestion is mainly stored in the gall bladder. When the acid chyme comes in contact with the duodenal mucous mem- brane, it excites afferent nerve endings that cause a reflexjiontraction of the gall bladder, and this expresses some of the bile into the duodenum. The secretin, which the acid at the same time produces, besides affecting the pancreas, acts on the liver cells, stimulating them to the increased secretion of bile. Thus, by a nervous reflex operating oil the gall bladder and later by a hormone mechanism operating on the liver cell, the increased secretion of bile is insured throughout digestion. Of the bile discharged into the intestine, a certain proportion of the bile salts is reabsorbed into the portal blood. When these arrive at the liver they also excite secre- tion of bile, thus assisting secretin in maintaining the secretion through- out the process of intestinal digestion. Intestinal Juice The secretion of intestinal juice, or succus entericus, can obviously be studied only after isolating portions of the intestine and connecting them PHYSIOLOGY OF THE DIGESTIVE GLANDS 477 with fistulse of the abdominal walls. It appears here again that both a nervous and a hormone mechanism exist. Mechanical stimulation of the intestinal mucous membrane causes an immediate outflow of intestinal juice, the purpose of which under normal conditions is evidently to assist in moving forward the bowel contents. This mechanically excited juice does not contain any enterokinase and only small amounts of the other enzymes. Further evidence for nervous control of the secretion of intes- tinal juice has been obtained by isolating three pouches of intestine be- tween ligatures, and then denervating the central pouch by carefully cutting all the nerves without wounding the blood vessels. On returning the pouches to the abdomen and leaving them several hours, it has been Fig. 152. Loop of intestine after tying off the portions, cutting the nerves running to the middle portion, and returning the loop to the abdomen for some time. (From Jackson.) found that the middle pouch becomes distended with secretion, whereas the two end pouches remain empty (Fig. 152). If the pouches are left for several days in the abdomen, however, the secretion from the denervated portion disappears again. The explanation of the result is possibly that the nerves under ordinary conditions convey impulses to the intestinal glands, which tonically inhibit their activity. The existence of hormone control is evidenced by the fact that no enterokinase is present in the intestinal juice unless pancreatic juice is placed in contact with the mucous membrane. Injection of pancreatic juice into the blood, however, does not cause any secretion of intestinal juice ; whereas the injection of secretin has such an effect. CHAPTER LIII THE MECHANISMS OF DIGESTION MASTICATION, DEGLUTITION, VOMITING Mastication By the movements of the lower jaw on the upper, the two rows of teeth come together so as to serve for biting or crushing the food. The resulting comminution of the food forms the first step in digestion. The up and down motion of the lower jaw results in biting by thei incisors, and after the mouthful has been taken, the side to side movements enable the grinding teeth to crush and break it up into fragments of the proper size for swallowing. The most suitable size of the mouthful is about 5 c.c., but this varies greatly with habit. After mastication, the mass weighs from 3.2 to 6.5 gm., about one-fourth of this weight being due to saliva. The food is now a semifluid mush containing particles which are usually less than 2 mm. in diameter. Some, however, may measure 7 or even 12 mm. Determination of the proper degree of fineness of the food is a func- tion of the tongue, gums, and cheeks, for which purpose the mucous membrane covering them is supplied with very sensitive touch nerve endings. The sensitiveness of the tongue, etc., in this regard explains why an object which can scarcely be felt by the fingers seems to be quite large in the mouth. If some particles of food that are too large for swallowing happen to be carried backward in the mouth, the tongue returns them for further mastication. The saliva assists in mastication in several ways: (1) by dissolving some of the food constituents; (2) by partly digesting some of the starch; (3) by softening the mass of food so that it is more readily crushed; (4) by covering the bolus with mucus so as to make it more readily transferable from place to place. The secretion of saliva is therefore stimulated by the chewing movements, and its composition varies according to the nature of the food (page 466). In some animals, such as the cat and dog, mastication is unimportant, coating of the food with saliva being practically the only change which it undergoes in the mouth. In man the ability thus to bolt the food can readily be acquired, not, how- ever, without some detriment to the efficiency of digestion as a whole. Soft 478 THE MECHANISMS OF DIGESTION 479 starchy food is little chewed, the length of time required for the mastica- tion of other foods depending mainly on their nature, but also to a certain degree on the appetite and on the size of the mouthful. It can not be too strongly insisted upon that the act of mastication is of far more importance than merely to break up and prepare the food for swallowing. It causes the food to be moved about in the mouth so as to develop its full effect on the taste buds; the crushing also releases odors which stimulate the olfactory epithelium. On, these stimuli depend the satisfaction and pleasure of eating, which in turn initiate the process of gastric digestion (see page 470). The benefit to digestion as a whole of a large secretion of saliva, brought about by persistent chewing, has been assumed by some to be much greater than it really is, and there has existed, and indeed may still exist, a school of faddists who, by deliberately chewing far beyond the necessary time, imagine themselves to thrive better on less food than those who occupy their time with more profitable pursuits. Deglutition or Swallowing After being masticated the food is rolled up into a bolus by the action of the tongue against the palate, and after being lubricated by saliva is moved, by elevation of the front of the tongue, towards the back of the mouth. This constitutes the first stage of swallowing, and is, so far, a voluntary act. Al^put this time a slight ^ap^t.QTy f* v nt T> a / *1n of the diaphragm occurs the so-called respiration) of swallowing and the mylohyoid quicklv__cpntracts. wit]LJ;hej?pnsequence that the bolus passes between the pillars of the fauces. This marks the beginning of the second stage, the first event of which is that the bolus, by stimulating sensory nerve endings, acts on nerve centers situated in the medulla oblongata so as to cause a coordinated series of movements of the muscles of the pharynx and larynx and an inhibition for a moment of the respiratory center (page 351). The movements alter the shape of the pharynx and of the various openings into it in such a manner as to compel the bolus of food to pass into the esophagus (see Fig. 153) : thus, (1) the soft palate becomes elevated and the posterior wall of the pharynx bulges forward so as to shut off the posterior nares, (2) the posterior pillars of the fauces ap- proximate so as to shut off the mouth cavity, and (3) in about a tenth of a second after the mylohyoid has contracted, the larynx is pulled up- wards and forwards under the root of the tongue, which by being drawn backwards becomes banked up over the laryngeal opening. This pulling up of the larynx brings its upper opening near to the lower half of the dorsal side of the epiglottis, but the upper half of this struc- 480 DIGESTION ture projects beyond and serves as a ledge to guide the bolus safely past this critical part of its course. (4) As a further safeguard against any entry of food into the air passages, the laryngeal opening is narrowed by approximation of the true and the false vocal cords. So far the force which propels the bolus is mainly the contraction of the mylohyoid, assisted by the movements of the root of the tongue. When it has reached the lower end of the pharynx, however, the bolus readily falls into the esophagus, which has become dilated on account of a reflex inhibition of the constrictor muscles of its upper end. This so- called second stage of swallowing is, therefore, a complex coordinated movement initiated by afferent stimuli and involving reciprocal action Fig. 153. The changes which take place in the position of the root of the tongue, the soft palate, the epiglottis and the larynx during the second stage of swallowing. The thick dotted line indicates the position during swallowing. of various groups of muscles: inhibition of the respiratory muscles and of those that constrict the esophagus, and stimulation of those that elevate the palate, the root of the tongue, and the larynx. It is purely an involuntary process. The third stage of deglutition consists in the passage of the swallowed food along the esophagus. The mechanism by which this is done de- pends very much on the physical consistence of the food. A solid bolus that more or less fills the esophagus excites a typical peristaltic wave, which is characterized by a dilatation of the esophagus immediately in front of and a constriction over and behind the bolus. This wave travels down the esophagus in man at such a rate that it reaches the cardiac sphincter in about five or six seconds. On arriving here the cardiac THE MECHANISMS OF DIGESTION 481 sphincter, ordinarily contracted, relaxes for a moment so that the bolus passes into the stomach. In many animals, including man and the cat, the peristaltic wave travels much more rapidly in the upper part of the esophagus than lower down because of differences in the nature of the muscular coat, this being of the striated variety above, and of the non- striated below. The purpose of more rapid movement in the upper part is no doubt that the bolus may be hurried past the regions where, by distending the esophagus, it might interfere with the function of neigh- boring structures, such as the heart. In other animals, as the dog, the muscular fiber is striated all along the esophagus, and the bolus of food correspondingly travels at a uniform, quick rate all the way. It takes only about four seconds for the bolus to reach the stomach in the dog. The peristaltic wave of the upper part of the esophagus in the cat and presumably in man, unlike that of the intestines (see page 501), is trans- mitted by the esophageal branches of the vagus nerves. If these are severed, but the muscular coats left intact, the esophagus becomes dilated above the level of the section and contracted below, and no peristaltic wave can pass along it; on the other hand, the muscular coat may be severed (by crushing, etc.) but the peristaltic wave will continue to travel, provided no damage has been done to the nerves. In the lower part of the esophagus, however, the wave of peristalsis, like that of the intestines, travels independently of extrinsic nerves. This has been observed in animals in which all of the extrinsic nerves have been cut some time previously. This difference between the upper and the lower portions is associated with the difference in the nature of the muscular fibers above noted (Meltzer). 14 The propagation of the wave by the nerves in, the upper part of the esophagus indicates that the second stage and the first part of the third stage of deglutition must be rehearsed, as it were, in the medullary centers from which arise the nerve fibers to the pharynx and the upper levels of the esophagus. It is thought that the discharges from these local centers are controlled by a higher swallowing center situated in the medulla just above that of respiration, the afferent stimuli to which proceed from the pharynx by the fifth, superior laryngeal, and vagus nerves. The exact location of the sensory areas whose stimulation is most effective in initiating the swallowing reflex varies considerably in different animals. In man it is probably at the entrance to., the pharynx; in the dog it is on the posterior wall. A foreign body placed directly in the upper portion of the- esophagus of man has been observed to remain stationary until the individual made a swallowing movement. The afferent fibers in the glossopharyngeal nerve exercise a powerful inhibitory influence on the deglutition center as well as on that of respira- 482 DIGESTION tion. Thus, if swallowing movements are excited by stimulating the cen- tral end of the superior laryngeal nerve, they can be instantly inhibited by simultaneously stimulating the glossopharyngeal, and the respiratory movements stop in whatever position they may have been at the time. When the glossopharyngeal nerves are cut, the esophagus enters into a condition of tonic contraction, which may last a day or so. This shows that the inhibiting impulses are acting continuously. This inhibition of the esophagus is indeed a most important part of the process when liquid or semiliquid food is swallowed. By contraction of the mylohyoid muscle fluids are quickly shot down the dilated esopha- gus, at the lower end of which, on account of the closure of the cardiac sphincter, they accumulate until the arrival of the peristaltic wave which iias meanwhile been set up by stimulation of the pharynx. As the per- istaltic wave approaches the cardia the sphincter becomes inhibited al- lowing the fluids to be passed into the stomach. If the swallowing is ^immediately repeated the esophagus remains dilated because peristalsis is inhibited and the fluid collects above the closed cardiac sphincter until the last mouthful is taken Avhen the peristaltic wave passes over the esophagus and sweeps the contents through the sphincter which is at the same time relaxed. When a series of swallows in rapid succes- sion, as in drinking, is made the cardiac sphincter may remain inhibited throughout, so that the fluid passes directly into the stomach unassisted bv \ the peristaltic wave. The Cardiac Sphincter The passage between the esophagus and the stomach is guarded by the cardiac sphincter or cardia. This exists in a permanently con- tracted state, or tonus, superimposed on which from time to time are rhythmic alternations of contraction and relaxation. This tonus is never very pronounced. In man it is said that a water pressure of from 2 to 7 cm. applied to the esophageal side of the sphincter will drive air or water into the stomach, this pressure being less than that of a column of fluid filling the thoracic esophagus in the erect position. During repeated deglutition the tonus becomes less and less marked, and after a number of swallows the sphincter may become completely relaxed. When this relaxation disappears, however, the sphincter becomes more contracted than usual and remains so for a longer time. The tonic condition of the sphincter is controlled by the vagus nerve, stimulation of which causes relaxation with an after-effect of strong contraction. Mechanical or chemical stimulation of the lower end of the esophagus increases the tonus of the sphincter. Forcing of the sphincter from the stomach side requires a higher pressure than from the esopha- geal. Eructation of gas, for example, does not take place until intra- THE MECHANISMS OF DIGESTION 483 gastric pressure has risen to about %Lc,m. of water. In deep anesthesia, however, intragastric pressure may rise considerably higher without forcing the sphincter. In animals fed with starch paste impregnated with subnitrate of bis- muth and then examined by means of the x-rays, the variation in degree of tone of the sphincter has been observed to be responsible for occasional regurgitation of some of the gastric contents into the esophagus up to the level of the heart or even to the base of the neck. The presence of the gastric contents in the esophagus starts a peristaltic wave, which pushes the material back again into the stomach. This peristaltic wave starts in the absence of any other phases of the deglutition process, indicating that it has been excited by the presence of the material in the esophagus itself, and belongs, therefore, to the lower order of peristaltic wave, as seen in the intestines but not in the upper half of the esophagus. Regur- gitation of food into the esophagus occurs only when the intragastric pressure is fairly high. It may last for a period of from twenty to thirty minutes after the meal is taken, and disappears when the tonus of the sphincter becomes increased as a result of the presence in the gastric contents of free hydrochloric acid. Much information has been secured by listening with a stethoscope to the sounds caused by swallowing and by observing with the x-ray the shadows produced along the course of the esophagus when food impreg- nated with bismuth subnitrate is taken. When a solid bolus is swal- lowed only one sound is usually heard, but with liquid food there are two, one at the upper end, due to the rush of the fluid and air, and the other at the lower end (heard over the epigastrium), four or six seconds later, due to the arrival here of the peristaltic wave with the accompanying opening of the cardiac sphincter and the escape of the fluid and air into the stomach. Sometimes, when the person is in the horizontal position, this second sound may be broken up into several, indicating that, unassisted by gravity, the fluid does not so readily pass through the sphincter. The x-ray shadows yield results in conformity with the above. After swallowing milk and bismuth, for example, the shadow falls quickly to the lower end of the esophagus and then passes slowly into the stomach. When the passage of a solid bolus is watched by the x-ray method, its rate of descent will be found to depend on whether or not it is well lubricated with saliva; a well lubricated bolus takes from eight to eighteen seconds. Vomiting Vomiting is usually preceded by a feeling of sickness or nausea, and is initiated by a very active secretion of saliva. The saliva, mixed with 484 DIGESTION air, accumulates to a considerable extent at the lower end of the esopha- gus, which it distends. A forced inspiration is now made, during the first stage of which the glottis is open so that the air enters the lungs, but later the glottis closes so that the inspired air is sucked into the esophagus, which, already somewhat distended by saliva, now becomes markedly so. The abdominal muscles then contract so as to compress the stomach against the diaphragm and, simultaneously, the cardiac sphincter relaxes, the head is held forward and the contents of the stomach are ejected through the previously distended esophagus. The compression of the stomach by the contracting abdominal muscles is assisted by an actual contraction of the stomach itself, as has been clearly demonstrated by the x-ray method. After the contents of the stomach itself have been evacuated, the pyloric sphincter may also relax and permit the contents (bile, etc.) of the duodenum to be vomited. The act of vomiting is controlled by a center located in the medulla, and the afferent fibers to this center may come from many different regions of the body. Perhaps the most potent of them come from the sensory nerve endings of the fauces and pharynx. This explains the tendency to vomit when the mucosa of this region is mechanically stimu- lated. Other afferent impulses come from the mucosa of the stomach itself, and these are stimulated by emetics, important among which are strong salt solution, mustard water and zinc sulphate. Certain other emetics, particularly tartar emetic and apomorphine, act on the vomit- ing center itself, and can therefore operate when given subcutaneously. Afferent vomiting impulses also arise from the abdominal viscera, thus explaining the vomiting which occurs in strangulated hernia, and in other irritative lesions involving this region. X-ray observations have been made on the movements of the stomach of cats after the admin- istration of apomorphine (Cannon). The first change observed is an inhibition of the cardiac end of the stomach, which becomes a perfectly flaccid bag. About the midregion of the organ, deeper contractions then start up, which sweep towards the pylorus, each contraction stopping as a deep ring at the beginning of the vestibule, while a slighter wave con- tinues. A very strong contraction at the incisura angularis finally develops and completely divides the gastric cavity into two parts. On the left of this constriction the stomach remains completely relaxed, but at the right of it waves continue running over the vestibule. It is while the stomach is in this condition that the sudden contraction of the dia- phragm and abdominal muscles shoots the cardiac contents into the relaxed esophagus. As these jerky contractions are continued, the gastric walls seem to reacquire their tone. Afferent impulses from the duodenal mucosa are even more potent for the initiation of the vomiting reflex than are impulses arising in the stomach itself. CHAPTER LIV THE MECHANISMS OF DIGESTION (Cont'd) THE MOVEMENTS OF THE STOMACH The Character of the Movements Even from the earliest days it has been recognized that the stomach performs two important functions: (1) receiving the swallowed food and then discharging it slowly into the intestine, and (2) initiating the chemical processes of digestion. In order to understand the mechanism by which the stomach collects and then discharges the food, it is neces- sary first of all to recall certain anatomical facts concerning the organ, and for this purpose it is most convenient to accept the description given by Cannon, which is illustrated in the accompanying figure. The organ is divided into a cardiac and a pyloric portion by a deep notch in the lesser curvature, called the incisura angularis. The cardiac portion is further subdivided into two by the cardiac orifice. The part which lies, in man, above a line drawn horizontally through the cardia is the fundus. The part lying between the fundus and the incisura angularis is known as the body of the stomach, which, when full, has a tapering shape. The pyloric portion lying on the right of the incisura angularis is further divided into three parts: the pyloric vestibule, the antrum and the pyloric canal, the latter of which lies next the pyloric sphincter and in man measures about 3 cm. in length (see Fig. 154). From a study of its development and structure and from a consideration of its behavior when examined radiographically Cole 30 believes that the ascending or first part of the duodenum virtually belongs to the stomach and should be so considered when the gastric movements are under investigation. Since this portion of the duodenum receives and retains the chyme for a short time after its egress from the stomach and in radiographs appears to surmount the pyloric sphincter, he has termed it the " reservoir cap" or pilleus ventriculi. The filled stomach of a person standing erect is so disposed that the greatest curvature forms its lowest point, which may be considerably below the umbilicus. As digestion proceeds and the stomach empties, the greater curvature becomes gradually raised, so that ultimately the pylorus comes to be the most dependent part of the stomach. From 485 486 DIGESTION these and many other observations it is certain that the emptying of the stomach does not at all depend on the operation of the force of gravity. Indeed, that this can not be the case is perfectly clear when we con- sider the disposition of the stomach in quadrupeds. Exact observation on the movements which the stomach performs from the time it is filled with food till it empties, have been made by the x-ray method, first introduced by Cannon. 15 The method consists in feed- ing the animal with food that has been impregnated with bismuth sub- nitrate, then exposing him to the x-ray and either taking instantaneous photographs of the shadows or observing them by means of a fluorescent screen. In the accompanying figure (Fig. 155) the outline of the shadow cast by the stomach is shown at intervals of an hour each during diges- tion. Soon after the stomach has become filled, peristaltic waves are seen to take their origin about the middle of the body of the viscus, and to course toward the pylorus. The region above the origin of these waves that is, the cardiac half of the body of the stomach and all the fundus, often called the cardie pouch is free from peristaltic Avaves but is the seat of a tonic contraction which as digestion proceeds presses steadily with increasing force upon the mass of food and delivers it slowly to the lower and more active por- tion of the stomach. As the food passes into the lower part of the stomach, the cardiac pouch becomes gradually reduced in size until finally when the stomach is empty its outline, as re- vealed by radiographs, departs from the pyri- form shape characteristic of the full organ to assume a more or less tubular form. From this description it is evident that the function of the cardiac portion of the stomach is to serve as a reservoir for the food which by a slow contraction of the gastric wall is gradually delivered into the lower and more motile portion of the stomach. The motor phenomena of this portion are of a complex nature and will now be considered. Cole, 30 by the employment of serial x-ray photographs taken at short intervals has shown that this portion of the stomach undergoes a succes- sion of rapid changes in shape (Fig. 156), due to the peristaltic waves which course over this region toward the pyloric sphincter. The waves Fig. 154. Schematic outline of the stomach. At C is the cardia: F, fundus; I A, incisura angularis; B. body; PC, pyloric canal; P, pylorus. The antrum is the portion from IA to PC inclusive. (From Cannon.) THE MECHANISMS OF DIGESTION 487 in their passage do not progress as an unvarying band of constriction but exhibit alternately phases of increased and diminished tone phases of contraction and relaxation. The period of gastric activity intervening between a phase of complete relaxation and the reappearance of this phase is termed a gastric cycle. In some types of gastric motility a peristaltic wave takes but one gastric cycle to complete the journey from its origin to the pylorus. Such a wave would show in consequence one phase each of complete relaxation and maximal contraction. In other types two, three or four gastric cycles may occur during the passage of the wave. The waves therefore are spoken of as one, two, three or Fig. 155. Outlines of the shadows cast by the stomach at intervals of an hour each after feeding a cat with food impregnated with bismuth subnitrate. (From Cannon.) four cycle types of peristalsis respectively. It follows from this that at any moment the stomach will show one, two, three, or four waves accord- ing to the particular type of peristalsis which is present. The four cycle is the most common type ; the rapid one cycle type is very rare. The duration of a gastric cycle is from 2-3 seconds. The time required for the passage of a four cycle type of peristaltic wave from its origin to the pyloric sphincter therefore will be from 8-12 seconds. It rarely exceeds 10 seconds. Following the cardiac analogy Cole divides the gastric cycle into a systole and a diastole. In the former which occupies 7/10 of the cycle the constrictions produced by the peristaltic waves are most marked. Diastole occupies 3/10 of the cycle and represents the period during which relaxation is taking place. (Fig. 156, VII-X.) 488 DIGESTION The adaptation of the capacity of the normal stomach to the volume of its contents is remarkable. When the stomach of a living animal is dis- tended by fluid the intragastric pressure shows very little change even up to the point of rupture of the viscus (Grey 16 ). After excision, the stomach loses in great part this adaptive power which seems to be con- trolled not through the extrinsic nerves, but by the nervous elements residing in the gastric wall itself. The Effect of the Stomach Movements on the Food This has been studied: (1) by dividing the food into portions that are differently colored and, after some time, killing the animal, freezing Fig. 156. Skiagrams of human stomach at intervals after food illustrating a gastric cycle. (From Cole.) the stomach and making sections of it (see Fig. 157) ; (2) by mak- ing little pellets of bismuth subnitrate with starch and observing their behavior under the x-rays; or (3) by removing samples of the stomach contents by means of a stomach tube (Rehfuss tube) inserted so that its free end lies in either the cardiac or the pyloric region. By the first of the above methods it has been found that the first mouthfuls of food lie along the greater curvature, where they form a layer over which that subsequently swallowed accumulates, with the last por- tions next the cardia. The pepsin and hydrochloric acid of the car- THE MECHANISMS OF DIGESTION 489 diac end, therefore, act soonest on the first swallowed portion of a meal, and the more recently swallowed central masses are not affected by the secretions for some time, so that opportunity is given for the saliva mixed with the food to develop its digestive action. As has been shown by removing the stomach contents with a tube at various periods after feeding with starchy food, considerable amylolysis may occur for some time. When separate samples are removed in this way from the cardiac and pyloric parts, it has been found that after half an hour the contents of both have about the same percentage of sugar, but that for some time after this interval the cardiac contents contain considerably more sugar than the pyloric. Later the percentages of sugar again become about equal, no doubt on account of diffusion. The diastatic action in the fundus is finally brought to an end when the contents become completely permeated by the hydrochloric acid. In this connection it is worthy of note that the addition of hydrochloric Fig. 157. Section of the frozen stomach (rat) some time after feeding with food given in three differently colored portions. (From Howell's Physiology.") acid up to the point of neutrality greatly accelerates the rate of diastatic digestion. As the outer layers of food in the stomach become partly digested on account of the action of the pepsin and hydrochloric acid, the food is slowly pressed into the active right half of the stomach, where by the action of the peristaltic waves it is moved on to the pyloric vestibule. By observing the x-ray shadows cast by two pellets of bismuth subni- trate it has been noted by Cannon that, as the peristaltic wave approaches a pellet, it causes it to move forward more rapidly for a short distance, but soon overtakes it and in doing so causes the pellet to move back a little towards the fundus. This backward movement is less than the forward movement, so that after the wave has passed, the position of the pellet is a little forward of that which it would have occupied had there been no wave. The behavior of the pellet, and, therefore, of the stomach contents, is very like that of a cork floating at the edge of the sea; as each wave approaches, it hurries the cork on a little, but after 490 DIGESTION its passage the cork recedes again until the second wave carries it still a little farther forward. As the peristaltic wave approaches the pyloric vestibule and becomes more powerful its effect on the pellets becomes more marked. These observations made on cats and other laboratory animals no doubt also apply in the case of man. Removal of the contents of the cardiac and pyloric regions separately with a stomach tube after feeding with a test meal part of which was colored with carmine or charcoal, has shown that none of the coloring material was present in the contents of the pyloric end up to twenty minutes or so after the food had been taken. It then appeared but at first only in traces. Another important distinction between the food in the two portions of the stomach relates to its consistency. In the pyloric end it is semifluid and homogeneous in character; in the cardiac end, on the other hand, it is a lumpy, rather incoherent mass. The gastric movements must greatly facilitate the digestive processes in the stomach. In the cardiac part the undisturbed condition of the food will, as we have seen, facilitate the digestive action of ptyalin, whereas in the body of the stomach the peristaltic waves, besides mov- ing the food onward, will tend to bring fresh portions of mucous mem- brane and food in contact, so that the latter becomes more thoroughly mixed with the pepsin and hydrochloric acid. In the pyloric part, where no hydrochloric acid is secreted, the contents, already sufficiently acid in reaction, become more thoroughly churned up with the local pepsin secretion, so that proteolytic action progresses very rapidly. The peristaltic waves also facilitate absorption from the stomach of such substances as glucose in concentrated solution and, probably, of hydro- lyzed protein ; water, however, is not absorbed. The fact that the mucosa of the vestibule has, relatively to the cardiac end, few secreting glands is in harmony with the view that absorption is an important function of this part of the stomach. The observations of Carlson, 17 Ginsburg 18 and others indicate that the usually accepted explanation of the pains of gastric and duodenal ulcers, namely, corrosion and irritation of exposed nerve endings in the gastric and duodenal mucosae by highly acid stomach contents, is not correct. The pains of ulcer may be present when the contents of the stomach are alkaline and they may be absent when marked hyperacidity exists. Ac- cording to these authors the pains are analogous in origin to those of hunger, and are the result of contractions of the stomach and duodenum, the nerves of which are in a hyperexcitaBIe~stateV^ Hyperacidity then, will affect the pain of ulcer in so far only as it increases the motility of the stomach. THE MECHANISMS OF DIGESTION 491 THE EMPTYING OF THE STOMACH The Control of the Pyloric Sphincter Recent experimental work demands that the view originally advanced by Pavlov and elaborated by Cannon, namely that the acidity of the chyme constitutes the chief factor in the control of the pyloric sphincter, be abandoned. This conception of the pyloric mechanism fails to account for the rapid passage of w^ater and egg white into the duodenum, and leaves unexplained the normal emptying time of the stomach in certain pathological states in which the secretion of acid is suppressed or greatly reduced. From the study of serial radiographs Cole 30 showed that in man a def- inite relationship existed between the movements of the antrum and those of the pyloric sphincter. The activities of the sphincter were found to be directly proportional to the magnitude of the antral contractions; Avhen the motility of the antrum was low the sphincter contractions were feeble, powerful antral waves on the other hand were associated with vigorous sphincter movements. Chyme passed into the duodenum during gastric systole but not during diastole. Luckhardt, Phillips and Carl- son 31 combining fluoroscopic observations with graphic records, obtained by means of a balloon inserted into the stomach, have shown that the passage of chyme into the duodenum coincides with peristaltic waves travelling over the stomach. The gastric contents collected from a duode- uostomy made immediately below the pyloric sphincter rarely showed the presence of free acidity as tested by congo-red. It has been noted that water placed in the stomach passed through the sphincter in gushes of varying intervals apart, but the emissions on the whole showed evidence of rhythm and it was assumed on this account that they were dependent upon peristaltic waves (Ivy). The water as it left the stomach was neutral in reaction. These observations in the main have been confirmed and extended by other investigators. The more precise study of the interdependence of the antral and sphincter movements has been made by Wheelon and Thomas 32 who combined the study of radiographs with graphic records obtained by balloons placed respectively in the antrum and pyloric canal. In this manner tracings could be taken of movements occurring simultaneously in the two regions. These observers find that when the peristaltic wave commencing in the body of the stomach and trav- elling over the vestibule reaches the antrum the sphincter becomes relaxed so that any material swept forward by the constricting wave is free to pass through the pyloric opening. The wave is sufficiently broad to involve the whole antrum at one time and indeed it involves 492 DIGESTION the sphincter before it has begun to disappear from the antrum with the result that for a short period the antrum and sphincter together are in the contracted state. Soon after this, the antrum enters upon its neg- ative phase (relaxation) the sphincter being still contracted. These facts are shown in Fig. 158. Fluoroscopic examination of the movements of the human stomach showed that some chyme escaped through the sphincter during the passage of every peristaltic wave over the antrum. On the other hand the reaction of the contents of the stomach or duo- denum did not have any influence on the emptying. When, for exam- ple, tenth-normal hydrochloric acid was poured through a tube into the antrum, in sufficient quantity to render its contents distinctly acid, no effect upon the sphincter movements or their relation to the antral waves could be demonstrated. One to five per cent sodium bicarbonate solu- tions likewise were without effect upon the opening of the sphincter. Fig. 158. Tracings showing the relationship between contractions of the antrum (lower tracing) and the pyloric sphincter (upper tracing). Note that the antrum precedes the sphincter but that the latter contracts before the antrum has relaxed. (From Wheelon and Thomas.) Acids introduced through a tube into the duodenum in quantities suf- ficient to render the chyme in this situation of an H-ion concentration considerably in excess of that normally present was unable to prevent relaxation of the sphincter in response to an antral wave. Alkaline solutions in the same region failed to produce sphincter relaxation. The various foodstuffs, carbohydrates, proteins and fats took approximately the same time to leave the stomach, and each type of food commenced to pass through the pyloric opening within a few minutes (3-12) after its ingestion. (McClure, Reynolds and Swartz. 34 ) The foregoing account of the movements of the antrum and pyloric sphincter indicates that the "law of the intestine" (page 501) with some minor modifications, directs the activities of these portions of the stom- ach. The experiments also appear to establish definitely the paramount importance of the movements of these regions as a factor in gastric THE MECHANISMS OF DIGESTION 493 evacuation. The sphincter opens in response to every peristaltic wave arriving at the antrum and apparently does so blindly without regard to the reaction of the gastric or duodenal contents. That some factor other than gastric peristalsis, however, may influence the emptying time is probable. It is difficult, otherwise, to account for the rapid passage from the stomach of water and egg-white and the retention of other substances until they reach a more or less fluid consistency. The later work of Wheelon and Thomas 33 points to the existence of an intimate relationship between the activities of the duodenum below Fig. 159. Diagram of the relationships of the contractions of antrum, sphincter and duo- denum and of the passage of chyme from the antrum into the duodenum 3, Ci, Cz. The seg- mental waves of the duodenum are marked A, B, C t D, and E and it will be noted that A oc- curs when the antrum is completely relaxed and the sphincter contracted. It will therefore assist to empty the "reservoir cap" of the duodenum. The duodenum is seen to be relaxed to its greatest extent after the antrum has ceased contracting and when the sphincter is contracted. (After Wheelon and Thomas.) the " reservoir cap" and the movements of the antrum and pyloric sphinc- ter. When simultaneous tracings were taken of the antrum and sphinc- ter and of the duodenum below the "cap" definite relationships were found to exist between the activities of these portions of the digestive tube. These relationships are shown diagrammatic ally in Fig. 159. The duodenum enters upon its positive phase 2% seconds after the commence- ment of the sphinster contraction and at this moment the antrum is relax- ing and remains relaxed throughout the sphincter and duodenal contrac- tions. The sphincter enters upon its negative phase a short time prior to the maximal contraction of the duodenum and the greatest relaxation 494 DIGESTION of the duodenum occurs a short time after the commencement of the sphincter contraction. Superimposed upon the duodenal peristaltic waves are rhythmical segmentary movements (A, B, C, D, E) . The first of these occurs at the commencement of the peristaltic wave and coin- cidently with the maximal height of the sphincter contraction. The sec- ond segmentary wave surmounts the crest of the peristaltic wave in the duodenum and occurs during relaxation of the sphincter. The peristaltic waves in the duodenum by the withdrawal of material from the " reservoir cap" will aid in the evacuation of the latter. This receives the chyme during the contraction of the antrum but none can return to the antrum when this relaxes since now the sphincter is closed. The motility of the stomach appears to influence duodenal peristalsis and in this way indirectly aids in the withdrawal of material from the ''cap" which it is the purpose of the antral and sphincter activities to fill. On the other hand duodenal motility influences the activity of the sphincter for stimulation of the duodenum below the "cap" in addi- tion to a contraction at the stimulated point and relaxation below, pro- duces a sharp contraction of the sphincter followed by a prolonged nega- tive phase. Sphincter motility unassociated with antral activity may in this way be induced. From these considerations it may be seen that the peristaltic waves in the duodenum which hasten the emptying of the cap by the withdrawal of its contents are probably, through their influ- ence upon the sphincter, a factor also in the filling of the "cap" from the stomach side. That the degree of receptivity of the duodenum is an important factor in emptying the cap is indicated by the observation that a dog starved for 18 hours emptied its stomach of an 8 oz. barium meal in 80 minutes while a second meal of the same amount was not entirely removed after 2% hours. Influence of Pathological Conditions on the Emptying An important surgical application of these facts concerns the behavior of food after gastroenterostomy. It has been thought that this operation would cause the food to be drained from the stomach into the intestine and thus leave the region of the stomach between the fistula and the pylorus inactive. This assumption is based on the idea, which we have seen to be erroneous, that gravity assists in the emptying of the stomach. As a matter of fact, it has been found that, if the gastroenterostomy is fnade when there is no obstruction at the pylorus, the chyme takes its Aiormal passage through the sphincter and, almost without exception, '/none leaves by the fistula. When the pylorus is partly occluded, the food sometimes passes in the usual way, and sometimes by the fistula. The cause for this predilection for the pyloric pathway depends on the THE MECHANISMS OP DIGESTION 495 pressure conditions in the gastric contents. Gastroenterostomy, there- fore, is efficient only when gross mechanical obstruction exists at the pylorus. The operation should never be performed in the absence of demonstrable organic pyloric disease. Another objection to gastroenterostomy in the presence of a patulous pyloric sphincter rests on the fact that the food, after passing the sphinc- ter and moving along the intestine, mav__again enter the stomach through the fistula. This is most likely to occur when the stomach is full of food, for under these conditions the stretching of its walls separates the edges of the opening, the intestine being drawn taut between the edges, so that the opening between the stomach and the intestine assumes the form of two narrow slits, which act like valves permitting the food to enter but preventing its escape from the stomach. Only seldom under these circumstances can any food pass into the intestine beyond the stomach opening. Repeated vomiting after gastroenterostomy has been observed in experimental animals only when obstructive kinks or other demonstrable obstacles were present in the gut, the obstruction being lo- cated in that part of the intestine beyond its attachment to the stomach. When the pyloric obstruction is complete, food must, of course, leave by the fistula, digestion by the pancreatic juice and bile being still car- ried on because of the fact that for a considerable distance down the intestine, secretin, which we have seen is essential for the secretion of these fluids, is still produced by the contact of the acid chyme with the intestinal mucosa. Further provision for adequate digestion of food in such cases is secured, as some of the food after leaving the fistula passes back for a certain distance into the duodenum, where, however, it soon excites peristaltic waves, which again carry it forward. This insures thorough mixing with the digestive juices. From their experi- mental experience Cannon and Blake 19 recommend that, when the fistula has to be made, it should be as large as possible and near the pylorus, and that the stomach afterwards should not be allowed to become filled with food. To avoid kinking of the gut, they also recom- mend that several centimeters of the intestine should be attached to the stomach distal to the anastomosis. The consistency of the food appears to have little influence on its rate of discharge from the stomach at least in the case of potatoes. Distinctly hard particles in the food retard the stomach evacuation. There is usually a considerable amount of gas in the part of the stomach above the entrance of the cardia, on account of which this part of the stomach has sometimes been called the stomach bladder. In the upright position this gas forms a bright area in the x-ray plate (Fig. 155), but when the person reclines it spreads to a new location. Its presence may 496 DIGESTION influence gastric digestion by preventing the contact of the food with the mucous membrane, and by interfering with the efficiency of the peri- staltic waves in moving the food. Considerable gas therefore retards the emptying of the stomach, as has been shown experimentally by x-ray observations on animals fed with the standard amount of food followed by the introduction of air. It was noted that the air did not diminish the frequency or strength of the peristaltic waves, but that these could not efficiently act on the food. When along with gas there is also atony of the stomach w r alls, the retardation in the discharge will, of course, be still more pronounced. The temperature of the swallowed food does not appear to have much influence on the stomach movements or on the the rate of discharge from the organ. CHAPTER LV THE MECHANISMS OF DIGESTION (Cont'd) THE MOVEMENTS OF THE INTESTINES The length of the small intestine and the size of the cecum of the large intestine vary considerably in different animals. In the carnivora, such as the cat, the small intestine is relatively short; in the herbivora, relatively long. Thus, it is three times the length of the body in the cat, and four to six times in the dog; whereas in the goat and sheep, it may be nearly thirty times the length of the body. In the carnivora the cecum is either absent or rudimentary, whereas in those herbivora which do not have a divided stomach the cecum is very large and sacculated, as is also the colon. The reason for the great size in herbivora is that practically the whole of the digestion of cellulose takes place in this part of the gut. This digestion, as we shall see later, does not depend on any secretion poured forth by the animal itself, but upon the action of bacteria and of certain enzymes (cytases) that are taken with the vegetable food. Movements of the Small Intestine The movements of the small intestine have been studied (1) by the bismuth subnitrate and x-ray method, (2) by observing them after open- ing the abdomen of an animal submerged in a bath of physiologic saline at body temperature, (3) by observing the changes in pressure produced in a thin-walled rubber balloon inserted in the lumen of the gut and connected with a recording tambour (Fig. 160), and (4) by excising portions of the intestine and keeping them alive in a bath of saline solu- tion at body temperature, through which oxygen is made to pass. THE SEGMENTING MOVEMENTS When a suitably fed animal is placed on the holder for examination )y the x-ray method, no movement in the intestinal shadows is generally )bserved for some time. The first movement to appear is the breaking of me of the columns of food into small segments of nearly equal size. ich of these segments again quickly divides, and the neighboring lalves suddenly unite to form new segments, and so on, in a manner 497 DIGESTION which will be made clear by consulting Fig. 161. This rhythmic seg- mentation, as Cannon has called it, continues without cessation for more than half an hour, and the food shadow does not meanwhile seem to change its position in the abdomen to any extent. The splitting up of the seg- ment and the rushing together of the neighboring halves proceed as a rule with great rapidity; thus, if we count the number of different seg- Fig. 160. Apparatus for recording contractions of the intestine. (From Jackson.) inents during a definite period, we may find the rate of division in the cat to be as high as 28 or 30 a minute. In man the divisions occur at a frequency of approximately 10 per minute, which corresponds to the fre- quency with which sounds can be heard when the abdomen is auscultated. Although half an hour is the period which this process usually oc- cupies, it may last considerably longer. In certain animals, such as the rabbit, segmenting movements have not been observed, but instead THE MECHANISMS OF DIGESTION 499 of them a rhythmic to-aiid-fro shifting of the masses of food along the lumen of the gut, rapidly repeated for many minutes. When the intestines are floated out in a warm bath of saline solution, it is seen that the rhythmic segmentation is caused by narrow rings of contraction. Under such conditions also it is often noted that the loops of intestine sway from side to side. The balloon method also re- veals the presence of slight waves of contraction that pass rapidly along the gut, and follow each other at the rate of twelve to thirteen per minute. Both of the muscular coats of the intestine are involved, and it is believed that the contractions are responsible not only for the pendular move- ments but for the rhythmic segmentation observed by the x-ray method. According to this view these movements are constantly passing along the intestine, and become exaggerated by the mechanical stimulus -which is offered by the masses of food to such an extent that they divide the masses into portions. The evidence for this belief rests on the fact that [/ *v-r< >T^\ f~r\ >-^ s ti>Z *s*~\. C : J vA^ vj_J ( fS /^~K v Fig. 161. Diagrammatic representation of the process of segmentation in the intestine. An unbroken shadow is shown in I and its segmentation in 2. The dotted lines across each mass show the position of division and in 3 is shown how new masses are formed by the split portions coming together. (.From Cannon.) when the contraction is studied by the balloon method, it becomes marked over the middle of the balloon, where the greatest tension exists. Several functions can be assigned to these movements. They cause intimate mixture of the food with the digestive juices, and by bringing ever new portions of food in contact with the mucosa, they encourage absorption. They also have an important massaging influence on the blood and lymph in the vessels of the intestinal walls. Indeed, the pas- sage of lymph from the lacteals into the mesenteric lymphatics seems to depend very largely upon these movements. The investigations of Alvarez 20 and his coworkers show that the rhyth- mic intestinal contractions are not of uniform rate throughout the entire length of the small bowel, but vary in frequency inversely with the dis- tance from the pylorus. For example, the contractions of a segment >f the duodenum proceed at a more rapid rate (17-21 per minute) than do the contractions of an ileal segment (10-12 per minute) under the 500 DIGESTION same experimental conditions. Associated with the variations in fre- quency are also differences in the amplitude of the contractions of the in- testinal muscle. As the contractions become less frequent their ampli- tude increases to the extent that the ratio between the amplitude of the contractions of duodenal and ileal segments is as 3 to 20. Tone and irrita- bility diminish progressively from duodenum to ileum. According to Alvarez the underlying cause of the decline in contraction rate is to be found in a study of the metabolism of the intestinal muscle in the differ- ent regions. The determination of the metabolic changes in different parts of the intestinal muscle from pylorus to the lower end of the ileum showed a gradual descent in the curve of energy output (metabolic gra- dient) which ran a course parallel to that of the intestinal contractions, a slow or a rapid metabolism being associated with a slow or a rapid contraction rate, respectively. This direct relationship between the fre- quency of rhythm and the metabolism rate of intestinal muscle is in favor of the view that the intestinal contractions are myogenic, moreover, the graded activity of the muscle with regard to rhythm, tonus and ir- ritability is probably an important factor in the production of peristalsis and in the determination of the direction which this movement shall take. THE PERISTALTIC MOVEMENTS The other movement observed in the small intestine is that known as the peristaltic wave. It occurs in two forms: (1) as a slowly advancing con- Fig. 162. Intestinal contractions (balloon method) after excision of the abdominal ganglia and section of both vagi. Mechanical stimulation above (/) and below (2) the balloon causes relaxa- tion and contraction respectively. (From Starling.) traction (1 to 2 cm. per minute), preceded by an inhibition of the walls, and proceeding only through a short distance in a coil (4 to 5 cm.); and (2) as a swift movement called the peristaltic rush, which sweeps with- out pause for much longer distances along the canal. Further analysis of the peristaltic wave can readily be made by the THE MECHANISMS OF DIGESTION 501 balloon method (Fig. 162). If the gut is pinched above the balloon, a marked relaxation occurs over the latter, and this relaxation extends for about two feet down the intestine. If, on the other hand, the gut is pinched a little below the situation of the balloon, a long-continued contraction occurs over the latter. The conclusion that we may draw from this result is that the stimulation of the gut causes contraction above the point of the stimulus and relaxation below, this being known as "the law of the intestine" (Bayliss and Starling). We have seen that it applies also in the case of the cardiac and pyloric sphincters. THE PHYSIOLOGICAL NATURE OF THE EHYTHMIC AND PERISTALTIC MOVE- MENTS Interesting information in this connection has been gained by obser- vation of the behavior of the movements after the application' of drugs to the gut or after cutting the nerve supply. The rhythmic movements are not affected by the application of nicotine or cocaine. Since these drugs paralyze nervous structures it has been concluded_tha,tJJae^ rhythmic movements, are jnvjagsftie in origin. The question is not a settled one, however, for it has been found by Magnus that, although strips of the longitudinal muscle, isolated in oxygenated saline solution, will continue to beat, they do not do so if the adherent Auerbach's plexus of nerves is stripped off from them. The nature of the peristaltic contractions is more definite; they must clearly depend upon a local nervous struc- ture, since they are paralyzed by the application to the gut of cocaine or nicotine. This local nervous system no doubt also resides in Auerbach's plexus, which must therefore be considered as complex enough to be (see page 830) endowed with the power of directing nervous impulses so as to bring about relaxation of the gut in front of the stimulus and contrac- tion over it. NERVOUS CONTROL OF MOVEMENTS The influence of the central nervous system on the intestinal movements has been studied by the usual methods of cutting and stimulating the extrinsic nerve supply. Through the splanchnic nerves tonic inhibitory impulses are conveyed to the intestine (except the ileocolic sphincter), for after these nerves are severed the movements become more distinct. Indeed, in many animals after opening the abdomen no intestinal move- ment can be observed until these nerves have been cut. Stimulation of the peripheral end of the nerve also inhibits any movement which may mean- while be in progress. The impulses through the vagus nerve are of an opposite character. Section of these nerves has little effect, but stimula- tion causes contraction. (Figs. 163 and 164.) ^ 502 DIGESTION By observing the rhythmic contractions of an isolated strip of the small intestine suspended in a bath of oxygenated saline solution at body tem- perature, it can readily be shown that the presence of even a minute trace of epinephrine is sufficient to produce complete inhibition of the movement. The parallelism between the effects of splanchnic stimulation and those of Kig. 163. The effect of excitation of both splanchnic nerves on the intestinal contractions. Starling.) (From Fig. 164. The effect of stimulation of right vagus nerve on the intestinal contractions. (From Starling.) epinephrine injection is very significant, for in this way the marked inhi- bition of intestinal movement which occurs during fright may possibly be explained (see page 787). The circular muscular coat of the last two or three centimeters of the ileum before it joins the cecum is definitely thicker than the rest of this coat, indicating that it has a sphincter-like action. This ileocolic THE MECHANISMS OF DIGESTION 503 sphincter, as it is called, opens when food is pressed against it from the ileum, but remains closed when food is pressed against it from the cecum. It therefore obeys the law of the intestine. That it is physiologically distinct from the musculature of the rest of the ileum is indicated by the fact that the splanchnic and vagus nerves do not affect it in the same way; thus, stimulation of the splanchnic causes a strong contraction of the sphincter, whereas this is unaffected by stimulation of the vagus. Peristalsis is much more rapid in the duodenum than in other parts of the small intestine. During the first stages of digestion, the food ordi- narily lies mainly in the right half of the abdomen, and later in the left half. There is considerable variation in the time that elapses before it enters the colon. In the cat, carbohydrates reach this part of the gut in about four hours. Movements of the Large Intestine On account of the great differences which we have already seen to exist in the size and relative importance of the colon as a digestive organ in different classes of animals, it is not surprising that the movements observed are very different according to the dietetic habits of the animal. Apparently the movements are much the same in the cat as in man. As the food passes through the ileocolic sphincter into the cecum and accumulates there, it gradually sets up, by its pressure, a contraction of the muscular walls of the gut somewhere about the junction between the ascending and transverse colon. This wave of contraction then begins to travel slowly toward the cecum, without, however, being pre- ceded by any relaxation of the wall of the gut, as is the case with a true peristaltic wave. This first wave is soon followed by others, with the result that the food is forced up into the cecum, against the blind end of which it is crowded, being meanwhile prevented from passing into the ileum by the operation of the ileocolic sphincter and by the oblique manner in which the ileum opens into the cecum. As the result of the distention of the cecnm set up by tjlfinr iff finllH antiperistaltic waves, a true coordinated peristaltic wave is occasionally initiated, and passes along the ascending colon preceded by the usual wave of inhibition. These waves, however, disappear before they reach the end of the colon, so that the food is again driven back by the so- called antiperistaltic waves. The effect of the movements is to knead and mix the intestinal contents, and thus encourage the absorption of water from them. The resulting more solid portions then collect toward the splenic flexure, and become separated from the remaining more fluid portion by transverse waves of constriction, which develop into peri- staltic waves carrying the harder masses into the distal portions of the 504 DIGESTION colon, where they collect chiefly in the sigmoid flexure. The descending colon itself is never distended with contents and merely serves as a tube for transferring the masses from the transverse colon to the sigmoid flexure. The time taken for a capsule of bismuth to reach the various parts of the large intestine is shown in Fig. 165. After a certain mass has collected in the sigmoid flexure and rectum, the increasing distention causes a reflex evacuation of this portion of the gut through centers located in the spinal cord. The impulses from these centers, besides contracting the rectum, etc., also coordinate the contrac- tion of the abdominal muscles and the relaxation of the sphincter ani so as to bring about the act of defecation. By the skiagraphic method it Fig. 165. Diagram of time it takes for a capsule containing bismuth to reach the various parts of the large intestine. has been found that the pelvic colon gradually becomes filled with feces from below upward, and that the rectum remains empty until just before defecation. The bulbosacral autonomic fibers to the large intestine are transmitted by the pelvic nerve (nervi erigentes) and their stimulation causes increased contractions. The thoracic autonomies, on the other hand, diminish the contractions. EFFECT OF CLINICAL CONDITIONS ON THE MOVEMENTS Observations of practical value have been made on the behavior of the peristaltic wave after various intestinal operations. After an end-to-end anastomosis of the gut, no evidence can be obtained by the x-ray method that any hesitation occurs in the movement of the shadows at the anas- THE MECHANISMS OF DIGESTION 505 tomosis. On the other hand, when a lateral anastomosis is established, stagnation of the food in the region of the junction may occur, this having been found, on opening the gut, to be caused by the accumu- lation of hair and undigested detritus at the opening between the op- posed loops. Another objection to lateral anastomosis is the fact that in performing the operation a considerable amount of the circular muscle is cut, which interferes with peristaltic activity. Moreover, the end of the proximal loop beyond the opening is in danger of becoming filled up with hardened material, and the end of the distal loop may become invaginated and induce obstruction in the region of the anastomosis. Observations have also been made by the x-ray method on the be- havior of the intestinal contents following intestinal obstruction. It has been observed that, as the material collects in the gut just above the obstruction, strong peristaltic waves are set up, which move the food toward the obstruction so powerfully as to cause the walls of the canal in front to become bulged, until at last the pressure causes the con- tents to be squirted back through the advancing ring of peristaltic con- traction. These waves were observed to succeed one another rapidly. When a portion of gut is reversed in position, the peristaltic waves con- tinue to travel in their old direction toward the duodenum. The effect of this is to produce a partial obstruction at the upper end of the re- versed gut. The type of peristalsis known as the peristaltic rush can be induced experimentally in animals by intravenous injection of ergot. It prob- ably also occurs in conditions of abnormal irritation of the gut in man, and is believed to be the characteristic acuity of the gut after a strong purge. CHAPTER LVI HUNGER, APPETITE AND THIRST The sensations of hunger and appetite are due to different causes, the former being definitely correlated with contraction of the empty stomach, and the latter being a complex of sensations operating in the nervous system along with memory impressions of the sight, taste, and smell of palatable food. Appetite is therefore a highly complex nervous integra- tion, whereas hunger is a much simpler process. It is particularly with hunger that we shall concern ourselves at present. Hunger When a thin- walled rubber balloon of proper size is placed in the stomach and connected by a rubber tube with a water, bromoform or chloroform manometer (made of wide glass tubing 1.5 cm. in diameter and provided with a suitable float on the free limb) a tracing may be taken of the movements of the stomach (Fig. 166). For use on man the capacity of the balloon should be from 75 to 150 cubic centimeters. The record thus obtained when the balloon is placed in the empty stomach of a normal person shows four types of wave. Two of these may be discounted, being due to the arterial pulse and the respiratory movements. The third is known as the tonus rhythm, and is caused by tonic contractions of the fundus of the stomach of varying amplitude. The periods of tonus in- crease during the powerful rhythmic contractions to be immediately described. While these changes in tone are occurring, no subjective sen- sation of hunger is experienced. (See Fig. 167.) The fourth and most significant type consists of powerful_r%#/imic contractions, alternating with periods of quiescence. These contrac- tions occupy a period of about twenty seconds, and are superimposed upon the tonus rhythm. They gradually increase in amplitude and fre- quency; and, in the case of young and vigorous persons, may pass into a condition of incomplete tetanus, after which they suddenly subside, leaving only a faint tonus rhythm. The rhythmic contractions are defi- nitely associated with the sensation of hunger, and are '~ more marked the more intense the sensation is. When tetanus occurs the hunger sen- sation is continuous, but it instantly disappears when the tetanus gives place to relaxation. 500 HUNGER AND APPETITE 507 When the contractions are comparatively feeble, the length of the period during which they occur is about twelve minutes. When the contractions are powerful, the periods are always initiated by weaker contractions with long intervening pauses; finally the pauses disappear and the contractions become more and more pronounced until, as above mentioned, a virtual tetanus lasting from two to five minutes, may supervene. The duration of the entire hunger period varies from one-half to one and a half hours, with an average of from thirty to forty-five minutes, and the num- ber of individual contractions in a period varies from twenty to seventy. Between the hunger periods, intervals of from one-half to two and one-half hours of quiescence may supervene. (See Fig. 168.) Similar contractions, often passing into incomplete tetanus, have been observed in the stomach of healthy infants, some of the observations hav- ing been made before the first nursing. The intervals of motor quies- cence between the hunger periods are shorter than in adults. In obser- Fig. 166. Diagram of method for recording stomach movements. B, rubber balloon in stomach. 1), kymograph. F, cork float with recording flag. M, manometer. L, manometer fluid (bromo- form, chloroform, or water). R, rubber tube connecting balloon with manometer. S, stomach. 7', side tube for inflation of stomach balloon. (From Carlson.) vations made during sleep, it was observed that, when the contractions were very vigorous, the infant would show signs of restlessness and might awake and cry. As in the adult, the contractions are evidently associated with subjective sensations of hunger. Contractions of the empty stomach have also been recorded on a large variety of animals, including the dog, rabbit, cat, guinea pig, bird, frog and turtle. They vary somewhat in type in different animals. With regard to the time of onset of the tonus and hunger contractions, it has been observed that the only period during which the fundus is free of them is immediately after a large meal. After a moderate meal the tonus rhythm begins to appear in about thirty minutes. It gradually increases in intensity, until by the time the stomach has nearly emptied 508 DIGESTION itself the tonus has become conspicuous, and the stronger hunger con- tractions usually begin to appear. Superimposed upon those of the tonus rhythm, hunger pangs may appear in man when the stomach still contains traces of food. By studying the shadow of the outline of the stomach produced by Fig. 167. Tracing of the tonus rhythm of the stomach (man) three hours after a meal. (From Carlson.) having a person or animal swallow two balloons, one inside the other and with a paste of bismuth subnitrate between them, it has been ob- served that the weaker type of hunger contraction begins as a con- striction involving the cardiac end of the stomach, and moving toward the pyloric end as a rapid peristaltic wave. When the contractions are Fig. 168. Tracings from the stomach during the culmination of a period of vigorous gastric hunger contractions. One-half original size. (From Carlson.) very vigorous, this wave spreads so rapidly over the stomach that it is difficult to determine whether it really occurs as a very rapid peristalsis or as a contraction involving the fundus as a whole. These contractions resemble very closely the movements that have sometimes been observed HUNGER AND APPETITE 509 after a bismuth, meal, and which, have been thought by clinical observers to indicate a hyperperistalsis of the stomach. The fundus is therefore not entirely passive during digestion; for, although early in this act there may be no evidence of contraction, yet the contractions of the tonus rhythm may appear and become pronounced before the stomach, is en- tirely empty. In other words, the digestion contractions of the filled stomach (see page 485) pass gradually over into the hunger contractions of the empty organ. Remote Effects of Hunger Contractions. It is well known that during hunger certain general subjective symptoms are likely to be experienced, such as a feeling of weakness and a sense of emptiness, with a tendency to headache and sometimes even nausea in persons who are prone to headache as a result of toxemic conditions. Headache is likely to be more pronounced or perhaps present only in the morning before there is any food in the stom- ach. These symptoms indicate that hunger contractions are associated with ititttttttfttfttttttttniitftttt' Fig. 169. Showing augmentation of the knee-jerk (upper tracing) during the marked hunger con- tractions (lower tracing). (From Carlson.) hyperexcitability of the central nervous system, and it is of considerable interest that objective signs of this association can be elicited. If the knee-jerk be recorded along with a record of the gastric contractions, it will be found that it is markedly exaggerated simultaneously with the strong hunger contractions of the empty stomach, this augmentation being greatest at the height of the stomach contractions, when the hun- ger pangs are most intense, and falling off again to normal when these disappear (Fig. 169). Further changes occurring during the hunger period include an increase in the pulse rate and vasodilatation. By comparing plethysmo graphic tracings of the arm volume (see page 209) and stomach contractions, it has been found that the increase in volume occurs pari passu with the increasing tonus of the stomach, but that it begins to shrink before the stomach contraction has reached its maximum. Occasionally, however, as in acute hunger, a somewhat different rela- 510 DIGESTION tionship obtains, vaso constriction being more prominent. During each hunger contraction there is also increased salivation A _ll}e degree of which varies with different individuals. This salivation is independent of the more copious "watering of the mouth" that accompanies the thought or sight of appetizing food. Hunger During Starvation. During enforced starvation for long periods of time, it is known that healthy individuals at first experience intense sen- sations of hunger and appetite, which last however only for a few days, then become less pronounced and finally almost disappear. It is of interest to know the relationship between these sensations and the hunger contractions in the stomach. This has been investigated by Carlson and Luckhardt, who voluntarily subjected themselves to complete starvation, except for the taking of water, for four days. During a great part of this time records of the stomach contractions w r ere taken by the balloon method, and it was found that the tonus of the stomach and also the frequency and intensity of the hunger contractions became progressively more pronounced as starvation proceeded. Towards the end of the period it was also noted that incomplete hunger tetanus made its appearance where ordinarily, as in Carlson's case, this type of hunger contraction was infrequent. Sensations of hunger were present more or less throughout the period, being therefore probably due to the persistently increased tonus. The onset of a period of hunger contraction could usually be foretold by an increase in the hunger sensation, and as these contractions became more marked, the hunger sensations became more intense. On the last day of starvation a burning sensation referred to the epigastrium was added to that of hunger. The appetite ran practically parallel with the sensa- tion of hunger, and both of these sensations became perceptibly dimin- ished on the fourth or last day of starvation, this diminution being, however, most marked in the sensation of appetite. Indeed, instead of an eagerness for food, there developed on the last day a distinct repugnance or indifference towards it. Accompanying these sensations of hunger and appetite a distinct mental depression and a feeling of weakness were experienced during the latter part of the starvation period. On partaking of food again the hunger and appetite sensations very rapidly disappeared, and also practically all of the mental depression and a great part of the feeling of weakness. Complete recovery from the latter, however, did not take place until the second or third day Wfter breaking the fast. From this time on both men felt unusually well; indeed they state that their sense of well-being and clearness of mind and their sense of good health and vigor were as greatly improved as they would have been by a month's vacation in the mountains. They further point out that, since others who have starved for longer periods HUNGER AND APPETITE 511 of time unanimously attest to the fact that, after the first few days, the sensations of hunger become less pronounced and finally almost dis- appear, they must have experienced the most distressing period during their four days of starvation. Although the hunger sensation was strong enough to cause some discomfort, it could by no means be called marked pain or suffering, and was at no time of sufficient intensity to interfere seriously with work. Mere starvation cannot therefore be designated as acute suffering. It is of further interest to note that dur- ing the starvation period a continuous flow of .secretion of acid gastric juice was found to be occurring in the stomach, to the presence of which acid or burning sensation experienced in the epigastrium on the last days is probably to be attributed. Control of the Hunger Mechanism. The control of the hunger mecha- nism, like that of any other mechanism in the animal body, may be ef- fected through the nervous system or it may depend on the presence of chemical substances or hormones in the blood. As a matter of fact, it can readily be shown that both those methods of control are employed, and we will now consider briefly some of the facts upon which this con- clusion depends. Although many facts are now known with regard to the nervous control of the hunger mechanism, it is difficult to piece these together in such a way as to formulate a simple theory which fits in with all the observed facts. We know that the stomach possesses in itself a local nervous mechanism by which, like the heart or intestine, it can automatically perform many of the movements which are exhibited in the intact animal. These local movements may, however, be considerably influenced by impulses transmitted to the stomach along the vagus and splanchnic nerves. We have therefore to seek for evidence indicating the relative importance of the local nervous mechanism in the stomach itself and of the impulses transmitted to this organ by the extrinsic nerves. We must then seek the position of the center which perceives the sensation of hunger. It will be simplest to consider first the effect of section of the extrinsic nerves in observations made on lower animals. Section of the splanchnic nerves increases gas- trie tonus and augments the gastric hunger contractions. Section of both vagus nerves, performed of course below the level of the heart, leaves the stomach in a more or less hypotonic condition. The tonus is not entirely abolished; it varies somewhat from day to day, and may become quite pronounced even though the vagi are cut. In this hypotonic state the hunger contractions are diminished in rate and regularity. Sec- tion of both the splanchnic and vagus nerves throws the stomach into a permanent hypotonus, except in prolonged starvation, when hunger contractions develop that are usually of great amplitude and with particularly long intervals between the contrac- tions. The general conclusion to be drawn from these experiments is that, although completely isolated from the central nervous system, the stomach still exhibits typical hunp-er contractions, which must therefore be essentially dependent upon an automatic mechanism in the stomach wall itself. Over this mechanism, extrinsic nerve impulses have merely a regulatory control. Variations and Inhibitions of the Hunger Contractions. The afferent 512 DIGESTION stimuli that may set up impulses traveling by the extrinsic nerves to the stomach are conveyed by the nerves of sense or are of psy- chic origin. Stimulation of the gustatory end organs in the mouth, as by chewing palatable food, always causes an inhibition of the tonus and a diminution or disappearance of the hunger contractions. Even the chewing of indifferent substances, such as paraffin, suffices to produce distinct inhibition, unless in a case in which the contraction has passed into a tetanus. It is of interest that swallowing movements, in the ab- sence of any food substance in the mouth, are sufficient to produce a transitory inhibition of the gastric tonus a receptive relaxation of the stomach, as it has been aptly called. The diminution in tonus and hunger contractions in these various ways is accompanied by a diminu- tion in the hunger pains. Afferent nerve stimulation affecting the hunger contractions may also originate in the stomach mucosa itself, as has been shown by Carlson on his patient by introducing the various substances to be tested through a tube into the stomach. A glassful of cold water introduced in this way inhibits the tonus and the hunger contractions for from three to five minutes unless these are severe, this inhibition being followed by no augmentation either of the tonus or of contractions. Ice-cold water has a greater effect than water at body temperature. This result is somewhat different from that which most men experience as the result of drinking a glass of cold water. Weak acids of strengths varying up to that found present in the gastric juice itself 0.5 per cent cause a marked inhibition of the hunger movements, but this inhibition does not persist until all the acid has escaped from the stomach or been neutralized, which explains why hunger contractions should still occur when an acid secretion is present in the stomach, as in starvation. Normal gastric juice itself produces an in- hibition, which is no doubt dependent upon the acid which it contains, and it is prob- able that, at the same time that it leads to inhibition of the hunger contractions, the acid initiates peristalsis of the pyloric region (see page 487). Weak alkaline solu- tions have no greater effect on the hunger contractions than an equal volume of water. Weak solutions of local anesthetics, such as phenol or chloretone, are without effect. With regard to alcoholic leverages interesting results were obtained. Wine, beer, brandy, and diluted pure alcohol inhibit both the tonus and the contractions. The duration of this inhibition varies directly with the quantity of the beverage intro- duced into the stomach and with its alcohol percentage. These observations are ap- parently not in harmony with the experience of most men that the taking of alcoholic beverages serves to awaken or increase the appetite, the difference being no doubt due to the fact that appetite and hunger contractions of the stomach are not dependent on each other, appetite being, as we have seen, a complex psychic affair, whereas the hunger contractions depend upon a local mechanism in the stomach wall itself. As the inhibition produced in one or other of these ways passes off, the hunger contractions are resumed at their previous intensity and not in an augmented form. From the promptness of the inhibition, it would appear that the stomach contractions are affected, not reflexly through the central nervous system or by changes in the chemical composition of the blood, but by a direct action on the neuromuscular mechanism HUNGER AND APPETITE 513 in the stomach walls, and it is important to bear in mind that the inhibitory effects 011 the stomach contractions of the fundus may proceed quite independently of the changes in the pyloric region that are con- cerned with the mechanical processes of digestion. After one or both of the extrinsic nerves of the stomach were severed in dogs, a certain degree of inhibition could still be induced by the above methods, indicat- ing that, although section of the extrinsic nerves depresses the inhibitory reflex, it does not abolish it. Various mitigations of the hunger contractions have been discovered. Smoking: has this effect, and compression of the abdomen by tightening the belt also inhibits the contractions provided they are not of marked intensity. Considerable muscular exercise, such as brisk walking or running, causes inhibition, which usually persists until after the exer- cise is discontinued. When the tonus and contractions return, in this case, they seem to be somewhat more pronounced. Application of cold to the surface of the body as by placing an ice pack on the abdomen or taking a cold douche, procedures which are well known to induce increased neuromuscular tonus, in general causes an inhibition of the gastric tonus and hunger contractions, the degree of which is roughly proportional to the intensity of the stimulation. There is certainly never an increase in the gastric tonus or hunger contractions. If such stimula- tion is maintained, the inhibitory effects on the stomach gradually diminish, even though the individual be shivering intensely. With regard to the nerve centers concerned in these phenomena, little that is definite is known. The sensory nuclei of the vagus nerve in the medulla must be considered as the primary hunger center, and through this center, not only influences affecting the stomach contractions, but also those associated with the hunger sensations, must be mediated. It Avould appear from observations on the hunger behavior of decerebrate animals that there can be no hunger center located on the cerebral cortex itself, for such animals exhibit practically the same hunger effects as normal animals. It is in- teresting to note that, at least in the case of decerebrate pigeons, this hunger behavior entirely disappears on removal of the optic thalami, where important nerve centers hav- ing to do with the bodily responses of the animal to hunger impulses would therefore appear to be located. These observations support the suggestion that has been made by several neurologists that the sense of pain is located somewhere in the thalamic region. Concerning the influence of psychic states, Carlson says that in his own case the hunger contractions became weaker and the intervals between them greater when he was suddenly awakened during his fast and saw two of his friends partaking at his bedside of a "feast of porterhouse steak with onions, potatoes, and a tomato salad." These results are no doubt due to local inhibition dependent upon the psychic secretion of appetite gastric juice. When no such juice is produced, the sight and smell of good food does not appear to affect materially 514 DIGESTION the hunger contractions of the stomach. No doubt it stimulates the appetite, but that, as we have seen, is a psychic affair. In the diagnosis of gastric and duodenal ulcer stress has been laid upon the occurrence of pains 2-3 hours following a meal and relieved by the ingestion of food. The possibility of the pains complained of being due to hunger contractions and consequently physiological must not be lost sight of. Surgical measures have unquestionably been need- lessly resorted to in cases where this possibility has not been borne in mind. Thirst The Sensation of Thirst has been believed by some observers to be a general one either dependent primarily upon dehydration of the tissues of the body, including those of the central nervous system, or resulting secondarily from a rise in the osmotic pressure of the blood the hyper- tonicity producing disturbances in the nerve cells. It is probable, how- ever, that the thirst sensation is a pureJyjQoaLima^ajnjeJy, drying of the mucous membranes of the mouth and pharynx. This view is supported by the fact, that if the pharynx of a person suffering from thirst be painted with a solution of cocaine all craving is relieved and it does not return until the effect of the anesthetic has passed off. That thirst may be produced through the drying of the interior of the mouth as by prolonged speaking, or mouth breathing, is common experience. Admin- istration of atropine acts similarly by inhibiting the oral secretions. According to Cannon 21 the salivary glands play the chief role in the mechanism for the production of the thirst sensations. He finds that the secretion of these glands, which normally moistens the interior of the mouth and pharynx, is reduced markedly (60 to 75 per cent) after a few hours abstinence from fluids. Since the composition of the sali- vary secretion is from 97 to 99 per cent water it is readily seen how a fall in the general water content of the body would bring about a re- duction of salivary flow with consequent drying of the oral and pharyn- geal mucous membranes, f This view ascribes to the salivary glands the important duty of keeping sentinel over the water content of the body, the sensation of thirst being the signal which warns the organism that the tissue fluids require to be replenished. | CHAPTER LVII THE BIOCHEMICAL PROCESSES OF DIGESTION In a book designed primarily for clinical workers, it would be out of place to enter into details concerning the biochemical processes taking place during the digestive process. There is, however, a certain amount of fundamental knowledge which it is essential that we should consider. In the first place it should be borne in mind that in the digestion of carbohydrates and proteins, various intermediate stages are passed through before the final absorption products are formed. The highly complex molecule of which protein, for example, is composed, is first of all broken down into several smaller but still highly complex mole- cules, each of which then undergoes further disruption, until ultimately the amino acids are set free. Certain enzymes, such as trypsin, can carry this process from the beginning through the greater part of its course without the assistance of other enzymes, but in the natural proc- ess of digestion, as it occurs in the gastrointestinal tract, the different stages of the disruption are controlled by different enzymes. One enzyme prepares the food for action by the next. This interdependence of their actions demands that some provision should be made whereby each en- zyme is secreted at the proper time; that is, when the foodstuff has al- ready been prepared for its action by that of its predecessor. Thus, it would be useless after food is taken for the gastric and pancreatic juices to be secreted at the same time. Instead, the gastric juice is secreted first, and the pancreatic only after the food has been prepared for its. action. This correlation in function we have already seen to be dependent largely on the action of hormones. DIGESTION IN THE STOMACH The gastric juice contains two important digestive agencies: (1) the enzyme, pepsin, and (2) hydrochloric acid. It is particularly in juices secreted in the cardiac end of the stomach that these two substances are found present; towards the pyloric end the hydrochloric acid entirely disappears, and the pepsin content becomes distinctly less. 515 DIGESTION The Functions of Hydrochloric Acid The functions of hydrochloric acid may be conveniently divided into physiological and biochemical. The former functions have to do with the control of the movements of the stomach, including the opening of the pyloric sphincter, and, after the chyme has entered the duodenum, with the secretion of pancreatic juice and bile. The biochemical functions are concerned: (1) in assisting the pepsin in the digestion of proteins, (2) in bringing about a certain amount of inversion of disaccharides, and (3) in having an antiseptic action on the stomach contents. Re- garding the last mentioned of these functions, it may be said that the chyme, as it is ejected from the stomach, is usually sterile, although it may contain spores and certain bacteria that are protected against the digestive agencies of the stomach. This protection is afforded by an outer covering of a chitinous nature (spores), or, as in the case of the tubercle bacillus, by a covering of waxlike material. It is believed that persons with strictly normal digestion are much less liable to infection by such bacteria, as those of typhoid and cholera, than persons with less active gastric secretion. When the acid of the gastric juice falls below the level at which it develops an antiseptic action, various bacteria and yeasts grow in the stomach contents, producing by the resulting fermen- tation irritating organic acids and gases. It is under these conditions that yeasts, sarcinse, and lactic and butyric acid bacilli find in the gastric contents a suitable nidus on which to grow. THE AMOUNT OF ACID It has long been known that considerable variations in the amount of hydrochloric acid in the gastric juice are associated with symptoms of indigestion. On this account a more or less elaborate technic has been developed for the purpose of determining the amount of hydrochloric acid in the gastric contents.* There are three things in connection with this activity that we may measure: (1) the total titrable hydrochloric acid; (2) the free hydrochloric acid; and (3) the actual hydrogen-ion concentration. The determination of the total available acids is made by titrating a measured quantity of gastric juice against a standard alkali, using phenol- phthalein as an indicator. By this method about 75 c.c. of decinormal alkali solution are required to neutralize 100 c.c. of normal gastric juice. The determination of the free hydrochloric acid is made by using special indicators, such as those of Giinzbcrg and Topfer, which change color at a hydrogen-ion concentration of about 10-"' (see page 27). To produce this hydrogen-ion concentration, a considerable quantity 0.05 per cent or more of an organic acid is necessary, whereas it requires only a trace of hydrochloric acid. Normal human gastric juice, when titrated with one of *The methods can be found in any volume on clinical diagnosis. THE BIOCHEMICAL PROCESSES OF DIGESTION 517 these indicators, gives a figure which corresponds to about 0.03 N. hydrochloric acid (see page 22). For the accurate determination of the hydrogen-ion concentration, it is necessary to use the gas-chain method (see page 29). When gastric juice is collected through a fistula from an empty stomach, very little difference Avill be found between the free hydro- chloric acid and the total acid; that is, between the results obtained by the second and the first of the methods described above. This is because in such juice there is no organic matter capable of combining with the hydrochloric acid, and there are no other acids, such as lactic or butyric, which might be produced by fermentative processes. The difference between the two titrations, however, becomes marked when protein food is undergoing digestion in the stomach, because at its different stages of digestion protein combines with increasing quantities of the hydrochloric acid. The pathological condition in which there is most definitely a diminution of the hydrochloric acid is cancer, either of the stomach itself or occasionally of some other part of the body. An in- crease is particularly marked in ulcer of the stomach. A considerable variation in hydrochloric acid may however be the result merely of func- tional (neurotic) conditions. THE SOURCE OF THE ACID A question that has puzzled physiologists for many years concerns the mechanism by which hydrochloric acid is secreted. The percentage of hydrochloric acid in the gastric juice is considerably above that at which any animal cells can live, and yet this acid is secreted by the lining membrane of the stomach, its source being, of course, the sodium fTiWirlp nf f.Tip blnnd . plffW"* 1 How then do the cells of the gastric glands bring about the separation of this powerful acid from the per- fectly neutral blood plasma? In the first place, it is significant that the mucous membrane of the stomach contains a higher percentage of chlorine than the average of other organs and tissues, indicating that it has the power of abstracting chlorine from the blood. The excess of chlorine in the mucosa must, moreover, be but a very small fraction of that actually secreted into the gastric juice. The chlorine content of the mucosa of the cardiac end is considerably greater than that of the pyloric. These facts indicate that chlorine is attracted by the gastric cells, but they throw no light on the question as to where the hydro- chloric acid is really formed. Is ifjmjibpi p^Hg, HT n-nly in t,h.ejj 1 ' mp ' n of the gland tubes? That is to say, is it formed before or after the gastric juice has been secreted from the cells? After intravenous injection of solutions of potassium ferrocyanide and some inert salt of iron, such as one of the scale preparations, examination of the gastric glands has 518 DIGESTION shown that the prussian blue reaction, which requires the presence of free mineral acid, is most pronounced in certain of the parietal cells. A considerable amount of the precipitate is, however, also visible in the lumen of the glands and in the stomach itself. Certain observers affirm that, although some of the parietal cells may take the stain, the vast majority of them do not do so; and, moreover, that cells incapable of forming hydrochloric acid (e. g., of the liver) may also become stained, and that the precipitation may occur in the blood and lymph. The confusion in the results by these methods prompted A. B. Macal- lum 22 and Miss M. P. Fitzgerald to investigate the distribution of the chlorine in the cells by a rnicrochemical method, in which the chlorides were precipitated with silver nitrate and the silver chloride then reduced by exposing the section to light. It was found that both kinds of gas- tric-gland cell, chief and parietal, but particularly the parietal, gave the chloride reaction. Using as a stain a substance (cyaninine) which reacts blue with acid and red with alkali, Harvey and Bensley, 23 however, aver that the secretion of the glands is practically neutral until the foveola is reached, where the stain becomes blue, indicating an acid reaction. This seems to show that the acid is not really secreted by the cells of the gastric gland, but is formed after secretion. According to the latter investigators, the chlorine is secreted by the cells into the fovea as some weak chloride, such as ammonium chloride, or it may be as an ester. Shortly after its secretion, this weak chloride undergoes a hydrolytic or other dissociation, during which free hydro- chloric acid is liberated and ammonia or some other weak base set free. Of these two products of the reaction the weak base is reabsorbed by the gland cells, but the hydrochloric acid is left behind because the cells are impervious to it. Indirect evidence in support of this view is afforded by certain other instances in which hydrochloric acid is pro- duced by the action of cells ; thus, the mould Penicillium glaucum when it is grown in a medium containing ammonium chloride absorbs the am- monia but leaves the hydrochloric acid. The high penetrating power of the ammonia ion in practically all cells, and the fact that the mucosa of the stomach contains a higher percentage of ammonia than any other tissue in the body, must also be considered as circumstantial evidence in favor of this view. Whatever be the mechanism by which hydrochloric acid is produced, there is no doubt that the epithelium is impenetrable by it. When the vitality of the epithelium becomes lowered, as in anemia or after partial occlusion of the arteries, the acid may penetrate the cells and cause digestion of the stomach walls. Hyperacidity may on this account become dangerous, as it lowers the resistance of the cell. THE BIOCHEMICAL PROCESSES OF DIGESTION 519 The digestive action of hydrochloric acid is closely linked with that of pepsin, with which it will, therefore, be considered. The Action of Pepsin It is commonly believed that before its secretion pepsin exists in the cells of the gastric glands as zymogen granules. The chief evidence for this belief appears to be that after considerable activity the amount of zymogen granules in the gland cells is found to be decidedly dimin- ished. By such an hypothesis it is easy to explain certain interesting results concerning the effect of weak alkali on the activities of extracts of the mucous membrane of the stomach. When the mucous membrane is extracted with weak acids, the extract is very active proteolytically. If this so-called pepsin solution be made faintly alkaline, or even only neutralized, and again made acid, it will be found to have lost much, if not all, of its activity. On the other hand, an aqueous extract may be rendered slightly alkaline for a short time and still display its digestive activity on subsequent acidification. The extract made with water is therefore much more resistant toward alkali than that made with weak acid, and the difference is explained on the supposition that the watery extract contains pepsinogen, whereas the acid extract contains pepsin. It is believed that there are several varieties of pepsin, because the optimum con- centration of acid in which pepsin, derived from the stomachs of different animals, acts is not always the same. Pepsin of the dog, for example, acts best in a hydrogen-ion concentration corresponding to that of a 0.05 N. hydrochloric acid solution, whereas that of the human stomach works best at a concentration of 0.03 N. Different pepsin solutions also show a difference with regard to the optimum temperature at which they act, and with regard to the nature of the protein which they most readily attack. Thus, the pepsin of a calf's stomach digests casein very rapidly, but coagulated egg white only slowly, whereas the pepsin of the pig's stomach acts on both these proteins at about the same rate. It is well known that the activity of pepsin can proceed only in the presence of acids, but this action of acids does not appear to depend on the hydrogen-ion concen- tration alone, for when equal quantities of the same pepsin are mixed with quantities of different acids so that the hydrogen-ion concentration of the mixtures is uniform, it is found that digestion proceeds most rapidly with hydrochloric acid and least rapidly with sulphuric acid. The SO 4 ion seems, therefore, to be unfavorable for peptic activities. The acid seems to combine with the protein before the pepsin attacks the latter; for, if we first combine the protein with acid and then wash away all traces of free acid, the protein can be digested in a neutral pepsin solution without the liberation of any free acid. There is evidence to show that pepsin itself also becomes combined with the pro- tein during the digestive process. If a piece of protein such as fibrin be immersed in a solution of pepsin and then taken out and washed thoroughly to get rid of all adherent pepsin, it will be found, on placing it in a hydrochloric acid solution of the proper strength, that peptic digestion proceeds. Advantage may be taken of this fact to separate pepsin from a solution, but the best protein to use for this purpose is 520 DIGESTION not fibrin but elastin. By such a method it has, for example, been shown that there is some pepsin in the intestinal contents, which indicates that when the chyme passes into the intestine, the pepsin is not, as used to be thought, immediately killed by tin- proteolytic enzyme. PRODUCTS OF PEPTIC DIGESTION With regard to the products of peptic digestion, little can be said here. The first product is a metaprotein known as acid albumin or syntonin. It is precipitated from the digestion mixture by neutraliza- tion. The next product is known as primary proteose, being precipi- tated by half saturation with ammonium sulphate. The third product is secondary proteose, produced by complete saturation with the above reagent; and after all these bodies have been separated out, there re- mains in solution the fourth product peptone which among other things is characterized by the fact that with the biuret test it gives not a violet but a rose-pink color. It has often been claimed that along with these products a certain amount of free amino acids may also appear in a peptic digestive mix- ture. This, however, may be due to the action of erepsin, which is usually present in pepsin preparations. It is important to note that the term proteose is a general one, and that there are probably many varieties of this substance, differing from one another according to the protein from which they are derived. The change produced by pepsin and hydrochloric acid is of the nature of an hydrolysis, for it has been found that the amount of hydrogen and oxygen in the digestive products is greater than that in the original protein. It is by a similar process of hydrolysis that the other proteolytic enzymes, such as pancreatin and erepsin, operate, but this does not imply that the exact grouping that is split apart by the hydrolytic proc- ess is the same for each of these enzymes. Indeed, there is considerable evidence that pepsin does not, like the other enzymes, break up the long chain of amino acids that are linked together to compose the polypep- tides, but that it only splits the big molecule of albumin or globulin into several large groups, each of which is composed of long ammo-acid chains. Its action appears to be analogous with that of amylase on starch, by which, it will be remembered, the big polysaccharide mole- cule is split into smaller polysaccharide molecules, which then become attacked by the dextrinase and split into disaccharide xmtfTeeules (see page 689). The evidence in support of this view is: (1) that pepsin is unable to digest polypeptides, and (2) that it is able to digest certain proteins upon which erepsin (see page 52-4) has no action. The hydrolytic splitting of large into smaller protein molecules, like that bv which the chains of amino acids in the polypeptides are subsc- THE BIOCHEMICAL PROCESSES OF DIGESTION 521 quently broken up, consists in a breaking of amino-carboxyl linkings (XHCO) (see page 634), with the consequent liberation of a large num- ber of unattached amino groups. The number of these free amino groups can be determined quantitatively by the formaldehyde titration method of Sorensen.* By this method it can be shown that from the very start of peptic digestion the number of free amino groups increases, and pari passu the power of the digestive products to combine with free hydro- chloric acid. Indeed, when, the experiments are done quantitatively and the digestion allowed to proceed for a considerable time, the increase in the formol titration is practically equal to the decrease in the free acids as determined by the Giinsberg reagent. The rate of peptic digestion is usually estimated by the law of Schiitz and Borissow^, according to which the amount of coagulated albumin that is digested in a Mett's tube is proportional to the square root of the amount of pepsin, f The pepsin which leaves the stomach in the chyme is not all destroyed in the intestine, as was at one time believed to be the case, for, as we have seen above, some pepsin can be detected in the gastrointestinal con- tents. A part of the pepsin may be absorbed into the blood and carried back to the gastric glands to be used again. This would account for the presence of antipepsin in the blood, and also for the presence of pepsin in the urine. It is probable, however, that most of the pepsin is de- stroyed after it enters the intestine. Clotting of Milk in the Stomach Besides its pOAver of digesting protein, the gastric juice is also endowed with the property of clotting milk. This action is commonly attributed to the presence of another enzyme besides pepsin, namely, rennin; but in recent years considerable controversy has raged around the question as to whether pepsin and rennin are not the same thing. One strong argument in favor of this view is that all digestive juices that are capable of digesting protein can also clot milk. In any case, when gastric juice acts on milk, it splits *VIP fflfifT 1 ! n ^ *^ A milk into two portions, one of which, called parjicasoin, immediately combines with calcium to form an insoluble f.nllmdpl compound, which is precipitated and, by entangling the fat of the milk, forms the clot; the other protein remains in solution *In this method the basic character of the amino acids is destroyed by the formaldehyde, so that a higher decree of acidity develops in the mixture. Bv determining the increased aciditv bv titration with alkali, an estimate is obtained of the number of amino groups. (See page 635.) tThe amount of coagulated egg albumin digested is ascertained by measuring the length digested away from the end of a column of coagulated egg white contained in a glass tube (Mett's method). (See Cobb, P. W. : Am. Jour. Physiol., 1905, xiii, 448.) Jin the above nomenclature casein is the same as rasc-inogcn, and paracasein the same as casein, of the Knglish physiologists. 522 DIGESTION and is known as whey ajbumose. From studies on molecular weight it is believed that the paracasein is produced from casein by the splitting of the molecule of the latter into two, from which it would appear that the action of this enzyme is nothing more than the first stage in the hydrolysis of the casein molecule. The whey albumose, according to this view, is a by-product. There are many investigators, however, who believe that rennin and pepsin are not identical, since an infusion of the stomach of a calf has a powerful clotting action on milk but a very weak digestive one on egg white, whereas a similar infusion from the stomach of a pig shows exactly the reverse properties. This question is one of so controversial a nature that it would be out of place to go into it further here. It should be pointed out, however, that, when the gastric contents are acid in reaction, milk will become clotted by the action of the acid itself quite independently of any pepsin or rennin the juice may contain. This acid clotting of milk is probably of a different chemical nature from that produced by the enzymes. On other foodstuffs than proteins the action of the gastric juice is relatively unimportant, although polysaccharides may be considerably broken down in the cardiac end of the stomach on account of the action of swallowed saliva (see page 489), and disaccharides, as we have seen, may become split by the hydrolyzing effect of the hydrogen ion. Fat digestion also takes place in the stomach when the fat is taken in an emulsified condition, as in milk and egg yolk, but not when in masses, as in meat or butter. This action is due to the presence of a fat-splitting enzyme, or lipase, in the gastric juice. CHAPTER LVIII THE BIOCHEMICAL PROCESSES OF DIGESTION (Cont'd) DIGESTION IN THE INTESTINES The further changes which the half-digested foodstuffs in the chyme undergo in the intestinal canal depend on the enzymes present in the secretion of the various glands and on the presence of bacteria. The most important of the digestive juices are the pancreatic juice and bile. The latter, however, does not contain any enzyme, its influence on diges- tion being entirely adjuvant. Pancreatic Digestion When we were considering the mechanism of secretion of the pan- creatic juice, we saw that the juice produced by the action of secretin on the gland cells does not contain any active proteolytic enzyme, although it contains one capable of acting on polysaccharides and another, on fat. THE ACTION OF TRYPSIN When pancreatic juice is mixed with the secretion of the duodenum or of the upper part of the small intestine, it immediately develops powerful proteolytic power. The same result may also be obtained by mixing it with an extract of the mucous membrane of the duodenum made with dilute bicarbonate solution. A very small amount of the extract is capable of increasing the digestive activity of- a very considerable quan- tity of pancreatic juice, showing that the action depends on the presence of an enzyme which has been called grrfg&lkWtP'Se. This influence of the intestinal secretion is readily destroyed by heating. Large quantities of alkali are contained in the pancreatic juice and bile, so that in the upper reaches of the intestine the acidity of the chyme is practically neutralized. A little lower down, however, an acid reaction may again develop (see page 539). On account of these facts it has been concluded that the activity of trypsin is most rapid in the pres- ence of a slight excess of hydroxyl ions; i. e., in a weakly alkaline solu- tion. It is interesting to note that, as a result of the great secretion of alkali by the pancreas, extracts of this organ after death show a very high degree of acidity in comparison with extracts from other organs 523 524 DIGESTION and tissues. It has also recently been shoAvn that the activity of trypsin does not depend on the presence of free hydroxyl ions, but that it may proceed in the presence of a decided amount of free acid. If pepsin is present together with trypsin in a distinctly acid solution, the pep- sin seems to destroy the trypsin, unless the mixture contains a con- siderable quantity of protein, when the tryptic activity may persist even for several hours. A practical conclusion that we may draw from these results is to the effect that preparations of trypsin the so-called pancreatin, for example if given with the food, may pass in an active condition into the duodenum, where, in the more favorable environment i created by the neutralization of the excess of acid, it will develop its Iproteolytic power. The therapeutic administration of pancreatin is, therefore, justified (Long 24 ). The activated trypsin acts on proteins in very much the same way as pepsin, except that the decomposition of the peptone and proteoses into polypeptides is the chief feature of the process. Thus, after tryptic digestion has proceeded for some time, only a trace of primary proteoses but considerable quantities of leucine, tyrosine and other amino acids will be found present. Some investigators believe that the thorough nature of the digestive action of activated pancreatic juice may depend on its also containing erepsin, an enzyme which we shall see to be pres- ent in considerable amount in the mucous membrane of the intestine and other tissues, and whose particular function is to split polypeptides into the amino acids. From the autolytic digestion which takes place in organs kept in a sterile condition after death, tryptic digestion differs in that it produces only small quantities of ammonia. The large quanti- ties of ammonia produced in autolytic digestion no doubt have a rela- tionship to the acids simultaneously set free during this process. In the products of tryptic digestion it is usually found that, although there has been considerable splitting of the protein into, amino acids, there are still a good many amino-carboxyl (NHCO) linkages left un- broken, indicating that certain polypeptides are still intact in the mix- ture. To split the polypeptides requires the aid of ._tlie wepsin, which is present in the mucous membrane of the intestine. Interesting inves- tigations have been made on the exact degree to which trypsin-entero- kinase can split up the various known polypeptides. This seems to depend on the structure of the polypeptide molecule and on the number of amino acids present in the chain. For example, alanylglycine, but not glycylalanine is hydrolyzed, although both contain the same amino acids but linked together in a different way; and tetraglycylglycine, which contains five glycine radicles, is hydrolyzed, whereas diglycylgly- cine, which contains only three, is not. THE BIOCHEMICAL PROCESSES OF DIGESTION 525 The importance of the presence of erepsin in the mucous membrane of the intestine is that it serves asjjjjarrifir t.n the passage of a.nv unsplit amino acids from the int.ftst.ina,] p.nr\t_pTTt^ into_ the blood. It insures the breaking up of the protein molecule into its ultimate units before absorp- tion. The further fate of the absorbed amino acids will be considered under the subject of protein metabolism. THE ACTION OF LIPASE Neutral fat is decomposed into fatty acids and glycerine by the lipase present in the pancreatic juice. This enzyme may also be extracted from the glands by means of 60 per cent alcohol. Its action is remarkably accelerated by the presence of bile, and considerably depressed by inor- ganic salts. It is also very dependent on the degree of alkalinity, the optimum being a hydrogen-ion concentration of H x 10' 8 . The favoring action of bile is undoubtedly owing to the bile salts (see page 528), and it is probable that this action is dependent upon the influence which these have in lowering surface tension and therefore bringing about a more intimate contact between fat and water. THE ACTION OF AMYLOPSIN The action of pancreatic juice on carbohydrates depends on the amylolytic enzyme called amylopsin. In animals having no active ptyalin in the saliva, amylopsin serves as the only diastatlc enzyme concerned in the digestive process. In any case, at least for the first stages of the disruption of the starch molecule that is, its conversion into dextrines amylopsin is a more powerful enzyme than ptyalin. It does not appear to be so efficient as ptyalin in the final stages of the hydrolysis, for it does not produce so much reducing sugar as ptyalin does. Indeed ex- tracts of pancreas will sometimes convert starch into soluble starch and dextrine with great speed, but produce scarcely any reducing sugar. On this account it is believed by many investigators that there are at least two distinct and separate enzymes in amylopsin and also perhaps in ptyalin, one a true amylase, which converts starch into dextrine, and the other a dextrinase, which converts dextrine into maltose. In the case of both ptyalin and amylopsin digestion proceeds best in a very weak acid reaction. Amylopsin, as it is secreted in the pancreatic juice, is fully activated; bile, apart from the alkali which it contains, having no influence on its digestive power. Besides amylopsin the pancreatic juice also contains ..maltose, and in the case of young animals or of those that take milk with their food throughout their lives, lactase also. After the suckling animal has dis- 526 DIGESTION continued taking milk, the lactase disappears from the pancreatic juice. Attempts have been made to bring it back by feeding the adult upon milk, but without success. Occasionally the pancreatic juice also con- tains invertase. The Bile Associated with the pancreatic juice in all its functions is the bile. When this fluid is prevented from entering the intestine, the digestive process becomes very imperfect, the absorption of fat being particularly interfered with (see page 722). Bile is also an excretory product, and its composition therefore is much more complex than that of the other digestive fluids. This varies very much, however, according to the method of collection. Bile from the gall bladder after death contains much more solid material, particularly bile salts and mucin, than that collected from a fistula of the bile duct or gall bladder during life. These differences will be evident from the accompanying table. Bile from Gall bladder Fistula 100 parts contain Water 86 . 97 Solids 14 3 Organic salts (bile salts) 9 0.9-1-8 Mucin and bile pigment 3 0.5 Cholesterol 0.2 0.06-0.16 Lecithin and fat.. 0.5-1.0 0.02-0.09 Inorganic salts 0.8 0.7-0.8 In general it may be said that bile obtained from a fistula in man contains only about 3 per cent of total solids, of which from one-fourth to one-half are inorganic, whereas bile from the gall bladder contains 10 to 20 per cent of total solids, of which only about one-twentieth are inorganic. The chief cause for this difference appears to be that when the bile goes to the intestine, a considerable proportion of its bile salts is reabsorbed into the portal blood and reexcreted by the liver. Some of the difference may also be caused by the fact that absorption of water takes place from the gall bladder, and that mucin and possibly cholesterol are secreted by this organ. These striking differences be- tween fistula and gall-bladder bile are observed only when the com- mon bile duct is occluded. If the bladder fistula is made with the com- mon duct left open, some of the bile gains entry to the duodenum and therefore becomes reexcreted. It is well known that a fistula of the gall bladder in man after a time closes up and the bile again takes its usual course along the bile duct into the duodenum. THE BIOCHEMICAL PROCESSES OF DIGESTION 527 Interesting observations have been collected on the amount of the secre- tion from a fistula, both in man and in the lower animals. In man it is commonly stated that about 500 c.c. of bile are secreted daily, the amount varying considerably during the different hours of the day. The secretion of bile is greatly reduced by hemorrhage. It is greater on a meat diet than on one of carbohydrates. It is reduced during starva- tion, but continues to be secreted up to the moment of death. FUNCTIONS OF BILE One of the main functions of the bile salts is that they greatly assist, not only in the digestion, but also in the absorption of fats. When bile is excluded from the intestine, the feces are loaded with fatty acids which have been split off partly by the now less effective lipase and partly by the action of bacteria. The fatty acid thus liberated in the absence of bile salts is not absorbed, because the bile salts serve as the carriers of fatty acids into the epithelial cells and lacteals. They com- bine with the fatty acids, probably by forming some chemical compounds in which they carry them into the endothelial cells where the compounds become disrupted, the fatty acid combining with glycerine to again form neutral fat and the bile salts being carried to the liver and reexcreted. The influence of bile salts in assisting the action of lipase is probably due to a lowering of the surface tension, thus bringing water and fat into closer union. This accelerating influence has also been demonstrated when synthetic bile salts have been used, showing clearly that it is really these and not any other constituent of the bile that are responsible for its accelerating influence. Bile also functionates as a regulator of intestinal putrefaction. This it does apparently because of its slight laxative properties, by which the intestinal contents are expelled before the bacteria have grown to any great extent in them. Bile itself is a favorable culture medium for certain bacteria, so that it can have no antiseptic action. Its assistance in the action of trypsin and amylopsin depends very largely upon the alkali which it contains. As an excretory vehicle, hilp is important, because it possesses the power of dissolving cholesterol. Toxins and metallic poisons of various kinds are also excreted in it. Although not directly concerned with the digestive function, it will be convenient to say something here concerning the chemical nature and derivation of the various biliary constituents. 528 DIGESTION THE CHEMISTRY OF BILE The Bile Salts In mostjuiimals the bile salts consist of the sodium salts of glycocholic and taurocholie acids. Each of these acids is composed of a"part called cholic acftt"Tvhich is more or less related to cholesterol, and of glycine (CHoNHoCOOH ammo-acetic acid) or taurine (C 2 H 7 NS0 3 ), a derivative of cvjstejfts,, which is a-amino-/?-thiopropionic acid (CH 2 HS.CHNH 2 . COOH). The exact form of cholic acid varies in different animals, that of the pig, for example, being different from that of man. Bile salts are an exclusive product of liver metabolism; i.e., they are not formed in any other part of the animal body. They give a very sensitive color reaction known as Pettenkof er 's, which however is not specific of bile acids, since it is also given by oleic acid and by many aromatic substances and alcohols. It must be remembered that the part of the bile salts that is characteristic of the liver is the cholic acid, the taurine and glycine being present in other tissues and organs. When cholic acid is given to animals mixed with the food, the amount of taurocholiB acid excreted with the bile is increased, indicating that there must be a store of taurine available in the organism. This store can not, however, be large, for if the feeding with cholic acid is repeated several times, it will be found that the taurocholie acid diminishes and glycocholic acid takes its place; and this increased excretion of glyco- cholic acid goes on just as long as cholic acid is fed. The reserve of taurine in the animal body appears therefore to be limited, although it is used in preference to glycine when there is an excess of cholic acid to be neutralized. On the other hand, the store of glycine seems to be inexhaust- ible. That there is no reserve of cholic acid itself in the body is indicated by the fact that no increase in taurocholie acid excretion by the bile results when cystine, the mother substance of taurine, is given with the food. If both taurine and cholic acid be fed, however, the excretion of tauro- cholie acid increases. The relative amounts of taurocholie and glycocholic acids in the bile of different animals differ considerably. Human bile contains relatively a small amount of taurocholie acid; on the other hand, the bile of the dog contains a large excess of it. Cholesterol In human bile the percentage of this important substance is not high (1.6 parts per 1000), but it is of great clinical importance because of the fact that it may separate out as a precipitate forming gallstones. The THE BIOCHEMICAL, PROCESSES OF DIGESTION 529 percentage of cholesterol in these varies from 20 to 90; the remainder being organic material such as epithelial cells, inorganic salts, pigment, etc. The origin of cholesterol is partly endogenous and partly exoge- nous. In the former case it comes from the envelope of red blood cor- puscles and from tl) ft rmrvnns tissues, where it is present in considerable amount. The latter source is, of course, the food. The increase in cholesterol esters in the blood after feeding with food rich in this sub- stance has been shown, particularly in rabbits. That the bile should be the pathway through which cholesterol is excreted depends no doubt on the fact that it contains bile salts, which along Avith their other properties hava..^- remarkable solvent action on cholesterol. This solvent property depends on the cholic acid part of the bile salts, which, as already remarked, is chemically very closely related to cholesterol; indeed, the relationship is so close that some have suggested that cholic acid is derived from cholesterol. This would mean that the cholesterol of blood is excreted in two ways, as cholesterol and as cholic acid. Other observers, however maintain that the cholesterol is excreted mainly by the lining membrane of the gall bladder, and that this explains why gall-bladder bile contains more of it than fis- tula bile. This evidence is, however, not very strong, for the greater excretion of cholesterol under conditions where the circulation of bile is proing on may be explained as due to the presence of bile salts, which serve to carry the cholesterol out of the blood. Many problems remain to be elucidated in connection with the metabolic history of cholesterol. That some of it is absorbed when cholesterol is contained in the food might seem to indicate that its source is entirely exogenous. Against this view, however, stand two facts: (1) that the cholesterol in the feces of herbivorous animals is of the same variety as that present in those of carnivorous animals and not the phytosterol which is present in plants; and (2) that the universal presence of cholesterol in cells indicates that it must be manufactured there. The Bile Pigments The pigments of bile are bilirubin and biliverdin. The latter is pro- duced from the former by oxidation. If the oxidation be carried a stage further, a blue pigment called bilicyanin is formed. This process of oxidation can be observed in the ring test for bile pigment with fuming nitric acid. When bilirubin is reduced, urobilin, one of the pigments in urine, is formed. Bilirubin must therefore be considered as the mother substance of all these pigments, and it is of interest in connection with its derivation to know that it has the same formula 530 DIGESTION as iron-free hematin or hematoporphyrin, which is produced by treating hemoglobin with concentrated sulphuric acid. Chemical investigation has shown that bilirubin is built up from sub- stituted pyrrols, probably four such being contained in the molecule. The pyrrol group is also present in indole and tryptophane, and con- sists of four carbon atoms and an NH group linked together as a ring (see page 639). Similar pyrrol derivatives can be produced by decom- posing chlorophyl, the green coloring matter of plants. It is important to remember that bilirubin is acid in nature, and, therefore, can com- bine with alkalies to form salts. The relative amounts of bilirubin and biliverdin vary in the bile of different animals. When these pigments enter the intestine they are reduced to urobilin, part of which passes out with the feces, another part being absorbed into the blood and excreted in the urine. Part of that excreted in the urine exists, however, as a so-called chromogen named urobilinogen. The urobilinogen is converted into urobilin by the action of oxygen. The method by which bile pigments are produced from blood pigment has been studied by histological examination of the liver particularly of birds and amphibia, in which destruction of blood pigment goes on rapidly. Increased destruction of blood pigment can be induced by poisoning with certain substances such as arseniureted hydrogen. After extra- vasation of blood in the subcutaneous tissues, as in a bruise, for example, a decomposition of hemoglobin proceeds quite like that occurring in the liver, and leads to the production of blue and brown and green pigments like those of the bile. When hemolysis is produced, as by inhalation of arseniureted hydrogen or the injection of inorganic or biological hemolysins, there is an immediate increase in the amount of bile pigment in the bile. Even the injection of hemoglobin solutions has this effect. Under these conditions of hemolysis, besides an increase in urobilin, there may be considerable quantities of hemoglobin secreted in the urine. From such studies it is usually believed that the bile pig- ments are a peculiar product of hepatic activity, being produced from blood pigments that are derived from erythrocytes which have been broken down either in the liver itself or in some other viscus (e.g., the spleen). Whipple and Hooper 25 have shown that bile pigments may also be formed in other tissues than the liver. They have found, for example, that the bile pigments are formed just as readily in animals in which the circulation of the liver was greatly curtailed, by anastomosing the portal vein with the vena cava (Eck fistula), as in normal animals. Even when the circulation was limited to the anterior end of the animal (head and thorax) bile pigment appeared in the blood when hemolyzed erythrocytes were injected, and it was also formed when hemoglobin was placed in the THE BIOCHEMICAL PROCESSES OF DIGESTION 531 pleural and peritoneal cavities. The endothelial cells of the blood vessels and elsewhere can evidently form the pigments, at least when the liver is absent. When such a process occurs under normal conditions, it is quite probable that the liver acts merely as an excretory organ for the pig- ments in the same way as the kidney does for urea. Possessed of endo- thelial cells, the liver might itself also produce some of the pigments, but no more than other organs with a similar number of those cells. The derivation of bile pigments directly from hemoglobin is not their only source, for the same workers have observed that, whereas the excre- tion of pigment from a biliary fistula is remarkably constant in a dog fed on a fixed mixed diet, it became increased, sometimes by 100 per cent, when the diet was changed to one of carbohydrates, and depressed on a diet of meat. The question arises as to whether all of the bile pigments are really derived from broken-down hemoglobin. May they not also be manufactured de novo out of other materials'? Whipple and Hooper have also shown that bile is a most important secretion, for dogs rarely survive on an ordinary diet if bile is perma- nently prevented from entering the intestine. Intestinal symptoms soon supervene, and become progressively more severe until the death of the animal. Feeding with bile does not relieve the condition, but feeding with cookedjiver seems to have a beneficial effect. Bile salts and pigments usually accompany each other when any- thing occurs to interfere with the free secretion of bile. For example, after ligation of the bile duct both bile pigments and bile salts accumu- late in the blood, in the serum of which they may be recognized by the ordinary chemical tests in from four to six hours after the operation. If the accumulation be allowed to proceed further, the bile pigments become deposited in the tissues, giving them the peculiar yellowish ap- pearance known as jaundice. Under these conditions the bile salts and pigments also appear in the urine. The accumulation of bile salts in the body affects certain physiological processes; for one thing, it causes a great lengthening in the clotting time of the blood. If the blood supply to the liver is interrupted by ligation of the portal vein and hepatic artery at the same time that the bile ducts are occluded, not a trace either of bile salts or of bile pigment appears in the blood during the six to eighteen hours that the animals survive the operation. The amount of obstruction of the bile duct necessary to produce these symptoms is very slight, since bile is secreted at a very low pressure. Even a clot of mucus or a swollen condition of the mucous membrane of the duct is sufficient to produce obstruction. In the discharge of bile from the gall bladder into the duodenum it is claimed by Meltzer 26 that a reciprocal relationship exists between the contraction of the bladder 532 DIGESTION musculature and the relaxation of the muscular fibers surrounding the duct in the duodenum. If this reciprocal innervation fails to operate properly, discharge of bile into the duodenum may become obstructed so that a certain amount passes back into the blood, as in cases of bile- duct obstruction. Bile also contains a certain amount of l&oithiii and other phospholipins. The amount varies considerably in the bile of different animals, even in animals of the same species. It is probably derived, as already men- tioned, like the cholesterol, from the breaking-down of red blood cor- puscles that goes on in the liver. It is no doubt digested by the ferments of the intestinal tract, the liberated cholin, since it is toxic if absorbed, being further attacked by bacteria so as to become converted into cer- tain substances of a nontoxic nature. CHAPTER LIX BACTERIAL DIGESTION IN THE INTESTINE On an average diet, in twenty-four hours the feces of man weigh about 100 grams, or after drying, about 20 grams. About one-fourth of the dry matter consists of the bodies of bacteria. If plated out by the ordinary bacteriologic methods, however, it will be found that only a small proportion of these bacteria are living. The greater number have been destroyed, probably by the action of the mucin in the large intes- tine. The nitrogen content of the feces amounts to about 1.5 grams a day, of which about one-half is bacterial nitrogen. If the diet contains large quantities of cellulose material, as in green vegetable food and fruit, the mass of feces as well as the bacterial content may be consid- erably greater. The foregoing facts indicate that very extensive bacteriologic proc- esses must be going on all the time in the intestinal contents, and the question arises as to whether such action is beneficial or otherwise to the animal economy. To answer this question interesting observations have been made on the growth and well-being of animals excised from the uterus under strictly sterile conditions and maintained thereafter on sterile food. Such observations made on guinea pigs have shown that the animals thrive and grow perfectly for a considerable time. Experi- ments carried out on chicks have not, however, yielded similar results. Chicks hatched out from the egg under strictly sterile conditions and then fed on sterile grain, do not thrive, but do so if the grain is mixed with a certain amount of fowl excrement. These experiments, appar- ently contradictory in their results, show that for certain groups of animals bacteria are required, but not for others. The difference is probably dependent on the nature of the foods. It will be remembered that the size of the large intestine varies consider- ably according to the nature of the diet (see page 497). Animals taking great quantities of cellulose foodstuffs have very large ceca and very long large intestines; whereas those which, like the cat, live practically entirely on cellulose-free food, have a rudimentary large intestine. The size of the lower intestine is obviously dependent on the presence or absence of cellulose in the food. It will be remembered also that the forward movement of the contents of the large intestine is very slow; indeed, special provision is made, by the presence of the so-called anti- peristaltic wave, to delay its movement. This suggests that an important 533 534 DIGESTION digestive process must be proceeding in this part of the gut. In these ways conditions become established in the cecum for the active opera- tion of bacteria. They attack the cellulose, and liberate the more diges- tible foodstuffs contained in the vegetable cells, also producing out of the cellulose itself materials of nutritive value. The acids that are also produced by this process are neutralized by the carbonates secreted by the mucosa. In certain herbivorous animals the ruminants this process in the cecum is not relatively of such importance, because it takes place mainly in the paunch. The animals swallow the food and it mixes in this part of the stomach with the saliva, so that bacteria and ferments called cytases, con- tained in it, attack the cellulose, liberating the more easily digested food- stuffs inclosed within the cell walls. As this process goes on acids accumu- late in the digestive mixture. The food is then returned to the mouth and chewed over again, after which it is swallowed into the main stomach, where it is digested. The aid which bacteria render to digestion depends therefore on the nature of the diet. Man, being omnivorous, stands mid- way between the two groups of animals discussed above. Although the cellulose contained in his food is not itself sufficiently digested to furnish nutriment, yet it is so far acted upon as to permit the rupture of the cell, the contents of which are then digested. The cellulose is, however, of value in furnishing bulk to the intestinal contents "intestinal bal- last," it is sometimes called. In the small intestine in man there are bacteria capable of acting on carbohydrates and producing from them organic acids, such as lactic, acetic, etc. So long as a sufficiency of carbohydrate exists to encourage the action of these bacteria, others having an action on protein do not seem to thrive. It may be that this is to be accounted for partly by the production of acid substances by the carbohydrate fermentation, and partly by the fact that, as soon as the protein molecule is broken down by the digestive enzymes, its building-stone amino acids are ab- sorbed. There are probably also bacteria in the small intestine capable of splitting fat into fatty acid and glycerine, but practically nothing is known of their action. In the large intestine of man, along with the cellulose-digesting bacteria already mentioned, protein-digesting bac- teria are also present. These bacteria belong to the class, Bacillus coli communis, the various members of which are known as facultative anae- robes because they can grow in the presence or absence of oxygen. If bacterial growth is excessive or there is an insufficiency of carbohy- drates in the small intestine, the bacteria attack the amino acids pro- duced by the digestive enzymes and decompose them into products that may be toxic if absorbed into the blood. BACTERIAL DIGESTION IN THE INTESTINE 535 Bacterial Digestion of Protein From a pathological standpoint, the most important action of bacteria is that which takes place on protein. Under anaerobic conditions the intestinal bacteria have in general the power of splitting off the amino group whereas under aerobic conditions they split off the carboxyl group. This splitting off of the carboxyl group as carbon dioxide is per- formed by the so-called carboxylase bacteria, and it may take place either before or after deamidization (see page 649). If it happens after this process, the products are not highly toxic and include phenol, cresoL indole ar^ ftfcat.nlA, whir*Vi a re partly absorbed into the blood and partly excreted with the feces. The fractions of those substances that are absorbed into the blood have their toxicity removed by conjugation mainly with sulphuric acid to form the so-called ethereal sulphates. A part is also combined with p'lycuronic acid (see page 665). In the case of phenol and cresol this conjugation occurs immediately after absorption, but in the case of indole and skatole it is preceded by an oxidative process, converting these substances into indoxyl and skatoxyl respectively. The_jleiaxica- tion process occurs in the liver, as has been shown by experiments in which this organ was artificially perfused outside the body. They are then removed from the blood by the kidneys and excreted in the urine. The proportion of ethereal sulphates in this fluid therefore gives an esti- mate of the extent of intestinal putrefaction of protein (see page 665). The indican, being readily detectable by the well-known color reaction of Jaffe, serves as an indicator of excessive intestinal putrefaction. The indole and skatole which are not thus absorbed and detoxicated are excreted with the feces, to which they give the characteristic odor. The source of the phenol is tyrosine and that of the indole is trypto- phane. The chemical processes involved are shown in the following equations, in which the by-products of the reactions are in brackets. COH COH HC CH HC CH C.OH COH COH HC CH /\ HC CH HC CH 1 II 1 II 1 II HC CH HC CH HC CH V V v C 1 * 1 > 1 CH 2 CH 2 CH 2 HC CH HC CH \/ \x C CH CH 3 (CO 2 ! (NH.) | (C0 2 +H 2 0) | (C0 2 ) CHNH 2 CH 2 COOH I I COOH COOH (tyrosine) (p-oxyphenyl- (p-oxyphenyl- (paracresol) (phenol) propionic acid) acetic acid) 536 DIGESTION Putrefaction of tryptophane is probably preceded by deamidization : CH C C CH 2 CH 2 .COOH (NH,) CH H/V I I HC C CH \X\X CH NH (indole-propionic acid) CH (C(X + H 2 0) -CH HC (+CH 3 ) \/ \/ CH NH (CO 2 +H.,0) HC C CH \X\X CH NH (indole-acetic acid) (indole) (skatole) If, however, the carboxylase bacteria remove the carboxyl group be- fore the amino group has been removed, highly toxic substances called amines are produced. They are the so-called ptomaines. From alanine, ethylamine is formed; from tyrosine, phenoleTTryTamine ; from histidine, which it will be remembered is an important protein building-stone, histamine, (imidazyl ethylamine) and so on. The process of formation is illustrated in the accompanying formulae: 1. CH 3 .CH(NH 2 ).COOH CO 2 -f- CH 3 .CH 2 (NH,) Alanine Ethylamine 2. C 6 H,(OH).CH,.CH(NH 2 ).COOH CO 2 -f C 6 H 4 (OH) .CH,.CH 2 .NH 2 Tyrosine Phenylethylamine 3. C s N,H 3 .CH 2 .CH(NH 2 ).COOHz=CO, + C 3 H 3 N 2 . CH 2 .CH 2 .NH, Histidine. Histamine. Similar substances are very common in the metabolic products of plants; for example, they constitute the active principle of ergot. They are also no doubt produced in the tissues of mammals, imidazylethyla- mine, commonly called histamine, being thus produced, as well as the closely related epinephrine, which is the active principle of the supra- renal gland (see page 773), and may be described as a methylated ethyla- mine derivative of tyrosine. Phenylacetic acid produced by a similar process from tyrosine may be excreted in the urine, where it forms the mother substance of homo- gentisic acid, to which the dark brown color of the urine in alkaptonuria is due. The great importance attached to these decomposition products of proteins depends on the fact that they have powerful pharmacological actions. These actions are developed very largely upon the vascular system; histamine, (pages 253 and 307) for example, produces marked vasodilatation and lowers the coagulability of the blood, whereas other substances of the same class, like epinephrine, have the property of raising the blood pres- sure. In larger doses, serious nervous symptoms and a condition of pro- BACTERIAL DIGESTION IN THE INTESTINK 537 found collapse are produced. These observations have led several inves- tigators to believe that the persistent occurrence of bacterial fermen- tation and the absorption of the resulting decomposition products of protein into the blood ultimately cause arteriosclerosis and the other symp- toms that accompany senescence. It is difficult at the present time to knoAv how much of this one ought to believe, although it can not be doubted that putrefaction has an unfavorable action on the arteries, and that an excessive degree of it causes the symptoms of ptomaine poisoning. If the ptomaines have formed in the food before it is eaten, the symp- toms develop in from one^to'five hours after the meal, but if the decomposi- tion occurs in the intestine on account of bacteria that are taken at the same time as the food, the ptomaines may not have developed sufficiently to cause symptoms until from twelve to forty-eight hours ; sometimes, how- ever, they develop in an hour or so. Prominent among the symptoms is usuallj_jli4rrhea, which develops for the purpose of getting rid of the offending bacteria and ptomaines. Actual infection of food with bacteria of the paratyphoid-enteritidis type is much more common than poisoning by substances (ptomaines) that have been generated in food before it is taken (Jordan 27 ). Meat, milk and other protein foods are usually the carriers of the bacilli, and in most of the accurately recorded cases the meat or milk was found to be derived from animals suffering from enteritis or some other infection. Sometimes, however, perfectly good food may become infected by handling. Although the symptoms are usually acute, they may closely simulate those of typhoid fever, and the effects of the attack may linger for weeks or months. BOTULISM The commonest type of poisoning by substances actually present in the food is that known as botulism. In this the gastrointestinal symptoms, as a rule are not pronounced, indeed, paralysis of the intestinal tract with con- stipation is the rule, but those affecting the nervous system, dizziness, diplopia and other visual disturbances, with difficulty in swallowing, are very prominent. The temperature is usually normal; the pulse some- times slowed. In practically all of the reported cases, the source of infec- tion has been food which after having been subjected to some preliminary treatment, such as smoj^nj^jrickling, or canning, had been allowed to stand for some time and then eaten without cooking. The Bacillus botulinus, which is responsible for the production of the poisons or toxins, is a strict anaerobe and is readily destroyed by cooking, as are also the poisons. Antitoxins are formed by snblethal injections. Another but 538 DIGESTION now very rare example of poisoning by products formed in food is that caused by "ergotoxin." The treatment in such cases is to encourage diarrhea by giving pur- gatives. If the intoxication is of a more chronic character, the symptoms are vague, consisting of drowsiness, lassitude, headache, and general de- pression. The treatment here also is to clear out the intestines by a good purge. There can be little doubt that many of the unhealthy condi- tions of the skin leading to the formation of pimples, acne, and boils, are also caused by chronic intoxication with protein decomposition prod- ucts. Again, purgation is the proper treatment. It is unnecessary in a work of this character to go further into these highly important questions. It is probable, however, that the importance of the relationship of excessive protein putrefaction in the intestine to many of the so-called minor diseases cannot be overemphasized. On the other hand, we must be careful not to attribute every sort of chronic condition to this putrefaction. Toxemia is often a shibboleth of the profession. When a chronic disease cannot be diagnosed, it is put down as a toxemia. This, however, is not medical science it is medical shirk- ing. It is certainly unsafe at the present time to conclude that the ordinary symptoms of senescence, such as hard arteries or increased blood pressure, are invariably to be attributed to this cause. It will be re- membered that Metchnikoff is largely responsible for such a view, and also that he suggested, as the surest way to ward off the chance of such intoxication, the taking of buttermilk, which would supply bacteria through whose growth in the intestine the protein-destroying bacteria would not be able to thrive. It is probable that the same result could be attained in patients showing undoubted signs of suffering from intestinal putrefaction by a change in diet in the direction of giving more carbo- hydrate, for, as we have seen, if there is a plentiful supply of this food- stuff in the small intestine, the bacteria do not tend to attack the protein. Before leaving this subject it is interesting to consider for a moment the cause of the severe symptoms that follow intestinal obstruction. This question has recently been diligently investigated by Whipple, 28 who found that the nonprotein nitrogen of blood (page 641) becomes greatly increased in intestinal obstruction. The cause for this increase in non- protein nitrogen is found to be an excessive breakdown of tissue protein caused by the absorption into the blood of a proteose. When this pro- teose isolated from obstructed loops of intestine was injected into fast- ing dogs, profound symptoms of depression were produced, followed, in cases in which the dose was sublethal, by recovery in from twenty-four to forty-eight hours. Along with these symptoms the nitrogen elimina- tion by the urine increased by 100 per cent. A very interesting fact is BACTERIAL DIGESTION IN THE INTESTINE 539 that animals can be rendered immune to this proteose by progressively increasing periodic administration. When they are thus immunized, the toxic symptoms do not follow upon its injection, nor are the symp- toms produced by artificially creating an intestinal obstruction. Con- versely, when a chronic toxic condition is kept up by a partial obstruc- tion, such as that produced by making a gastrojejunal fistula and occlud- ing the duodenum, the animals are less susceptible than normal ones to proteose injection. We have here and there incidentally referred to the reaction of various parts of the gastrointestinal contents, but we would call attention once again to this important subject, especially since many points of uncer- tainty have recently been cleared up by the accurate observations of Long and Fenger, 29 who used the electrometric method for measuring the hydrogen-ion concentration. The contents of the duodenum removed by means of the Eehfuss tube in man showed a reaction varying from dis- tinctly acid to slightly acid, depending upon the proximity of the tube to the pylorus or papilla, this position being determined by x-ray exam- ination. The slight degree of alkalinity is surprising. Lower down in the duodenum the reaction was as frequently acid as alkaline, the de- gree of acidity, however, being so slight as to favor rather than retard the digestive powers of the pancreatic juice. To determine the reaction lower down, the observations were made on recently slaughtered animals (pigs, calves, and lambs), the small intes- tine being tied off in loops of the upper, middle, and lower thirds. The contents of the last loop were often alkaline, but might be more acid even than those of the first, which were usually faintly of this reaction. Con- siderable variations were, however, the rule. The mixed intestinal con- tents of a recently fed dog, removed immediately after death, gave P H = 6.79 ; i. e., very faintly acid. DIGESTION REFERENCES (Monographs) iPavlov, J. P.: The Work of the Digestive Glands. Trans, by Sir W. H. Thomp- son, London, Griffin, ed. 2, 1910. 2Starling, E. H. : Recent Advances in the Physiology of Digestion, W. T. Keene & Co., Chicago, 1907. sCannon, W. B.: The Mechanical Factors of Digestion, Internat. Med. Monographs, London, Ed. Arnold, 1911. ^Carlson, A. J.: The Control of Hunger in Health and Disease, Univ. of Chicago Press, 1917. sTodd, T. Wingate: The Clinical Anatomy of the Gastrointestinal Tract, Manches- ter, Univ. Press, 1915. eMacallum, A. B.: Ergeb. der Physiol., xi, 598-657. 'Cannon, W. B., and Cattell, McKeen: Am. Jour. Physiol., 1916, xli, 39. E.: Proc. Am. Physiol. Soc., Am. Jour. Physiol., 1918, xlv, 559. 540 DIGESTION , H. H., and Laidlaw, P. F. : Proc. Phys. Soc., Jour. Physiol., 1912, xliv, 12 and 13. ioBabkin, B. P., Bubaschkin, W. J., and Ssawitsch, W. W. : Arch, f . mikr. Anat., 1909, Ixxiv, 68. "Miller, F. E.: Quart. Jour. Exper. Physiol., 1913, vi, 57. isKeeton, E. W., and Koch, F. C. : Am. Jour. Physiol., 1915, xxxvii, 481 ; also Popiel- ski, L.: Arch. f.d. ges. Physiol., 1901, Ixxxvi, 215. isEdkins, J. S.: Jour. Physiol., 1906, xxxiv, 133-144. "Meltzer, S. J. : Am. Jour. Physiol., 1899, ii, 266. ic-Cannon, W. B.: Am. Jour. Physiol., 1898, i, 359. isGrey, E. G.: Am. Jour. Physiol., 1917, xlv, 272. ^Carlson, A. J.: Am. Jour. Physiol., 1917, xlv, 81. isGinsburg, Tumpowsky, and Hamburger: Jour. Am. Med. Assn., 1916, Ixviii, 990. ^Cannon, W. B., Blake, J. B. : Ann. Surg., 1905, xli, 686, Cf . No. 3. soAlvarez, W. C.: Am. Jour. Physiol., 1918, xlvi, 238. 2iCannon, W. B.: Proc. Eoy. Soc., 1918, xc, B, 283. 22Macallum, A. B.: See Fitzgerald M. P.: Proc. Eoy. Soc., Ixxxiii, B, 56. ssHarvey, B. C. H., Bensley, E. E.: Biol. Bull. Woods Hole, 1912, xxiii, 225. 24Long, J. H., et al.: Jour. Am. Chem. Soc., 1917, xxxix, 162 and 1493; also ibid., 1916, xxxviii, 38. 25Whipple, C. H., Hooper, C. W.: Am. Jour. Physiol., 1916, xl, 332 and 349; ibid., 1917, xlii, 257 and 264; Hoope; ibid, p. 280. 26Meltzer, S. J.: Am. Jour. Med. Sc., 1917, cliii, 469. 27jordan, E. V.: Food Poisoning, Univ. Chicago Press, 1917. 28Whipple, G. H., Cooke, J. V., and Stearns, T.: Jour. Exper. Med., 1917, xxv, 479. Also Whipple, G. H., Stone, and Bernheim: Ibid., 1913, xvii, 286 and 307. 29Long, J. H., and Fenger, F. : Jour. Am. Chem. Soc., 1917, xxxix, 1278. soCole, L. G.: Jour. Am. Med. Assn., 1912, lix, 1947. Ibid., 1913, Ixi, 762; Arch. Eoentgen Eay, December, 1911, April, 1912, Am. Jour. Physiol., 1917, xlii, 61S. -iLuckhardt, A. B., Phillips, H. T., and Carlson, A. J.: Am. Jour. Physiol., 1919, 1, 57. 2Wheelon, H., and Thomas, J. E.: Jour. Lab. & Clin. Med., 1920, vi, 124. asWheelon, H., and Thomas, J. E.: Am. Jour. Physiol., 1922, lix, 72. a*McClure, C. W., Eeynolds, L., and Swartz, C. O.: Arch. Int. Med., 1920, xxvi, 410. PART VI THE EXCRETION OF URINE CHAPTER LX THE EXCRETION OF URINE (Partly contributed by R. G. PEARCE) It will be advisable to introduce the subject by a brief review of the essential structural features of the kidney, in so far as they apply to the excretory function of the orj^m. STRUCTURE OF THE KIDNEY The kidney is mainly derived from the surface of the celom, and is a mesodermal structure. In this respect it differs from ordinary secreting glands, which are endodermal in origin. Just as it is more or less unique in its development as a gland, it is also unique in its method of functioning. The physiological theories of the mechanism of urinarj^ secretion are closely related to the highly characteristic structure of the kidney. For this reason a ftrief survey of the structure of the different parts of the uriniferous tubules and the epithelial cells with which these are lined, is advisable. The uriniferous tubule, which is the secreting unit of the kidney, takes its origin in the capsule of Bowman, which may be likened to a hollow sphere of very delicate epithelium, one side of which is invaginated by a very much convoluted capillary mass, the glomerulus. The capsule opens up by a narrow twisted neck into a tubule, which is rather tortuous in the cortex (the proximal convoluted tubule), but soon takes a sharp descending course in the medulla towards the pelvis of the kidney, and doubles back (loop of Henle) in a straight course again to the cortex, where it again makes a twisted course (the distal convoluted tubule), and terminates in a collecting tubule, which, uniting with other tubules, collects the urine and conducts it to the pelvis of the kidney (Fig. 170). The capsule is lined with very thin epithelial cells, especially over the capillaries comprising the glomerulus. The proximal and distal tubules 541 542 THE EXCRETION OF URINE contain epithelium showing a prominent striation. These striations are rows of granules, which run towards the lumen of the cell, becoming less distinct as they approach it and apparently standing in close rela- tionship to the rather prominent internal (lumen) striated border of the cell. Some histologists believe that the striations at the border are Fig. 170. Diagram of the liriniferous tubules (C) , the arteries (A), and the veins (5) of the kidney. really cilia, which are described as being immobile. The cilia are shown in Fig. 171. The descending limb of Henle's loop is lined with a thin pavement epithelium with large bulging nuclei. The distal convoluted tubule is lined with cells not unlike those found in the proximal tubules, except that the inner border is not striated. The diameter of the lumen THE EXCRETION OF URINE 543 of the capsule varies with the activity of the kidney, as is shown in the following figures given by Brodie and Mackenzie. 1 RESTING KIDNEY MM. KIDNEY DURING DIURESIS MM. Mean diameter of capsule " " " glomerulus '' tl tl space of capsule Lumen of proximal convoluted tubule " " distal " " 93.4 90.4 3.0 0.0 7.2 123.8 100.0 23.8 17.6 20.6 The urinary tubule has jLj-emarl^able blood supply. The renal arteries arise directly from the abdominal aorta and are very short. They run through the medulla to the cortex, and join with neighboring arteries to B. Fig. 171. Cross sections of convoluted tubules from kidney of rat. A, during slight secretion; B, during maximal secretion. (From Sauer.) form arches from which proceed branches, that radiate into the cortex and give off smaller branches each of which very shortly breaks up into a small capillary tuft, the glomerulus, which lies in the invaginated sphere of Bowman's capsule. By concentration of a beam of light on the kidney of the living frog Richards and his co-workers have succeeded in rendering the capillary blood flow in the glomeruli readily visible under the microscope. The capillaries collect into an efferent vessel, which ap- pears to be smaller than the efferent artery, and this vessel in emerging from the capsule again breaks up to form a capillary network about the con- voluted tubules, forming their sole blood supply. These capillaries 544 THE EXCRETION OF URINE coalesce to form the renal vein. The blood of the kidney must, accord- ingly, pass through two sets of capillaries. The kidney is richly supplied with nerves, which are for the most part derived from the celiac ganglion and are in connection with the splanch- nic and the vagus. Other branches from plexuses in the region of the suprarenal body and the aorta join with those coming from the celiac ganglion to form what is known as the renal plexus, which is arranged in a network along the blood vessels and on the walls of the pelvis of the kidney. These fibers are distributed to the very smallest blood ves- sels, and nerve fibers have been observed among the cells of the tubules. THE MECHANISM OF THE EXCRETION OF THE URINE The great number as well as the variety of substances which are pres- ent in both the blood and the urine makes it appear improbable that urine excretion is dependent upon chemical combinations within the renal cells, and leads us to seek a physicochemical mechanism to explain the phenomenon. Can we discover the processes by which the kidney manufactures a highly concentrated solution of salts from a very dilute solution of the same salts in the blood plasma ? The problem is compli- cated by the fact that the ratios existing between the concentration of each urinary salt in the urine and the concentration of the same salt in the blood are different. In other words, the urine is not merely concentrated blood plasma freed from protein. The passage of water and salts through the capillary wall and through the basement membrane surrounding the renal cell probably takes place by simple diffusion. If it were otherwise, an expenditure of energy would be required, and it is difficult to understand how a basement membrane could bring about energy changes. Any substance to which the cell membrane is permeable will diffuse into the cell until an equi- librium is established between its concentration within the cell and that of the lymph or blood plasma. A nondiffusible substance will not enter the cell because it can not pass through the cell membrane, and if it exerts an osmotic pressure, it will also tend to keep the water in which it is dissolved from entering. If water does pass into the cell under these conditions, it is due to the expenditure of energy opposed to and greater than that which is offered by the osmotic pressure of the nondiffusible substances. Possible sources for such energy are the pres- sure of the blood in the renal capillaries, which would exert a force op- posite to that of its osmotic pressure, and the presence within the cell of a concentration of salts greater than is present in the blood, and able to exercise a sufficient osmotic force to draw fluid into the cell against the osmotic force of the nondiffusible salts. The passage of the urinary THE EXCRETION OF URINE 545 constituents through the cell might also be due to simple diffusion, the substances passing through the cell to be extruded on the other side in the same concentration as in the blood. In this case, the renal cells would act merely as a filter, the urine having the same concentration of each urinary salt as is present in the blood. A comparison of the concentrations of the urinary salts in the urine and the blood shows, however, that the urine is not merely a deprotein- ized blood plasma, so that other factors must be sought to explain the excretion. Since the concentration of the urine requires the expenditure of much more energy than is provided by the known physical factors, it is generally accepted that the renal cell in some manner supplies this energy by its metabolic activity. It is impossible at present even to surmise the nature of the process. Two possibilities may be considered. One is that the urine is a filtrate of the blood which has passed through a portion of the renal epithelium into the tubules as a very dilute fluid, resembling the blood plasma minus its colloidal substances, and that this dilute fluid is concentrated by the reabsorption of fluid and of salts by other cells of the kidney, and again replaced in the blood stream. The other is that the salts and fluid are each actively and individually ex- creted by the kidney. Whichever condition is the true one, the fact remains that the change in the concentration entails the expenditure of a great amount of energy on the part of the renal cells. The energy which the kidney must use in the actual work of concen- trating the urine from the fluid of the blood plasma can not be com- puted from a comparison of the concentration of the urinary salts as a whole in both the blood and the urine. Each constituent must be con- sidered apart. We can not, for example, determine the molecular con- centration of the blood plasma and the urine (by measuring A) (page 10) and estimate the work which is expended in producing the con- centration from the observed difference. On the basis of such comparisons, however, it is said that the excretion of 100 c.c. of urine requires at the minimum 500 kilogrammeters of work (Cushny 2 ). Even this conserva- tive estimate may be wrong, for it does not take into consideration the possibility that the excretion of water by the kidney requires energy expenditure on the part of the renal cells. Theories of Renal Function For many years two rival hypotheses have dominated the teaching of the mechanism of renal function. Bowman and Heidenhain postulated that the constituents of the urine are secreted by the vital activity of the epithelium of the capsule and the tubules. The glomerular capsule secretes the water and the easily diffusible salts in a dilute solution, and T)46 THE KXCRKT1OX OF l-KLN'K the uriniferous tubules add to this fluid the various organic and inor- ganic salts to bring the urine to the necessary concentration. This theory has been termed the -vital theory. Ludwig, on the other hand, advanced what is termed the physical theory, which holds that the glomerulus and capsule act simply as a filter, which allows the fluid of the blood plasma to pass through in a very dilute solution and in large amounts. This fluid is concentrated by physicochemical processes on its passage along the urinary tubules to the pelvis of the kidney. Both of these theories are inadequate and fall to explain the phenom- ena which research has shown to occur in the kidney, but they have served to develop what Cushny terms a modern theory of urinary excretion. The Modern Theory of Urine Formation. This theory accepts the general scheme offiltration and reabsorption of Ludwig, but pays due respect to the fact that the known physical forces are not adequate to explain the reabsorption which must occur in the tubules. It therefore supplements Ludwig 's theory by assuming a vital activity on the nart of the epithelium of the tubules in reabsorbing fluids and salts from the dilute filtrate coming from the glomerulus and capsule. A large amount of plasma fluid is filtered through the Avails of the glomerular vessels. This fluid has the same concentration of the salts to which the capsule is permeable as does the blood plasma, but it is free of the col- loidal substances normally present in the plasma. The blood leaving the glomerulus is therefore a somewhat concentrated solution of plasma col- loids, and must have returned to it the proper amount of water and salts to make it an optimum fluid for the body cells. This is accomplished by active absorption from the glomerular filtrate. The salts that are of no use to the body are not reabsorbed and therefore appear in highly concentrated form in the urine. These salts are termed nonthreshold sub- stances, and since their presence in the plasma is unnecessary, they con- tinue to be excreted as long as they are present in any concentration in the blood. The salts that are necessary for the plasma are termed threshold substances, and are reabsorbed until they are again present in the plasma in optimal strength. For example, urea continues to be ex- creted as long as any is present in the blood, while glucose is almost com- pletely reabsorbed so long as its concentration remains under a more or less fixed level. The volume of deproteinizcd blood plasma which the capsule would require to filter oft' from the blood in order to furnish the amount of the various salts excreted each day, and the volume of water which would have to be absorbed by the epithelium of the tubules to account for the concentration in which the salts are found in the urine, has been calculated as follows: in order to produce 20 grams of urea in 1000 c.c. of urine, 62 liters of the water of the blood-plasma, containing 0.033 per cent of urea, THK KXCKHT10N OF L'KINE 547 would have to be filtered through the capsule, and (51 liters of water returned to the blood from the uriniferous tubules. This amount of water would be derived from 67 liters of plasma (see table on page 551). Since the bloodflow through the kidneys is very great, at least 500 liters per day, only about 13 per cent of the fluid contained in the blood passing through the glomerulus would pass by nitration through the capsule of Bowman. The fact that such a large amount of fluid would have to be reabsorbed from the uriniferous tubules is a possible a priori criticism of the theory, but Cushney points out that the amount each tubule would have to absorb per hour would be very small (in his experiment on a cat amounting to less than 0.014 c.c. per hour). According to the modern view, there are therefore two fundamentally different processes occurring in the kidney; filtration in the capsule and selective reabsorption in the tubules. It is important to consider some of the evidence which is considered to indicate that both of these pro- cesses occur. The Filtration Process. The filtration of the protein-free blood fluid through the renal capsule, like that through any other membrane, depends on several factors. (1) There must be a difference in the pressure between the blood and the urinary filtrate. In the laboratory the pressure used in filtering is usually supplied by gravity, but in the case of the filtration of the urine through the capsule the force is furnished by the pressure of blood in the glomerular vessels. (2) The character of the filter determines what substances shall pass. The renal capsule is a membrane normally im- pervious to the proteins of the blood, but pervious to the other constitu- ents. Under certain conditions it loses this character. (3) The char- acter of the fluid determines how readily it will filter through the mem- brane. If the fluid contains a substance which can not pass through the filter and which exerts an osmotic pressure in opposition to the filtering force, the rate of filtration as well as the amount filtered, will be reduced. If the capsule acts as a filter it should be possible to alter the rate of urine excretion by varying any of the above mentioned factors, and experimentally this is true. The factors can be varied in several ways. If the blood pressure is raised by tying off several of the branches of the aorta, the urine is appreciably increased, or if the blood pressure is lowered, as can be done by cutting the spinal cord the amount of urine is decreased. In the artificially perfused kidney, the fluid exuding from the ureter increases as the pressure of the perfusion fluid is raised, and decreases as the pressure is decreased, and Richards and Plant 17 have recently shown that this urine formation may run parallel with the pressure without their being any alteration in blood flow. This strongly supports the view that filtration is the function of the glomerulus. Apparently, excre- tion can continue only as long as the colloids of the plasma are not notably increased, for, as the osmotic pressure due to indiffusible colloids rises, the pressure in the capillaries is no longer able to overcome it. The same point has been shown by Starling and his pupils, who found that the excretion of 548 THE EXCRETION OF URINE urine ceased when the capillary pressure in the glomerulus fell below that exerted by the osmotic pressure of the blood proteins, the critical pressure being from 30 to 40 mm. Hg. They also found that dilution of the blood with saline solution by reducing the osmotic pressure of the proteins in the plasma, was accompanied by an increase in the rate of excretion; excre- tion in such cases being maintained at a blood pressure below the normal critical pressure. If the dilution of the blood was made with saline con- taining gelatin or gum arabic, on the other hand, the diuretic effect was greatly diminished, and any fall in the blood pressure was followed by a suppression of the urine (Knowlton 9 ). These experiments evidently indicate that saline causes diuresis by diluting the plasma proteins and lowering their osmotic pressure, since no diuresis occurs when the os- motic pressure of the blood is maintained by the addition of colloids having an osmotic pressure. The significance of these facts, in connec- tion with the raising of lowered blood pressure after hemorrhage, has already been alluded to (page 140). The view that saline diuresis is caused by physical changes alone is confirmed by the experiments of Barcroft and Straub, 10 who showed that the oxygen consumption is often not appreciably raised during the diuresis which occurs after the injection of saline. If the diuresis were due to an actual increase in the work of the kidney, the oxygen con- sumption would have been increased. In the frog, the glomerulus and the tubules are supplied with blood by the renal artery, as is the case in the mammal, but the tubules are also supplied with some of the blood coming from the lower ex- tremities and the trunk through a vessel which has no counterpart in the mammal the renal portal vein. The blood, therefore, which is supplied to the tubule is a mixture from the glomerulus and the renal portal system. By ligating the renal vessels it is possible to cut off the blood supply of the glomerulus while leaving the tubules supplied by the renal portal vein. Normally the pressure in the renal portal system is not sufficient to force blood back through the glomerular vessels. Liga- tion of the renal vessels at once results in a suppression of the urine. If the glomerular vessels are perfused with Ringer's solution at a pressure equal to that found in the aorta, a considerable flow of fluid may be secured from the ureters, but no fluid is obtained when the renal portal vein is perfused at a pressure equal to that normally present in this vein. Rowntree and Geraghty 11 found that phenolsulphonephthalein added to the fluid perfused through the renal portal vein, did not cause secretion, but when urea was added, fluid containing the dye was obtained from the ureter. Unfortunately the pressure employed in these experiments may have allowed some fluid to be forced backward into the THE EXCRETION OF URINE 549 glomerulus, so that the results may be due to filtration through the capsule. Microscopical examination of the glomeruli has revealed the important fact that all are not equally active at the same time. As judged from the blood flow in them, the glomeruli would appear to work in relays. The Reabsorption Process, It is generally accepted that the proof that the capsule acts as a filter is fairly complete. Unfortunately such decisive experimental facts can not be offered to prove the assumption that the epi- thelium of the tubules reabsorbs the excess of water and salts which are fil- tered off through the capsule. If the modern theory of urine excretion is cor- rect, the cells of the tubules must not only absorb large amounts of water, Renal arfery Malpighian corpuscle Renal-portal vein Fig. 1/2. Diagram of blood supply of Malpighian corpuscle and of convoluted tubules in amphibian kidney. (Redrawn from Cushny.) but they must also allow for the reentrance into the blood, either completely or partially, of certain salts, while they must reject others entirely. We have called attention above to the fact that the glomerular filtrate is very different from the urine that is finally passed. The urine contains a very high percentage of small molecules, and the proportion in which they are present is entirely different from that in the blood plasma or in the glomerular filtrate. This is shown in the following table, in which the figures in the first two columns represent the average number of grams of urea, uric acid, chlorine, and glucose in 100 c.c. of protein-free blood plasma and in 100 c.c. of urine. In the third column is given the change in concentration which must occur in the kidney. 550 THE EXCRETION OF URINE 100 C.C. PKOTEIN- 100 C.C. URINE CHANGE IN FREE BLOOD CONTAINS CONCENTRATION PLASMA CONTAINS IN THE KIDNEY Urea .033 2. 60 Uric Acid .0022 .05 22.7 Chlorine .41 .6 1.5 Glucose .1 Among the experiments that have been offered in support of the absorption of fluid and salts by the tubules, are those in which the pressure of the urine in the tubules is slightly increased by partial closure of the ureter (Cushny). In these experiments the ureter of one kidney is partly closed with a clamp and the excretion obtained from this kidney is compared with that of the opposite normal kidney. Considerable ob- struction of the ureter results in a decrease in the amounts of water, chloride and urea excreted, but the urea content is decreased relatively less than is the chloride and water content. These results can be explained on the basis that a pressure that is sufficient to oppose the head of pressure producing filtration in the giomerulus will reduce the amount of the glomerular filtration, and accordingly the time allowed for the passage of this filtrate along the tubules is increased and absorption becomes more complete. Since urea is probably not absorbed at all and chloride is, the discrepancy in the effects on the excretion of urea and chlorine in the partially obstructed kidney can be explained. When the obstruction of the ureter is only slight, however, opposite results to that just mentioned are obtained (Brodie and Cullis). This observation is difficult to harmonize with the reabsorption hypothesis. When very large amounts of water are taken by mouth, it often happens that the urine excreted has a concentration of salts less than that present in the fluid of the blood. Some investigators believe that such a condition is possible only on the as- sumption that water is actively excreted, but a more plausible explanation based on the modern theory is that the water that is absorbed from the alimentary tract reaches the kidney as a dilute saline solution, and is rapidly filtered off in a form somewhat more dilute than the optimal solution which blood plasma must have for the well-being of the tissues. The tubules reabsorb the amounts of water and of substances, such as chlorides and sugar, that are necessary to restore the plasma to the optimal concentra- tion, but they do not reabsorb the nonthreshold substances, such as sulphates and urea. Many attempts have been made, by destroying the capsules or the tubules by means of poisons or by operation, to determine directly or indirectly the question of the function of the tubules. In such experi- ments, however, the number of factors involved confuses the issue and makes the results practically valueless so far as determining the normal function of the tubules. Progress is however to be expected in the near future since Richards and \Yeani have succeeded in developing a method by which sufficient jfiltraU 1 can be removed from the capsule of Bowman in living frogs, to make inicrocheinical analyses possible. Most important results supporting the reabsorption hypothesis have already been obtained in the case of glucose and chlorides. Thus, when glucose was injected subcutaneously some appeared in the capsular filtrate but none in the bladder urine; likewise chloride was found in the filtrate but none in the bladder. 19 THE EXCRETION OF URINE 551 Other experimenters have attempted to show absorption in the tubules by injecting diffusible substances, such as chemicals and dyes, into the ureter under what they deemed sufficient pressure to force the solution into the tubules, and by an examination of the blood or the tissues to determine whether or not the injected substances had been absorbed. The results obtained by this method are not convincing, probably chiefly because of the difficulty in reaching the tubules. Indeed, it is very questionable whether it is possible to inject a substance into the tubules from the ureter. Years ago Heidenhain, the exponent of the vital theory of excretion, believed that he had demonstrated the ability of the renal cells to excrete dye substances injected in- travenously. Since he failed to find evidence of dye excretion in the capsule, but found masses of dye in the tubules and stained granules in the cells of the tubules, he concluded that the cells of the tubules had the power to excrete the dye, and from analogy he believed that the tubules must likewise excrete the water and the various urinary salts. Subsequent work, however, has failed to confirm his belief that the capsule is not concerned in the excretion of the dye, and it is as reasonable to explain the results of the experiments with the dyes by assuming that the masses of dye sub- stances found in the tubules and in the cells are due to the reabsorption of water and perhaps of some of the dye from the dilute glornerular filtrate, as to accept Heiden- hain 's hypothesis. Iii the following table taken from Cushny the movements of the con- stituents of the plasma may be followed through the kidney. The ulti- mate destination of each is indicated in the enclosures. 67 LITERS PLASMA CONTAIN 62 LITERS FILTRATE 61 LITERS REABSORBED FLUID CONTAIN I LITER URINE CONTAINS PER CENT TOTAL CONTAIN IN ALL PER CENT TOTAL PER CENT TOTAL Water 92 62 1. 62 1. 61 1. 95 950 c.c. Colloids | 8 5360 gm.| Dextrose 0.1 67 gm. 67 gm. 0.11 67 gm. Uric acid Sodium Potassium Chloride 0.002 1.3 0.3 200 0.02 13.3 0.37 248 1.3 " 200 " 13.3 " 248 " 0.0013 0.8 " 0.32 196 " 0.019 11.8 " 0.40 242 " 0.05 0.05 gm. 0.35 3.5 " 0.15 1.5 " 0.6 6.0 " Urea Sulphate 0.03 20 0.003 1.8 20 " 1.8 " 2.0 2.0 " 0.18 1.8 " (From Cushny.2) It will be noted that the dextrose alone is completely absorbed, and that the urea and the sulphate are not absorbed at all from the glom- erular filtrate. The other salts are partly absorbed. Although at present it is probably most useful for practical purposes to accept Cushny 's hypothesis, it should be remembered that we are far from being in a position to explain all the known facts of the renal function by means of it. There can be no doubt that a process that is closely analogous if not identical with filtration plays an essential part in the formation of urine, and that it is assisted by a more obscure process depending on a selective action of the renal cells. But whether this latter process is essentially one of reabsorption of certain molecules 552 THE EXCRETION OF URINE from tubule to blood, or one of secretion from blood to tubule is problem- atical. As G-. N. Stewart points out, the reabsorption hypothesis is no simpler to comprehend than the older hypothesis of Bowman and Heiden- hain, for according to both views the renal cells are assumed to exercise discriminative powers. DIURETICS As already mentioned, Barcroft and Straub 10 have shown that the diuresis which results from the injection of saline into the blood is not accompanied by any increase in the oxygen consumption of the kidney. This observation, coupled with the fact that the total amount of chloride, urea, and sulphate which is excreted during saline diuresis is greater than under normal conditions, indicates that the excretion of these salts is not due to any active secretory process in the kidney, but rather to a greater filtration because of increased heart action. The diuresis which is caused by adding urea or sodium sulphate to the blood, on the other hand, is accompanied by an increase in the oxygen consumption of the kidney. Since there is no increase in oxygen con- sumption accompanying the increased excretion of practically the same salts during saline diuresis, the greater oxygen consumption must be due to more work being done in separating the water from the sulphate etc., to return it to the blood. The diuresis resulting from certain doses of adrenalin and from pituitrin is apparently due to constriction of the vasa efferentia for, in the isolated kidney perfused outside the body, the addition of these drugs causes the volume of theor^a^jtoJnjsrease, the perfusion pressure to rise and diuresis to occur? 15 "" According to the modern theory the polyuria in diabetes is caused by the excessive amount of water taken and by the inability of the kidney to concentrate the urine (by reabsorption) against the osmotic pres- sure offered by the concentrated sugar solution in the tubules. The diuresis following the injection of sugar is therefore of the same type as that pro- duced by sulphate and urea. The diuretic action of the digitalis group is dependent upon its influence on the circulatory system. If the circulation is already sufficient, digitalis does not cause diuresis. The cause of the diuresis produced by pituitary extract is not definitely known. It may be owing in part to its action on the circulation and in part to a direct action on the kidney (see page 811). ALBUMINURIA The plasma proteins ordinarily do not obtain entrance into the tubules of the kidney. In disease such as acute nephritis and cardiac fail- ure, the plasma colloids are filtered off through the capsule, proba- THE EXCRETION OF URINE 553 bly because of some change that has occurred in the permeability of its membrane due to inflammation or asphyxia. In these cases the urine is usually reduced in amount. Probably there is no purely glom- erular or tubular type of nephritis, both structures sharing in the dis- ability. When foreign proteins such as egg albumin gain entry to the blood, they appear in the urine. This is also the case when hemoglobin is liberated from the blood corpuscles. In both cases masses of the ex- creted protein in the capsule may be detected by microscopical examina- tion when the kidney is excised and hardened during the excretion. There is also some evidence that the capsule is damaged during the fil- tration of the foreign protein, for it has been found after injecting egg Si Fig. 173. Nerve supply of the kidney. K, kidney; Si, S 2 , major and minor splanchnic nerves; V, vagus; C.G., Celiac ganglion; A, aorta. (From Cushny.) albumin, that more protein may appear in the urine in 24 hours than the amount injected. Albuminuria is also readily induced experimentally by clamping the blood vessels of the kidney. After occlusion of the renal artery for 30 seconds, the urine for some time ceases entirely to flow, and when it returns (in about an hour) it is loaded with protein which gradually disappears. In all cases of albuminuria the albuminous nitrate in its passage along the tubules is concentrated by the reabsorption process, and this may occur to such an extent that the protein is precipitated so as to form a cast to which detritus from the tubular epithelium may become added, the exact type of cell composing this detritus depending on the point at which the protein solidifies. 554 THE EXCRETION OF URINE The Influence of the Nervous System on the Excretion of Urine. In spite of numerous and repeated attempts to demonstrate that a nervous mechanism governs the excretion of urine, no proofs which are above criticism have been forthcoming. Stimulation of the splanchnic nerves results in a diminution in the excretion of urine, probably because of a diminution in the blood supply of the renal vessels owing to the vasocon- striction. Stimulation of the vagus nerves below the level of the cardiac branches has been said to result in the augmentation of the rate of urine excretion (Asher and Pearce 12 ). The results are doubtful, however, since there is no increase in the oxygen absorption under the above conditions (Pearce and Carter 13 ). There is no doubt that the renalnerves profoundly affect the excretion of urine, but that they do so directly is very improbable, since perfectly adequate renal -function can be maintained in animals that have had the kidneys entirely removed and then replaced. There are numerous re- flexes that affect the rate of urine excretion by constriction of the renal vessels. Injury to the bladder or ureter, abdominal injuries to the kid- ney, or even cold applied to the skin, may result in incomplete suppres- sion of the urine. CHAPTER LX1 THE AMOUNT AND COMPOSITION OF THE URINE IN HEALTH AND DISEASE (Partly contributed by R. G. PEARCE) In the chapters on digestion and metabolism, we have followed the course which food takes with especial reference to the nutrition of the body. The excretion of these elements of nutrition is taken up under a number of the subdivisions of physiology, viz., respiration, digestion, kidney function and the skin. In the chapters on digestion attention was called to the fact that the feces, besides containing the indigestible resi- due of the aliment, contain several excretory products which at one time or another have actually been within the body proper. These in- clude normally the pigments of the body and many of the heavier mineral salts, such as iron, magnesium, lime and phosphates; and under abnormal conditions, as when the metals are given as medicine, bismuth and mer- cury. The respiratory system excretes most of the oxygen and carbon. In this chapter we shall take up the manner in which the body rids itself of the nitrogenous, and some of the mineral waste materials. Even at the risk of repetition, it will be advantageous to recapitulate certain facts concerning the essential chemical structure of the urinary constituents, so that we may be in a position to appreciate the kidney function in health and disease. We now know that the kidney does not form any of the specific con- stituents of its secretion (except hippuric acid). These substances are formed in the various tissues of the body, and are brought to the kidneys by the blood, where they are eliminated. But while the constituents in the urine are unchanged in chemical composition from that in which they are found in the blood, they do occur in greatly changed proportions. It is this variation in the concentration of the urinary constituents in the blood and the urine which presents the most important and at the same time the most difficult question in the physiology of the kidney. In the following table the percentage composition of the blood plasma is compared with that of an average sample of human urine. The third 555 556 THE EXCRETION OF URINE column gives the change in concentration which each constituent under- goes in passing through the renal filter. BLOOD PLASMA PER CENT URINE PER CENT CHANGE IN CONCENTRATION Water 90-93 95 Proteins, fats and other colloids 7-9 Dextrose 0.1 Urea 0.03 2 60 Uric acid 0.002 0.05 25 Creatinine Ammonia 0.001 0.04 40 Sodium 0.32 0.35 1 Potassium 0.02 0.115 7 Calcium 0.008 0.015 2 Magnesium 0.0025 0.006 2 Chlorine 0.009 0.27 30 Phosphates (PO 4 ) 0.003 0.18 60 Sulphates (SO 4 ) Amino acids The Amount of Urine The amount of urine passed in twenty-four hours varies with the amount of fluid ingested and the proportion of fluid retained by the body or excreted by other channels. Under ordinary conditions a twenty-four- hour sample amounts to from 1000 to 1800 c.c. of urine. On a constant water intake the volume of urine is extremely variable for any single day or part of the day (Addis and Watanabe 3 ). The average volume of urine excreted by twenty individuals on the third, fourth and fifth days of a constant diet in which the fluid intake was 2,070 c.c., varied from 1,013 to 1,712 c.c. for a twenty-four-hour period, from 684 to 1,195 c.c. for the first twelve hours of the day, and from 501 to 788 c.c. for the first eight hours of the day. In normal subjects the amount of urine excreted during the night is usually less than that during the day. This is such a constant finding that in cases where more than 50 per cent of the urine is excreted in the twelve hours of the night, suspicions of renal disease should be aroused. With a constant intake of food and water, the specific gravity of samples of urine collected at frequent intervals throughout the twenty-four hours exhibits considerable variations. The night urine is usually of higher specific gravity than that of samples passed every two hours during the day, the variation often amounting to as much as ten points. If the variation does not occur, but the different specimens show a fixed specific gravity, either at a high or a low level, it is usually indicative of renal trouble. This is illustrated in the following table: AMOUNT AND COMPOSITION OF THE URINE 557 DAY NIGHT 8-10 A.M. 10-12 A.M. 12-2 P.M. 2-4 P.M. 4-6 P.M. 6-8 P.M. 8-8 P.M. -A.M. Normal person In Hypertensive Nephritis In Myocardial Decompensation 1.016 1.010 1.018 1.019 1.009 1.020 1.012 1.010 1.019 1.014 1.009 1.018 1.020 1.009 0.020 1.010 1.010 1.021 1.020 1.009 1.022 (Compiled from Mosenthal's figures.) The proportion of water to total solids is often very similar in plasma and urine, but when water is taken in large quantities the urine shows much greater changes than does the blood, and the solids may sink to a very low concentration. On the other hand, when little fluid is taken or when the skin and bowel eliminate a large amount of fluid, the urine may become very concentrated without any change in the blood plasma. The total solids in urine can be determined with approximate accuracy by multiplying the last two figures of the specific gravity by the con- stant coefficient 0.233 (Haeser). The Depression of Freezing Point. While the solids of the blood con- sist, for the most part, of proteins and colloids, those of the urine are made up of inorganic salts and small organic molecules. The mole- cular concentration that is, the total number of molecules in a given quantity of fluid is under ordinary conditions much greater in the urine than in the blood. The molecular concentration may be deter- mined by the depression of the freezing point of a fluid below that of dis- tilled water (see page 10). Blood freezes almost constantly at -0.56 C., while urine may freeze at temperatures which vary between -1 C. and -2.5 C.; if very concentrated it may freeze at a temperature as low as -5 C., or if dilute the freezing point may be as high as -0.075 C. The variability of the freezing point and the specific gravity of the urine lead us to a consideration of the relationship of the urinary volume to its concentration. In the first place, the volume of water ingested is more frequently than otherwise in excess of the minimum absolutely re- quired by the body, and is subject to greater variation than the sub- stances excreted in the urine. The kidney is able to eliminate one con- stituent of the plasma which may be present in excess without involving any changes in others. For example, when salt is added to the food and excreted in the urine, the total chlorides are increased, but the amount of urine and the other constituents may remain unchanged ; or, again, as may happen, excess of salt leads to an increase in the volume of the urine, but the salt concentration remains constant while that of the other urinary bodies is decreased. Similarly, although the rate of urea excretion is not demonstrably augmented by an increase in the volume of the urine, an increase in the rate of urea excretion induced by the 558 THi: KXrKKTJOX OF I'HLNK ingestion of urea is accompanied by a larger volume of urine. That these two factors may not stand in a causal relationship to each other is sug- gested by recent work of Addis and Watanabe, 3 who find no quantitative relationship between the rate of increase in urea excretion and the increase in urine volume, and who believe that the apparent relationship is due to a common cause, such as alteration in the rate of circulation or change in the activity of the kidney cells. Nevertheless, there appears to be a limit set to the power of the kidney to take the urinary salts or water from the plasma and to place them in the urine in quite different proportions. The definite amount of water required to hold the urinary salts has been termed the " volume obligative" (Ambard 5 ). These limits of concentration may be fixed by the energy which the kidney can bring to act against the osmotic resistance. The inconstancy in the behavior of the kidney toward ingested salts is probably due to the fact that the salts reach the kidney in the concen- tration in which they are held by the blood plasma, and not as they were ingested. If salt is absorbed rapidly enough to disturb the salt equilib- rium of the tissues and plasma, then water will be abstracted from the tissues, and the plasma on reaching the kidney will eliminate the salt and water together. The difference in the reaction arises from the varied activity in the tissues in general rather than in the kidney itself. THE REACTION OF THE URINE In man and the carnivora the reaction of the urine is generally acid to litmus or phenolphthalein, but alkaline to methyl orange. The acid responsible for the reaction is phosphoric, not in a free state, but as a mixture of the salts Na 2 , HP0 4 and jaH 2 P0 4 , in which there is an excess of the latter. If alkali be added to a solution of H 3 P0 4 , containing a little methyl orange, the tint changes from red to yellowish when the H 3 P0 4 has all been changed into NaH 2 P0 4 ; if more alkali be added, and the indicator be now changed to phenolphthalein the tint turns red when the H 3 P0 4 has been changed to Na 2 HP0 4 . The reaction of urine lies be- tween these two points. In the herbivorous animals the alkaline re- action is due to the fact that vegetables and fruits contain salts of dibasic or polybasic acids, such as acid potassium malate, citrate, acetate, and tartrate. Oxidation of these in the body gives rise to carbonates. Some of the carbonic acid is excreted through the lungs, and hence the associated base, generally sodium or potassium, is com- bined so as to form a weak basic salt. The measurement of the acidity of the urine in terms of the H-ion con- centration like the same measurement in blood, requires the use of the AMOUNT AND COMPOSITION OF THK TRINE 559 rather difficult electrical or indicator method, the principle of which has been described in Chapter V. Expressed in terms of C H , the acidity varies between 4.7 x 10- 7 and 100 x 10" 7 . The total potential acidity that is, the number of H ions which will be formed in the face of a con- tinual neutralization of those in solution may be obtained fairly accu- rately by titrating the urine with Vio normal alkali in the presence of neutral potassium oxalate, using phenolphthalein as an indicator (Folin) . The results may be expressed in acidity per cent in terms of c.c. N/10 NaOH required to neutralize 100 c.c. of urine. If the ammonia excretion is added to the titration results, the total potential acidity is very closely measured. In normal subjects the acidity is high in the relatively scanty urine that is excreted during the night. In the forenoon more urine is excreted and the acidity is muchjeas, i- ft-, alkalinity Trmo.h greater. This alkaline tide in the forenoon is not dependent upon the accompanying diuresis, and it is the only alkaline tide during the 24 hours. The old idea of an alkaline tide following the ingestion of food is not correct. These facts are taken advantage of in a test of renal efficiency in which the conditions are standardized by having the patient, after awaking in the morning, drink a definite volume of water, but take no food. The night urine (from 11 p. M to 7 A. M.) and hourly specimens between 7 A. M. and 12 noon are collected and their volume and alkalinity (c.c. N/10 NaOH per 100 c.c.) measured. Typical results after drinking 500 c.c. water are exhibited in the following table: No. OF CASES FROM WHICH DIURESIS ALKALINITY PER CENT THE MEAN c.c. PER HOUR MAXIMUM FIGURES ARE MAXIMUM AT AT 10 OR CALCULATED BEFORE AFTER NIGHT 11 A. M. I 49 55 336 27 84 II 51 61 182 23 74 III 5(5 63 221 22 42 (Adapted from Leathes.) In this table, I represents the response of normal subjects in whom diuresis and alkalinity both increased; II, that of nephritic patients in whom the diuresis reaction was subnormal, but the alkalinity normal, and III, more severe cases in whom both the diuresis and the alkalinity were subnormal. Leathes has been able to show that this morning alkaline tide is de- pendent upon a greater excitability of the respiratory center on waking compared with that during the night. This causes pulmonary ventila- tion to become more thorough during the morning hours than during the night, so that more C0 2 is washed out of the blood, thus lowering its acidity (alkalosis) so that alkali is excreted by the kidney. The C0 2 con- 560 THE EXCRETION OF URINE tent of the alveolar air at night in a typical case was 7.4 per cent and after rising 6.63. Leathes also showed that forced breathing causes a marked increase in the alkalinity of the urine, a result which confirms those of Haldane, Meakin, etc., referred to elsewhere in this volume (page 381). THE SOLID CONSTITUENTS In a person living on an ordinary diet the most important organic and inorganic constituents of the urine are as follows : TOTAL SOLIDS (40 TO 60 GRAMS) IN ONE LITER OF NORMAL URINE ORGANIC CONSTITUENTS, 25-40 GM. INORGANIC CONSTITUENTS, 15-25 GM. Urea, 20-35 gm. Sodium chloride (NaCl), 8-15 gm. Creatinine, 1.0-1.5 gm. Phosphoric acid (P 2 O 5 ), 2.5-3.5 gm. Uric acid, 0.5-1.25 gm. Sulphuric acid, (SO 3 ), 2-2.5 gm. Hippuric acid, 0.1-1.7 gm. Potassium (KjO), 2-3 gm. Other constituents (ethereal sulphates, Sodium (Na 2 O), 4-6 gm. oxalic acid, urinary pigments, etc.), Calcium (CaO), 0.1-0.3 gm. 1.5-2.3 gm. Magnesium (MgO), 0.2-0.5. Ammonia (NH 3 ), 0.3-1.2 gm. Iron (in pigment), 0.001-0.010. (Compiled from Mosenthal's* figures.) These urinary salts are present in the blood, and are only excreted by the kidney. An investigation of the mechanism of renal excretion must therefore include a study of the relationship existing between the con- centration of the urinary salts in the blood and in the urine. We shall briefly review the chief biochemical relationships of the most important of these constituents and then give tables showing the quantitative changes which they undergo in various diseases. The Organic Salts of Normal Urine Nitrogenous Constituents. The greater number of the organic salts of the urine are made up of bodies which contain nitrogen, and which are derived from the protein element of nutrition. The proteins, which form the chief building material of the body, are broken up into their con- stituent amino acids in the intestinal tract and absorbed as such by the blood. Portions of these acids are taken up by the tissues to repair and to replace those proteins which have been discarded, and the remaining protein, in excess of the body need for amino acids, is deamidized, the major portion of the carbon, oxygen and hydrogen being oxidized to form C0 2 and water, and the lesser portion of these elements being com- bined with the nitrogen to form urea, ammonia, uric acid, etc. A similar fate later awaits the nitrogen moiety which found a place in the tissues, and which is replaced in turn by new nitrogenous bodies.* *For further details see page 645. AMOUNT AND COMPOSITION OF THE URINE 561 Since all the ingested nitrogen, except a small and rather constant amount which is lost by the feces and the sweat, is excreted in the urine, the total nitrogen of the urine has been taken as a measure of the nitro- gen or protein metabolism of the body. In normal conditions the protein metabolism is adjusted in such a manner that the nitrogen intake is equal to the nitrogen output, a condition known as nitrogenous equilib- rium. If the nitrogen intake is reduced below the actual body needs, the excretion of nitrogen is greater than the intake which indicates that the body protein is replacing the protein usually furnished by the food. The minimum amount of protein that the body must have to maintain equilibrium varies in individuals, and with the nature of the protein (page 605). With the ordinary mixed diet it is usually between 12 and 20 grams a day, corresponding to from 75 to 125 grams of protein. Ordi- narily, nitrogen is not retained and an increase in the protein ingested is followed by an increase of nitrogen in the urine. In periods of growth and after undernutrition, however, protein is stored in the body. For this reason, unless the amount of nitrogen ingested is known, the study of the total nitrogen of the urine gives no information concerning the nature of the nitrogen metabolism of the body. The total output of nitrogen per day usually amounts to 10 to 15 grams from 1 to 2 per cent of the urine by weight. Urea. The chifijLjo-tho nitrogcaeu&-b^4i^^ of the urine is urea, the origin of which has been fully described in the chapters on metabolism. No constituent of the urine is subject to greater variation both in abso- lute and in relative amounts. On an average diet containing 120 grams of protein per day, the absolute urea excretion may amount to about 30 grams ; on a low protein diet it may be only a few grams. When the pro- tein intake is high, the nitrogen eliminated as urea may be 90 per cent of the total nitrogen; but when the protein intake is low, this proportion may fall to 60 per cent. The difference is because on a low protein diet the greater percentage of nitrogen eliminated is endogenous in origin, and urea, which is the chief constituent of the exogenous nitrogen moiety of the urine, is accordingly decreased on low diets. In recent years the importance of the relationship between the con- centration of the urinary constituents in the blood and the urine has been much insisted upon, and since the estimation of the amount of urea in the blood and the urine is relatively simple, most of the work has been done by using these values. Ambard and Weil 5 believe that a quantitative relationship exists between the rate of urine excretion and the concentration of urea in the blood and the urine, since the urea in the blood acts as a stimulus to the renal cells. By comparing the rate of urea excretion and the concentration of urea in the blood and urine 562 THE EXCRETION OF URINE in a mathematical formula, they have obtained a value which they be- lieve is more or less fixed for the normal kidney. This expression is known as Ambard's coefficient and formula* and has been used as a means of evaluating the functional capacity of the kidney. Whatever may be the value of the formula in expressing the relation- ship existing between the rate of urea excretion and the concentration of this substance in the blood, it is certain that, in diseased conditions where there is impairment of the kidney the concentration of urea in the blood remains permanently at an abnormally high average level, al- though the amount of urea excreted during twenty-four hours may bo exactly the same as under normal conditions. Probably the increased con- centration of urea in the blood under these conditions is a compensatory measure to provide sufficient pressure to cause its excretion through a damaged outlet. It is this increase in iirea of the blood which is indicated by the term urea retention in nephritis. The upper limit of blood urea-nitrogen is about 20 mg. per 100 c.c., which would correspond to about 0.45 gm. of urea per liter of blood. The average figure is half of this amount. The maximum concentration of urea in the urine is seldom over 8 per cent, On this basis the kidney can raise the concentration of the urea in the urine, at a conservative estimate, from 100 to 200 times. Normally the daily output of urea nitrogen may range from 8 to 12 gm., and the nitrogen which it contains is roughly 80 per cent of the total excretion for the day. Ammonia. The chief source of ammonia in the body is the ni- trogenous portion of the deamidized ammo acids. The ammonia found in excess in the portal blood is derived from ingested ammonium salts and from ammonia resulting from bacterial action on proteins in the intestinal tract. The ammonia of the body is present chiefly in the form of ammonium carbonate, and it is this salt that is the precursor of urea. Because ammonium carbonate is so readily converted into urea by the tissues of the body, little ammonia is normally present in the systemic blood. The greater portion of the ammonia that finds its way into the urine serves as a base to transfer acid radicles either invested or formed *Ambard and Weil's formula is: Ui 70 D x x P V25 K r= coefficient of urea excretion (Constant of Ambard). Ur = grams of urea per liter of blood. D := output of urea in grams per 24 hours. P r= weight of the patient. C = grams of urea per liter of urine. 70 r= standard weight. 25 = standard concentration of the urine. The average value for this constant in normal individuals is said to lie between .06 and .09. Critical reviews of the work have been published recently by Maclean 6 and by Addis and Watanabe. 3 AM Of N'T AND COMPOSITION OF THE URINE 563 within the body. The amount of ammonia in the urine, therefore, is a measure of the acid bodies of the blood. For the latter reason the deter- mination of the ammonia excretion in urine is of some clinical importance. The ingestion of mineral acids increases the ammonia excretion, while alkalies tend to reduce it. During: fasting and in diseases such as diabetes, where there is an abnormal metabolism, the amount of ammonia in the urine is increased. Ordinarily the daily output of ammonia nitro- gen does not exceed 0.5-0.6 gm., constituting 3-5 per cent of the total amount of nitrogen. According to Nash 20 and Benedict the formation of the urinary ammonia occurs in the kidneys. Thus, there is more am- monia in the blood of the renal vein than in that of the systemic circula- tion, and the latter does not become increased by intravenous injection of acid. That the kidney produces ammonia is further shown by the fact that this decrease* in the blood after complete nephrectomy. Creatinine. On a meat-free diet the daily excretion of creatinine is remarkably constant, amounting to from^T to 11 nig. per kilogram of body weight. For this reason its determination is accepted as an in- dispensable feature in metabolism investigations involving urine analy- sis. Any gross variation from the normal amount indicates the certain failure of the attendants to collect all of the twenty-four-hour specimen of urine. The creatinine is one of the last of the urinary constituents to accumu- late in the blood during renal insufficiency, and for this reason affords a reliable prognostic indication concerning the patients' condition. A rise in the creatinine concentration of the blood is evidence of serious renal disease, patients with concentrations of 5 mg. never recovering (Chase and Myers). 7 The concentration of creatinine in the urine is about 100 times greater than in the blood, in which there is 1-2 mg. per 100 c.c. In adult man creatine does not appear in the urine save during starva- tion or wasting diseases. In woman it is absent save after postpartum resolution of the uterus. Children commonly excrete creatine along with creatinine until the middle years of childhood. The Purine Bodies and Uric Acid. The most important purine in human urine is uric acid. Xanthine is the next in importance, and small amounts of hypoxanthine, guanine, and adenine are found. The human body has the almost unique distinction among mammals of not being able to destroy any of the uric acid it produces, and hence all the uric acid formed during metabolism must be excreted in the urine. Unfortunately the kidney appears to be less competent to rid the body of this waste than it is of the other urinary metabolites, and one of the 564 THE EXCRETION OF URINE earliest signs of renal insufficiency is now held to be a failure of the kidney to prevent the uric acid of the blood from increasing. Perhaps the reason for the inability of the kidney to excrete uric acid readily lies in the fact that its salts are among the least soluble of those in the urine. It is on this account that when the urine cools, a red sediment of urates containing certain pigments often separates out. The uric acid of the urine is possibly derived entirely from the purine metabolism of the body, in which the nucleins either of the body cells or of the exogenous food take part. It is decreased during starvation and increased L _by eating food rich in nucleins, such as liver and sweet- breads. Under ordinary conditions the excretion of uric acid amounts to from 0.3 to 1.2 gm. per day (0.02 to 0.10 per cent), the variation being de- pendent upon the state of health, diet, or personal idiosyncrasy. The blood of a normal individual contains on the average 1.8 mg. of uric acid per 100 c.c. The kidneys are therefore able to concentrate the uric acid in the urine from 30 to 60 times over its concentration in the blood plasma. The purines found in coffee and tea (caffeine, etc.) are excreted in the urine as salts not of uric acid but of methylated xanthines. Hippuric Acid. This is a constanj^ constituent of the urine of her- bivorous animals, and is usually present in small amounts in human urine. The amount rarely exceeds 0.7 gm. a day, but on a diet rich in fruits and vegetables it may exceed 2 gm. It is interesting, since it is the only urinary constituent that is synthesized by the renal cells. Amino acids are always present in small amounts in the urine, con- stituting, according to D. D. Van Slyke, about 1.5 per cent of the total nitrogen. The estimation of the amino-acid nitrogen of the urine has not been found to be of any clinical significance. 8 The aromatic oxyacids are normally present in the urine in varying amounts. These include phenol, indoxyl, skatoxyl, and phenylacetic, paraoxyphenyl, propionic, oxymandelic and homogentisic acids. These bodies are derived from phenylamino acids, such as tyrosine, tryptophane, and phenylalanine. It is believed that the putrefactive decomposition of proteins in the large intestine results in the production of these toxic bodies. The body protects itself by oxidizing them and uniting them to sulphuric acid to form the ethereal or conjugated sulphates, which are found in the urine in the form of sodium or potassium salts. The determination of the amounts of these bodies in the urine has therefore been taken as an index of the putrefaction going on within the bowel. The chief of these bodies is urinary indican, which is found usually as AMOUNT AND COMPOSITION OF THE URINE 565 a potassium salt. The test for indican in the urine consists in oxidiz- ing the indoxyl in an acid solution by means of ferric chloride to indigo blue, and shaking out the indigo blue with chloroform. The depth of the color of the chloroform affords a rough means of determining the amount of indican present. The fact that the indican test is nega- tive must not be taken to mean that the intestinal processes are normal, for if the intestine fails to contain phenylated amino acids, or the proper bacteria are not present, no indican will be found. On the other hand, the putrefactive process of the large bowel may not be very extensive, yet the amount of indican in the urine be increased, because of greater absorption due to constipation. Skatole, ajejsaLsmetHng substance, is formed by certain kinds of bac- teria. The greater proportion of this substance is excreted by the bowel, but if the person is constipated, some of it may find its way into the blood to impart a fecal odor to the breath and urine. Its presence therefore has some diagnostic importance. A very interesting body which is sometimes found in the urine is homogentisic acid. It is thought to be an intermediate step in the metab- olism of tyrosme, and is found in the urine of people suffering from alkaptonuria. The disease is remarkable in that it appears to run in families and produces no ill effects. Homogentisic acid is a strong reducing agent, and for this reason may be confused with sugar in Fehling's test. The inorganic constituents of the urine include the acids: chlorides, sulphates and phosphates; and the bases: sodium, potassium, magnesium, and calcium. The Salts of the Normal Urine The Chlorides. The chlorides compose the bulk of the acid rad- icles in the urine. Although they appear to be necessary constituents of the living cell, they do not, so far as known, enter into combinations with the organic constituents. The tissues appear to require a rather definite concentration of sodium chloride in order to carry on their work, for reduction in the sodium-chloride intake of the body results in a reduction in the chloride excretion by the urine. In salt starvation the chlorides may disappear entirely from the urine, the amount of chloride excreted appearing to be closely related to the amount of salt ingested. When the intake is constant, the rate of excretion is likewise more or less constant, but a sudden reduction in the salt of the diet may be accompanied by a slight decrease in the salt content of the blood, with an attendant loss of water. On the other hand, when the salt is 566 THE EXCRETION OF URINE again taken, there is a retention of salt and of water, with a consequent increase in body weight, until equilibrium is re-established on the old level. While the above is the usual reaction, a considerable retention of salt without an increase in the water content of the body may occur i some apparently normal cases. This is due probably to the deposition of salt in the tissues. Careful studies fail to confirm the idea that there is a fixed relation- ship between the salt and the water of the body. As with the nitroge- nous constituents, however, there appears to be a relationship between the rate of excretion of chlorides and the amount of chloride in the blood. Ambard believes that this relationship, like that of the excretion of urea to the blood urea, is capable of being expressed mathematically (see page 562), if allowance is made for the fact that NaCl is not excreted after it falls below a certain concentration in the blood equal to about 5.62 gm. per 1000 c.c. This level is more or less constant for normal individuals, but is considerably increased in disease of the kidney. This is known as the threshold of chloride excretion. The amount of sodium chloride excreted in the urine in twenty-four hours varies between 8 and 20 gm. a day, according to the intake. It is therefore apparent that the kidney is able to concentrate the salts of the plasma from ten to twenty times. The Sulphates. Since the inorganic sulphates do not form an im- portant constituent of the food, the greater portion of the sulphates of the urine are derived from the sulphur found in the protein molecule. For this reason the sulphates of the urine, like the nitrogen, are a meas- ure of protein metabolism. An increase in the nitrogen excretion is accompanied by an increase in the sulphur excretion, the ratio being about 5 to 1. The daily output of sulphur is between 1 and 3 gm. The greatest output is in the form of the alkaline sulphates, about 10 pe v cent in combination with aromatic bodies, and a small amount in com- bination with amino acids and neutral organic salts. The phosphates of the urine are derived from the food and from the oxidation of phosphorus-containing bodies in the tissues such as nuclein, lecithin, etc. The daily excretion varies between 1 and 5 gm., calculated as P 2 :V When calcium or magnesium is present in the food, they are excreted by the bowel as phosphate, and proportionately less is found in the urine. The amount usually excreted in the feces canals about 30 per cent of the total. Since phosphates in the urine exist as a mixture of the mono- and di- sodium hydrogen phosphates, they have an important bearing on the AMOUNT AND COMPOSITION OF THE URINE 567 reaction of the urine, the amount of each varying with the degree of the acidity of the urine (see page 558). On a heavy protein diet the urine is acid on account of the sulphuric and other acids formed from the meat, and in this case there is a greater amount of phosphoric acid and the mono-sodium hydrogen phosphate. When the urine is alkaline or less acid, as it is on a vegetable diet, there is a large amount of the disodium hydrogen phosphate. Since calcium and magnesium phosphates are more soluble than the diphosphates of the same metals, deposits of the earthy phosphates are often found in neutral or alkaline urines. When the urine is heated, the diphosphate of calcium breaks up into the mono-calcium and a tri-calcium phos- phate, which accounts for the fine turbidity often taken for albumin in the flame test. Addition of acid will cause this to disappear. The crys- tals of triple phosphates which occur in alkaline urine are ammonium magnesium phosphate, NH 4 MgP0 4 . Quantitative Changes in the Blood and Urine in Disease The precise diagnosis of many diseases is being greatly assisted by quantitative determinations of the various nitrogenous metabolites, of sugar and of inorganic salts, not only in the urine but also in the blood. Although the details of this work must be sought for in the texts deal- ing with clinical diagnosis, it may be of value, as indicating the practical nature of the work if we give one of the tables (page 568) recently pub- lished by Myers 16 which illustrates the nature of the results that have been obtained. COMPARATIVE NITROGEN PARTITION OF URINE AND BLOOD. (In per cent of total nonprotein nitrogen) FLUID URIC^ACID UREA CREATININE N N AMMONIA N REST N Normal Urine < X ^L5 Normal Blood 2 Blood in Gout and Early Nephritis 6 Blood in Parenchymatous Nephritis (Nephrosis) 2 Blood in Terminal Interstitial Nephritis 2 to 3 85 50 50 55 75 5 2 o 2 2.5 4 0.3 0.3 0.3 0.5 4.5 46 42 40 20 In the foregoing table is given a comparison of the partition of the non- protein nitrogenous constituents of the blood and urine as well as that of the blood in nephritic conditions. A useful description of .the most prac- tical and simple of the methods employed for making the necessary an- alyses will be found in the papers of Myers. 568 THE EXCRETION OF URINE s ^ c> o to CM 10 rt< tr- ? j g 10 N 10 10 rH CM CO o Ii5 1 1L 02 r-. W ts | Q o Q _C C3 Q 4*1 ^*XJ ^C^ r-. W M -*-* t** CD *^ ^ H g rH OO co o c ^ 1 - 9 a 9 CO p' o co o ^s J r~ *~i o o o rH +S 1-1 o O w 'P^O *** ^""^ ^^ 1-1 tn o3.2 ' g o 11 W o o> IQ J ^ t- IO ri JJ^ O j^ 1 ^ i o co IO O ,) 1 r^ ^^ r 1 S c ^* z ^L o .2 o *^ to ^* ft w^-o n 03 Q 9 S ^O ^ ^^ ^5 J? ^ T 1 rH CJ I 1 !3 to N 10 10 p i; ^ M ^V LO 10 CM M 7-1 co c/3- -M C C g 10 w>^ g -M IO OO co' "5 > H I5f\p CO* cq 9 CM CM CO GO ^. i i 1J 'S 10 ft W 03 S tf S w 2 K S S CM CM rH ? ^ a ^ \P IO o 00 ^_rt to 82 T| ^ 10 - o r^i ^ ^2 fej bb o .T 1 o o II rH CO <^> TJH i o-i C/3 5 . D IO TH 1 O m ' o CM co 3 5 cb ^ rH 1$ '- 1 ^1.1 1 1? t ^ fl 3 1 d o CJ Sir CONDITION "3 t 1|| Hi a | ^ 1 g S 'r 1 - C3 rrj S -(-a 1 1 IIII Early interstitial Acute nephritis . Parenchymatous (Nephrosis) Terminal Intersti nephritis . Bichloride poison Double poly cystic kidney Prostatic obstruc' Acute intestinal obstruction , Eclampsial Cholelithiasis Pernicious Anemi jSI ^3 X x \ x x x x V ^ ~\ \ \ \ \ \ \ N \ \ \ X X ^J \JO \ \ \ \ v \ s \ x x x \ ^ IR zu \ \ s, \ \ ^ ^ s. x . \ . \ \ \ \ ^ s \ \ x x \> $>* * \ \ \ \ \ X . 1 4 Lb , . SB ^ \ k X I \ \ \ \ sID I.I 30 40 50 60 70 80 90 100 t WEIGHT-KILOGRAMS Fig. 175. Chart for determining surface area of man in square meters from weight in kilo- grams (Wt.) and height in centimeters (Ht.) according to the formula: Area (Sq. Cm.) = Wt. 0.425 XHt. 0.725 X71.84. (From Dubois and Dubois, Arch. Int. Med., 1917, vol. 17.) termined at a glance (Fig. 175). Another method recently employed by Benedict is based on measurements made from photographs of the subject in various poses. By the use of these more accurate measurements of body surface, it is now known that, although the surface-area law gives us constant results for the energy output of different individuals of similar build, and offers us a much more accurate basis than body weight for comparing those of dif- ferent laboratory animals, yet it breaks down when applied to men in widely differing states of body nutrition. Thus, in the case of a man who starved for a month, the calorie output per square meter of surface decreased to- wards the end of the fast by 28 per cent. Obviously, therefore, it would be incorrect to draw conclusions regarding possible changes in energy output METABOLISM 577 of a series of emaciated or corpulent individuals by comparison of their calorie output per square meter of surface with that of normal individuals. The determining factor of energy output is undoubtedly the general condition of bodily nutrition the active mass of protoplasm of the body (Benedict). That there is a relationship between the body surface and metabolism is undoubted, but the relationship is not a causal one. At present, therefore, the only safe method to employ in comparing the metabolism of normal and diseased individuals is that called by Benedict "the group method," in which the metabolism of groups of persons of like height and weight is compared, it being assumed that such individuals have the same general growth relations. . Benedict, in collaboration with Harris, has carried this idea into effect by the preparation of standard tables which give the values for basal metabolism for both men and women for the commoner ranges of weight, stature and age. The values in the tables are based on the statistical constants of a sufficient number of accurate normal data so that they can be used as the standards for comparison with results obtained under various physiological and pathological conditions. Two sets of tables are employed, one for body weight and the other for stature and age, the values of the two tables being added together, and, since women have a lower metabolism than men, sets of tables for both sexes are necessary. To determine whether there is a significant modification of metabolism in clinical cases, it is necessary to compare the actually measured calories with those calculated from the tables on the assumption that the patient is in normal health. It has been found by this method that athletes have a somewhat higher metabolism than untrained persons, and that living on a vegetarian diet does not funda- mentally alter it. Influence of Age and Sex The energy output is low in the newly born ; it increases rapidly during the first year, reaching a maximum at about three to six years of age, and then rapidly declining to about twenty, after which it declines much more slowly. The decline in the earlier years does not proceed steadily, how- ever, for at the period just preceding the onset of puberty a decided in- crease becomes evident, indicating that at this period the metabolism of the growing organism is being stimulated. Females have a lower energy output than males, and the stimulating influence of puberty is less marked in them. In round numbers, 40 C. per square meter of surface per hour is the energy output of normal men, a 15 per cent deviation being considered as decidedly abnormal. The average metabolism of fat and thin subjects is 578 METABOLISM the same, but that of women is 6.8 per cent lower than that of men. The basal metabolism of a group of men and women between the ages of forty and fifty was 4.3 per cent below the average for the larger group between the ages of twenty and fifty; and that of a group between fifty and sixty years was 11.3 per cent lower. Influence of Diseases The measurements have been made by the direct method which has just been described, but since the much simpler indirect method (page 589) yields comparable results, it is being adopted for clinical purposes. These results were obtained by making parallel determinations of energy out- put by both methods, in disease as well as in health. Some of the ob- servations that have been made on the energy output in various diseases are as follows: In very severe cases of exophthalmic goiter, heat produc- tion may be increased from 50 to 75 per cent over the normal. The warmth of the skin and the sweating, which are prominent symptoms of this disease, are therefore accounted for by the increased elimina- tion of heat, and it is considered possible that the other symptoms would be caused in any normal individual were his metabolism maintained for months or years at the high level which it occupies in goiter. In the opposite condition of myxedema, the energy output is markedly reduced, but rises slowly during treatment with thyroid extract, or much more rapidly with the very active thyroid hormone recently iso- lated by Kendall (page 798). In diabetes it has often been thought that the rapid emaciation and loss of strength were dependent upon an excit( state of metabolism, or a useless burning up of the energy material. Al- though an increase could not be shown when the surface area was used as a basis for calculation (Du Bois) the group method shows an increased energy metabolism in many cases of diabetes amounting sometimes to 12 per cent (Benedict). In uncompensated cases of cardiorenal dis- 'ease, there is increased energy output. In pernicious anemia metabolism is normal, although in severe cases there may be an increased demand for oxygen. Even at the risk of repetition, it is important to point out that in all these diseases the energy output is the same whether measured directly or by the indirect method about to be described. The value of following the basal metabolism in the therapy of exoph- thalmic goiter has been demonstrated in recent work by Means and Aub, who have followed the metabolism and clinical condition of a series of cases over a period of several years. They found in the majority of cases that the results with the x-ray treatment are as good as those se- cured by surgical methods. The basal metabolism shows a rapid pre- METABOLISM 579 liminary fall after surgical removal, but a secondary rise occurs, whereas with x-rays the metabolism gradually declines throughout the treatment. There is practically no mortality with the x-ray treatment, and the risks of surgical interference in very acute cases is decidedly amelio- rated by a preliminary treatment with the rays. They conclude that "in the management of exophthalmic goiter periodic determination of the basal metabolism should be quite as much a routine as the examination of the urine for sugar in diabetes mellitus." THE MATERIAL BALANCE OF THE BODY We must distinguish between the balances of the organic and the in- organic foodstuffs. From a study of the former we shall gain information regarding the sources of the energy production whose behavior under various conditions we have just studied. From a study of the inorganic balance, although we shall learn nothing regarding energy exchange for such substances can yield no energy we shall become acquainted with several facts of extreme importance in the maintenance of nutrition and growth. To draw up a balance sheet of organic intake and output requires an accurate chemical analysis of the food and of the excreta (urine and ex- pired air). Methods for Measuring Output The principle by which the output is measured will be understood by referring to Fig. 176, from which it will be seen that the calorimeter is connected with a closed system of tubes provided with an air-tight rotary blower or pump to maintain a con- stant current of air, as indicated by the arrows. Following the air stream as it leaves the chamber, we note a side tube connecting with a meter to indicate changes in vol- ume of the air in the system. Beyond this and the pump is a specially constructed bottle containing concentrated H 2 SO 4 , then one containing soda lime, and lastly another H 2 SO 4 bottle. The first H 2 SO 4 bottle absorbs all the water vapor contained in the air coming from the chamber; the soda lime bottle absorbs the CO 2 , and the second H,S0 4 bottle absorbs water that is produced in the chemical reaction involved in the absorption of the CO 2 by the soda lime (2NaOH + CO 2 = H 2 O + Na 2 CO 3 ). By weigh- ing* these absorption bottles before and after an animal has been for some time in the chamber, the weight of H O and of CO given out can be determined. Another side tube leads to an oxygen cylinder, the valve of which is manipulated so as to cause oxygen to be discharged into the system at such a rate as to compensate exactly for that used up by the animal, as indicated by the behavior of the meter. The amount of oxygen required is determined either by weighing the oxygen cylinder before and after the observation or by measuring the volume of oxygen used by passing it through a carefully calibrated and very sensitive water meter inserted on the side tube that con- nects the 2 cylinder with the main tubing of the system. Since muscular activity causes pronounced changes in the rate of metabolism, means are usually taken to secure graphic records of any movements made during the observation. 580 METABOLISM The growing importance in clinical investigations of measurements of the respiratory exchange and the necessity for having methods that are as simple as is consistent with accuracy, have led to the introduction of several other forms of apparatus, of which those of F. G. Benedict and of Tissot are the most important. In these methods no calorimeter is em- ployed but the energy exchange is calculated from the amount of 2 inspired in a given time. In Benedict's method a tightly fitting mask is applied over the nose and mouth and connected, by a short T-piece, with the same tubing as that used in the respiration calorimeter. The patient I V/////////////////////////////^^^ I -2 is delivered from the weighed (^-cylinder. The increase in weight of bottles II and III gives the COal that of I, the water vapor. thus breathes in and out of the air stream that is passing along the tubing without any of the obstruction experienced when the breathing has to be performed through valves, as in the older (Zuntz) forms of portable respiratory apparatus. It is particularly for studies on man that this apparatus has been devised. The Tissot and Douglas methods are de- scribed in Chapter LXIV, where the method used for calculating the re- sults is also outlined. This is called the method of indirect calorimetry, and it has been clearly established by numerous observations that the results agree exactly with those secured by the method of direct calorimetry described above. For METABOLISM 581 most purposes the indirect method is quite satisfactory, and it is espe- cially valuable in cases in which there are considerable and sudden changes in body temperature. That the results by the two methods should agree shows clearly that the law of the conservation of energy must apply in the animal body, for it is evident that if any energy were derived from outside the body other than that taken with the food, the results by the direct method would be higher than those by the indirect. CHAPTER LXIII THE CARBON BALANCE Before proceeding to discuss the special metabolism of proteins, fats and carbohydrates, it will be advantageous to consider briefly some gen- eral facts concerning the excretion of carbon dioxide and the intake of oxygen. In the first place, it is important to note that the extent of the combustion process in the animal body is proportional to the amount of oxygen absorbed and of carbon dioxide produced, whereas the nature of the combustion is indicated by the ratio existing between the amounts of carbon dioxide expired and of oxygen retained in the body. An investi- gation of the carbon balance, in other words, is partly quantitative and partly qualitative quantitative in the sense that it indicates how in- tensely the body furnaces are burning, and qualitative in the sense that it tells us what sort of material is being burned at the time. THE RESPIRATORY QUOTIENT Influence of Diet. The respiratory quotient is determined by com- parison of the volume of carbon dioxide expired with the volume of oxy- gen meanwhile retained in the body or, as a formula, Vol. C0 2 expired ' VoT 2 retained* For the sake of brevity the respiratory quotient is often written R. Q. That it serves as an indicator of the kind of combustion occurring will be evi- dent from the following equations: 1. Carbohydrate: C fi H 12 O 6 -K6O 2 = 6CO 2 + 6H 2 O (Dextrose.) 2. Fat: C 3 H.(C ]8 H 33 O 2 ) 3 + 80O 2 57CO 2 + 52H 2 O (Olein.) 3. Protein : C 72 H 112 N 18 O 22 S + 77O 2 = 63CO 2 + 38H,O + 9CO (NH 2 ) 2 + SO, [Empirical formula for albumin (Lieberkiihn).] . T? A ^O 2 _ ^ n Q9 B -Q-= -57-77- 582 THE CAEBON BALANCE 583 4. Conversion of fat into carbohydrate: 2C 3 H 5 (C 18 H 33 2 ) 3 -f 640 2 = 16C 6 H 13 O 6 + 18CO 2 + 8H 2 O (Olein.) 5. Conversion of carbohydrate into a mixed fat : 13C 6 H 12 O 6 = C 55 H 104 O 6 + 23CO 2 + 26H,O. (Oleostearopalmitin.) Taking carbohydrates first, the general formula may be written CH 2 0, from which it is plain that, to oxidize the molecule, oxygen will be re- quired to combine with the carbon alone, according to the equation, CH 2 + 2 = C0 2 + H 2 0. In other words, the volume of carbon dioxide pro- duced by the combustion will be exactly equal to the volume of oxygen used in this process, in obedience to the well-known gas law that equi- molecular quantities of different gases occupy the same volume. The respiratory quotient is therefore unity (Equation 1). With fats and pro- teins, however, the general formula must be written CH 2 -{-0, indicating therefore that for its complete oxidation the molecule must be supplied with oxygen in sufficient amount to combine not only with all of the car- bon, but also with some of the hydrogen, forming water; so that the vol- ume of C0 2 produced will be less than the volume of oxygen retained, and the respiratory quotient will be less than unity. As a matter of fact, as the above equations show (2 and 3), the respiratory quotient for fats is 0.7 and for protein 0.82.* That the conditions hypothecated in the equations exist in the animal body during the combustion of the foodstuffs can easily be shown by ob- serving the respiratory quotient of animals on different diets. An her- bivorous animal, such as a rabbit, when it is well fed gives invariably a respiratory quotient of about 1, whereas a strictly carnivorous animal, such as the cat, gives a respiratory quotient of about 0.7. Even more striking perhaps is the comparison of the respiratory quotients in an herbivorous animal while it is well fed and after it has been starved for a day or two. In the latter case the respiratory quotient will fall to a low level because, by starvation, the animal has been compelled to change its combustion material from the carbohydrate of its food to the protein and fat of its own tissues. As already explained (page 582), it is from the respiratory quotient that we are enabled to tell what proportions of fat and carbohydrate, respectively, are undergoing metabolism. A useful table showing the percentage of calories produced by each of these foodstuffs, after allow- ing for protein, is given by Graham Lusk (see page 598). ^Vlore recent calculations based on accurate metabolism observations give 0.707 for fats and 0.801 for protein (Lusk). 584 METABOLISM Influence of Metabolism. Apart from diet, the respiratory quotient may often be altered by changes in the metabolic habits of the animal. These are most conspicuously exhibited in the case of hibernating animals. In the autumn months, when the animal is eating voraciously of all kinds of carbohydrate food and depositing large quantities of adipose tissue in his body, the respiratory quotient may be considerably greater than unity, indicating therefore either that relatively more carbon dioxide is being discharged or less oxygen retained. As a matter of fact, it can easily be shown that it is the former of the causes that is responsible for the higher quotient, the explanation for the increased production of C0 2 being that, as the carbohydrate changes into fat, the relative excess of carbon in the former is got rid of in the form of C0 2 , as indicated in Equation 5. On the other hand, if the animal is examined while in his winter sleep, it will be found that the respiratory quotient is extremely low, often not more than 0.3 to 0.4, which may be interpreted as indicating either an excessive absorption of oxygen or a markedly decreased excretion of carbon dioxide. As a matter of fact, there is a great diminution in both the excretion of carbon dioxide and the intake of 2 , because the whole metabolic activity of the animal is extremely depressed, but this diminution affects the oxygen to a much less degree, indicating therefore a relative increase in the oxygen retention. The explanation is that the oxygen is being used in the chemical process in- volved in the conversion of the fat back into carbohydrate. Whatever may be the relationship between fat and carbohydrate in the nonhibernating animal, there is no doubt that during hiberna- tion, before the fat stores are burned, fat is converted into something closely related to carbohydrates, the equation for the process being rep- resented as given above (No. 4). In man and the higher mammalia, the only condition apart from diet which can affect the nature of the combustion process is disease; thus in total diabetes (page 709) the organism loses the power of burning carbohydrate, so that whatever the diet may be, the respiratory quotient is very low, never higher than that representing combustion of fat and protein. It has been claimed by certain investigators that in diabetes the respiratory quotient may fall considerably below 0.7, indicating, as in hibernating animals, that fat is being converted into carbohydrate. The most recent and carefully controlled observations, however, deny this claim, and for the present we must assume that in the body of man fat is not converted into carbohydrate (see page 696). In numerous other diseases investigated by Du Bois and others 6 no qualitative change in the combustion processes in man has been brought to light. THE CARBON BALANCE 585 THE MAGNITUDE OF THE RESPIRATORY EXCHANGE It is evident that the amount of carbon dioxide expired and of oxy- gen retained will be proportional to the energy liberation in the animal body. Even at the risk of repetition it should be noted that the energy exchange can be very accurately calculated from the result of the material balance sheet indirect calorimetry, as it is called (page 580). On account of the comparative simplicity of measuring the carbon dioxide output and oxygen intake, it is natural that many of the obser- vations that have been made on energy production in the animal body depend on the use of this method, justification for which is found in the complete agreement between the results of direct and indirect calorim- etry in a great variety of diseases and conditions in man (Du Bois 6 ).* In the first place, it is interesting to compare the respiratory ex- changes of different animals computed per kilo body weight. This is shown in the following table. ANIMAL WEIGHT GM. OXYGEN AB- SORBED PER KILO AND HOUR GM. CARBON DIOXIDE DISCHARGED PER KILO AND HOUR GM. VOL. C0 2 VOL. 2 TEMPERA- TURE OF AIR Insecta Field cricket 0.25 2.305 Amphibia Edible frog 0.063 0.060 0.69 15-19 (44.2 c.c.) (30.76 c.c.) 0.105 0.1134 0.78 (73.4c.c.) (57.7 c.c.) Aves Common hen 1280 1.058 1.327 0.91 19 (740 c.c.) (675 c.c.) Pigeon 232-380 3.236 Sparrow 22 9.595 10.492 0.79 18 (6710 c.c.) (5334.5 c.c.) Mammalia Ox 638,000 0.389-0.485 660,000 Sheep 66,000 0.490 0.671 0.99 16 (343 c.c.) (341 c.c.) Dog 6213 1.303 1.325 0.74 15 (911 c.c.) (674 c.c.) Cat 2464 1.356 1.397 0.75 -3.2 3047 (947 c.c.) (710 c.c.) ) f 0.645 0.766 0.86 29.6 (450 c.c.) (389 c.c.) Rabbit 1433 1.012 1.354 0.97 18-20 Guinea pig 444.9 1.478 1.758 0.86 22 Rat (white) 80.5 3.518 7 (1789 c.c.) Mouse " 25 8.4 17 Man 66,70 0.292 0.327 (Modified from Pembrey.)" *For the convenience of those who may desire to know more about the methods of analysis that are suitable in the clinic, a chapter on the subject will be found beginning on page 589. 586 METABOLISM Several factors operate to explain these differences, and of these the following are of importance: 1. The Body Temperature. Increase in body temperature entails in- creased combustion. This explains why the metabolism of a bird is greater than that of a mammal of the same size, for, as is well known, the temperature of a bird is two or three degrees centigrade above that of other animals. Rise in body temperature also explains, in part at least, the increased metabolism observed in fever. 2. The Temperature of the Environment. In considering this we must distinguish between the effect produced on warm-blooded and on cold- blooded animals. Since the body temperature of a cold-blooded animal is only one or two degrees Centigrade above that of its environment, it follows that the metabolic activity will be directly proportional to the temperature of the latter. In a warm-blooded animal, on the other hand, the body temperature remains constant whatever changes may occur in that of the environment, this constancy of body temperature being dependent on the fact that the intensity of the combustion processes is inversely proportional to the cooling effect of the atmosphere. Thus, suppose the external temperature should fall, then the loss of heat from the body will tend to become greater, and to maintain the body tempera- ture at a constant level, the body furnaces must burn more briskly, with the result that an increased excretion of carbon dioxide and intake of oxygen will occur. This influence of the surrounding atmosphere on the metabolic activ- ity of warm-blooded animals has, as already pointed out, been used by several investigators to explain the greater combustion per kilo body weight of small as compared with large animals. The argument is that, since the surface of small animals relatively to their mass is much greater than in large animals, the cooling of the small animals will be proportion- ately greater. The relationship between surface and mass is shown by tak- ing two cubes and putting them together; the mass of the two cubes is equal to double that of either cube, whereas the surface is less than double, since two aspects of the cubes have been brought together. To prove the contention, the respiratory exchange has been computed per square meter of surface instead of per kilo body weight, with the result that a very close correspondence in the metabolism of different animals has been observed ; but this question has already been discussed, and we now know that the law of cooling can not be the only one that determines the extent of the respiratory exchange (see page 577). 3. Muscular Exercise. This has a most important influence on the ex- change and it is particularly in connection with it that studies in carbon- dioxide output and oxygen intake have been of great practical value, par- THE CARBON BALANCE 587 ticularly when the investigations are undertaken on men doing ordinary types of muscular exercise, such as walking or climbing. It is true that the influence of muscular exercise on the energy metabolism may also be studied by having a person in the calorimeter do exercises on an ergometer, but the results thus obtained are in many ways not nearly so valuable as those which can be secured by observing the respiratory exchange of persons doing ordinary types of muscular exercise in the open. The following table of observations on horses is of interest in this connection. CONDITION AIR EXPIRED CARBON DIOlXIDE OXYGEN ABSORBED C0 2 IN LITERS DISCHARGED IN IN LITERS PER 2 PER MINUTE LITERS PER MINUTE MINUTE Best 44 1.478 1.601 0.92 Walk 177 4.342 4.766 0.90 Trot 333 7.516 8.093 0.93 It will be observed that the metabolism increases extraordinarily for even a moderate degree of work, but that at the same time the respiratory quotient remains constant. From observations on the respiratory ex- change of working men and animals, extremely important facts concern- ing the efficiency of muscular work have been secured. The form of respiratory apparatus (Zuiitz or Douglas) employed for this purpose must be capable of being strapped on the man's back without causing any embarrassment to his bodily movements. By a comparison of the respiratory exchange with the amount of work done, the efficiency of the work can readily be determined. It has been found, for example, that the efficiency is much greater after the man or animal has got into the swing of the work, his energy expenditure per unit of work being much greater during the first half hour's work in the morning than it is later on. This indicates that after a little practice the muscles can ex- ecute a given movement and perform a given amount of work much more smoothly than when they are not in training. Another interesting outcome of the investigations has been to show that work done under ab- normal conditions that tend to produce any kind of muscular strain is done inefficiently. It has been found in marching soldiers, for example, that the slightest abrasion of the foot greatly increases the energy expenditure, for the man, in trying to avoid the pain produced by the abrasion, brings into operation muscular groups that are really not required for the efficient performance of the movement, but are used in the attempt to avoid pressure on the sore. Fatigue also causes inefficient performance of work; that is to say, the fatigued person, on attempting 588 METABOLISM the same amount of work as he performed before becoming fatigued, will do so at a much greater expenditure of energy. There is a diurnal variation in the respiratory exchange, which is in (general parallel with the body temperature; it rises during the day, the (time of activity and work, and falls during the night, the time of rest 'and sleep. Foodjilso affects respiratory exchange, but it will be unnec- essary to go into this further after what has been said on page 582. CHAPTER LXIV A CLINICAL METHOD FOR DETERMINING THE RESPIRATORY EXCHANGE IN MAN* (Contributed by R. G. PEARCE) Principle. Since the determination of the respiratory exchange in man is of some importance in the study of certain diseases of the respira- tion, circulation and metabolism, and also because directions for carry- ing out the necessary procedures are not generally available, we have thought it might be of assistance to include here brief directions for the Tissot and the Douglas methods. These methods have been found to compare favorably in accuracy with others in use at present,! and be- cause of their adaptability and simplicity they are specially suited for clinical work. By these methods the energy metabolism of the body is calculated from oxygen consumption or carbon dioxide excretion per minute (indirect calorimetry) (page 580), the figures for which are determined from the volume and percentile gaseous composition of the expired air. The subject breathes through valves which automatically partition the inspired and expired air. The expirations from a number of respirations are collected in a spirometer or bag, and the volume of the respirations per minute is determined. The gaseous composition of the expired air is determined by gas analysis, and the oxygen consumption and energy output of the body are calculated from the data obtained. Description and Use of Parts of the Apparatus: 1. THE MOUTHPIECE AND VALVES. The mouthpiece is made of soft pure gum rubber, and consists of an elliptical rub- ber flange having a hole in the center 2 cm. in diameter. The flange is attached on one side to a short rubber tube. On the other side at right angles to the rubber flange, are attached two rubber lugs. The rubber flange is placed between the lips, and the lugs are held by the teeth. The rubber tube of the mouthpiece is connected to the tube carrying the valves. The nose must be tightly closed if mouth breathing is used. This is accomplished by a nose clip, which consists of a V-shaped metal spring, the ends of which are provided with felt pads. A toothed rachet is attached to the ends of the spring, and serves to hold the spring tightly clamped on the nostrils in the proper position (see Fig. 177). Some individuals experience great distress when made to breathe through the mouth. *This chapter is added for the convenience of workers in this subject. tCarpenter: Carnegie Institution of Washington Reports, No. 216, 1915. 589 590 METABOLISM For these it is best to use a face mask. Unfortunately at the present time no mask is entirely satisfactory. Perhaps the best is one sold by Siebe, Gorman & Co.,* which is pictured in the cut. After being placed in position the face mask should be tested for leaks, which can be done by putting soap around the edges. Fig. 177. A, Nose clip; B, Face mask; C, Mouth piece. Fig. 178. Diagram of respiratory valves. 2. THE VALVES. The valves of Tissot are probably the best for the purpose, but they are expensive and difficult to obtain. We have made perfectly satisfactory valves from the prepared casings used in the manufacture of bologna sausage. These can *This mask has been used extensively by Carpenter. The agent on this continent is H. N. Elmer, 1140 Monadnock Bldg., Chicago. METHOD FOR DETERMINING RESPIRATORY EXCHANGE IN MAN 5S1 be obtained preserved in salt, and they will keep indefinitely on iee. When needed a short piece is taken, washed free from salt by allowing water from the tap to run through it, and softened in a weak glycerine solution. The gut becomes very soft and pliable, and does not dry quickly. A piece of the easing about 10 cm. long is threaded through a glass tube of about 15 mm. bore and 4 to 6 cm. long. One end Fig. 179. The Tissot spirometer. In actual experiment, subject is reclining or lying down and the valves and mouthpiece are held with a clamp. of the casing is brought around the outside of the tubing and secured by means of a thread. The lower end of the membrane is pinched off and the casing is then cut a little more than half way across its middle, so that the opening will lie just within the free end of the tube when the casing is drawn back through it. The loose end of the casing is slightly twisted an essential procedure and is then secured by a thread on the outer side of the tube. If properly made, the valve will work freely without 592 METABOLISM vibration, and the opening be sufficiently large to allow a good current of air to pass. It should collapse instantly and be air-tight when the current of air is reversed. The back lash, or lag of closure, of these valves is extremely small, and they will open or close with a pressure of air not exceeding the pressure changes in normal respiration. When not in use, the valves should be kept in glycrine water on ice. Valves prepared in this way have been in use a month without loss of efficiency. They are, however, made with so great ease that new valves are provided for each subject, and they are therefore especially adapted to ward work (Fig. 178). The valves are inserted in reverse order into a supporting metal T-piece, and the joints made air-tight by tape. The stem of the T is connected with the mouthpiece* Fig. 180. The Douglas bag method for determining the respiratory exchange. The arrange- ment of mouthpiece, valves, and connecting tubes shown here has been found to be more con- venient than that recommended by Douglas. Through a rubber tube of about % inch bore, the expired air is collected in the spirom- eter, or Douglas Bag. 3. THE TISSOT SPIROMETER is pictured in Fig. 179. We have found the 100-liter size to be very serviceable in the clinic. This instrument is mounted on a platform Having rubber wheels, and can be moved about the wards with ease. The bell of the spirometer is made of aluminum and is suspended in a water-bath between the double walls of a hollow cylinder made of galvanized iron. The height of the bell is 72 cm. and the diameter 42 cm. An opening at the bottom of the cylinder connects through a three-way stopcock with the rubber tube leading from the expiratory valve of the mouthpiece (see Fig. 177). The bell is counterpoised by means of a weight. In the original Tissot spirometer an automatic adjustment permitted water in amount equal METHOD FOR DETERMINING RESPIRATORY EXCHANGE IN MAN 593 to the water displaced by the bell to flow from the spirometer cylinder into a counter- poise cylinder as the bell ascended out of the water. The bell, being heavier out of water than when it is immersed, is accordingly counterpoised in any position, although Carpenter has shown that this refinement is unnecessary. An opening in the top of the spirometer permits the insertion of a rubber stopper, through which are passed a thermometer, a water manometer, and a stopcock with tube for drawing the sample of air. A scale on the side of the instrument gives the volume of the air. During an observation the subject sits in a reclining position or lies upon a couch. When the bell of the spirometer is placed at zero, the mouthpiece adjusted in the A B. Fig. 181. Haldane gas apparatus (A) and Pearce sampling tube (B). mouth, and the nose clamped, respiration is started, the expirations being passed through the stopcock, which is so turned as to allow them to pass to the outside air. After a few minutes the stopcock is turned so that the expirations are passed into the spirometer for a definite length of time. At the end of the period the cock is again turned, and after the barometric pressure, temperature, and volume of air have been noted, the composition of the air is determined in the Haldane gas analysis ap- paratus. 4. THE DOUGLAS BAG. The Douglas bag is made of rubber-lined cloth, and is capable of holding from 50 to 100 liters. It is especially useful for investigations during exercise, since it is fitted with straps so that the bag can be fastened to the 594 METABOLISM shoulders (Fig. 180). It is then connected with the valves, the mouthpiece of which is placed between the lips. Eespirations are commenced with the three-way valve turned so as to allow the expirations to pass directly outside. After respiratory equi- librium is established, the three-way valve is turned during an inspiratory period so that the succeeding expirations may pass into the bag. The time required to fill the bag comfortably is determined with a stop-watch. The air which has been collected in the bag during the period is thoroughly mixed and passed through a meter, the temperature and barometric pressure are noted, and a sample analyzed in the Haldane gas-apparatus. The bag should be emptied completely by rolling it up when nearly empty. 5. THE HALDANE GAS-ANALYSIS APPARATUS. PRINCIPLE. The Haldane method of analysis of expired air is simple and easily learned. The apparatus (Fig. 181) consists of a gas burette, a control burette of the same size (both surrounded with a water jacket), and bulbs containing dilute caustic potash or soda solution for the absorp- tion of the carbon dioxide and an alkaline pyrogallate solution for the absorption of the oxygen. The gas burette is connected with the bulbs by a two-way stopcock, which allows a sample of gas to pass into either bulb. A control tube (10) is put into con- nection with the burette through a manometer tube, which is connected with the alkali bulb, and can be made to compensate for any changes in temperature that may occur during the course of the analysis. For an analysis the gas is transferred to the burette from the sampling tube, saturated with water vapor over mercury, and then measured, after which it is transferred into the caustic solution to free it from CO 2 , and returned to the burette to determine the loss of volume due to CO absorption. It is then trans- ferred into the alkaline pyrogallate solution, which frees it from oxygen, after which it is again brought back to the burette to determine the loss in volume due to the absorption of the oxygen. THE APPARATUS. The detail of the Haldane apparatus is shown in the accompany- ing cut. The measuring burette (1) holds 21 c.c. The bulb is of 15 c.c. capacity, and the graduated stem, which is about 4 mm. in bore and 60 cm. in length, is grad- uated to 0.01 c.c. from 15 c.c. to 21 c.c. The stopcock at the top of the burette is double-bored, so that in one position air can be drawn in from a gas sampler (2) and in another sent into the absorption bulbs (3). The lower part of the burette extends through the rubber cork at the bottom of the water jacket (4). A piece of rubber tubing is attached to the bottom of the burette and is passed through a metal tube furnished on its inside with a metal disc which presses against the rubber tubing, the pressure being controlled by means of a fine adjusting screw (). Below this a glass stopcock (7) connects with rubber tubing to the mercury leveling bulb (5). The absorption bulb for CO 2 , containing 20 per cent NaOH or KOH (5), is put in con- nection with the burette by suitably turning stopcocks (3 and 8)* The control burette (10) is also in connection with this bulb through the manometer tube (11).^ Any variation in temperature which may occur during the analysis will cause the level of the alkaline solution in the manometer to change. When final readings of the shrinkage of volume are made, the level of the caustic solution is returned to the level of that in the manometer. By so doing any error due to temperature changes is avoided, since change in temperature must be equal in the two burettes. The absorption bulb for oxygen (12) is filled with a solution made by dissolving 10 grams of pyrogallic acid in 100 c.c, of a nearly saturated KOH solution. The *The stopcock (5) is double-bored, so that the tube leading from the burette can be brought into connection with either 9 or 12. fThis tube also has a three-way stopcock (79), so that it may be opened to the outside. METHOD FOR DETERMINING RESPIRATORY EXCHANGE IN MAN 595 specific gravity of the KOH should be 1.55, which is obtained approximately by dis- solving the sticks (pure by alcohol) in an equal weight of water. The mark (13) on the stem of the bulb indicates the level at which the solutions should stand. Enough pyrogallate solution is introduced through tube 15 to fill bulbs 12 and 14 two-thirds full. Then pyrogallate solution is poured into tube 16 until the difference in level of fluids is sufficient to produce enough pressure to raise the level of the pyrogallate solution in 12 to the level 13 on the stem. Stopcock 8 must be open during this pro- 'cedure. It may be necessary to add or take away a little pyrogallate solution through ''15 to attain the above level. Care must be taken to allow for complete absorption of oxygen from the air that AS entrapped between 14 and 16 before an analysis is made; otherwise changes will be produced in the level of the pyrogallate solution. The air in the capillary tubing connecting the burettes with the absorption bulbs must also be freed of CO 2 and O 2 . This can be accomplished by making a dummy analysis of atmospheric air before the real analysis. Great care must be taken to have atmospheric pressure in all the tubes at the start of the analysis. This is accomplished by opening the stopcock in the burette first to atmospheric air and then to the absorption bulbs, until no further change in the level of the fluids in the stems of the absorption bulbs occurs. This level is then marked and used as the standard. A small amount of water in the burette over the mercury assures saturation of the air with water vapor. Time for drainage must be allowed before making readings. A very serviceable sampling tube for the transfer of air can be made from a 30 c.c. ground-glass syringe, to which is attached a two-way stopcock. A cut of this is shown in Fig. 181. The dead space in these syringes is washed out by working the piston back and forth several times. A thin coating of vaseline prevents leakage of the gas. We have found that these sampling tubes will retain a sample of expired air without change up to eight hours. MANIPULATION OF APPARATUS. The sampling syringe (20} is attached to opening 2 of the burette, and its stopcock (17) opened to atmospheric air. The level of the mercury is raised to the level of the stopcock of the syringe and is then turned so that syringe and burette are in communication. The bulb of mercury is lowered so that the mercury falls in the burette. This draws the piston of the syringe with it, and fills the burette with air from the syringe. It is advisable to put a little positive pressure on the piston of the syringe in the maneuver to prevent possible leakage. When all of the air is in the burette a slight positive pressure is produced in the burette by gently pressing on the piston, and immediately thereafter the stopcock on the syringe (17) is again turned to the original position. This allows the pressure of air in the burette to come to that of the atmosphere. The height of the mercury is now adjusted to a convenient height in the burette by closing cock 7 and turning the milled screw 6. The cock 18 is now made to communicate with the absorption bulbs. If the air in the burette is at atmospheric pressure, no change will occur in the level of the fluids. The reading is then taken on the burette. The next step in the analysis consists in turning stopcock 8 to communicate with the caustic soda solution in bulb 9, and the leveling tube (5) is raised, forcing mercury into the burette and the air into bulb 9. The gas is passed back and forth several times until absorption is complete, as can be determined by the fact that the level of the mercury in the burette remains constant when the fluid in the bulb is returned to its original level (13) on the stem. In this adjustment it is convenient to make the gross leveling by the mercury bulb and the fine leveling by closing 7 and turning 6 until the fluid in 9 is at the original height. The reading on the burette indicates the loss in volume due to the CO 2 absorbed. 596 METABOLISM The oxygen is removed by a similar procedure, the gas being passed into the alkaline pyrogallate solution by turning cock 8 to communicate with bulb 12. The absorption of oxygen is slower than for CO 2 , and more care must be taken to get complete absorption. The air in the tubing between the fluid in 9 and stopcock 8 must be washed out several times in order to get the oxygen which is left in it after the absorption of the CO 2 . When this is complete, the final reading on the burette is made and the loss in volume from the second reading represents the oxygen. THE CALCULATIONS The calculation of the percentage composition of the air and of the respiratory quotient is represented in the following example of an actual analysis: (The temperature and barometric pressure as taken at the time of the experiment were 20 C. and 747 mm. Hg.) C0 2 analysis 1st reading of burette 20.00 2nd reading of burette after absorption of CO 2 19.20 CO 2 absorbed O80 0.80 -^- 20 = 4.0 per cent CO 2 in expired air. 2 analysis 2nd reading of burette 19.20 3rd reading of burette after absorption of O 2 15.90 O 2 absorbed 3^30 3.30 -f- 20 = 16.50 per cent of 2 in expired air. Determination of E.Q. O 2 in atmospheric air = 20.94% O, + CO 2 in expired air (16.50 -t- 4) = 20.50% 100 - 20.94* = 79.06%, N in atmospheric air. 100-20.50 =79.50%, N in expired air. Since nitrogen is an inert gas in the body, the last figure shows that more oxygen must have been taken in during inspiration than O 2 + CO 2 has been given back in expira- tion. This obviously must be taken into account in the calculations. The amount of O 2 actually inspired for each 100 c.c. of air expired is found as follows: 20.94 (% O 2 in atmospheric air) 79.06 (% N 2 in atmospheric air) < 79 ' 50 <% N * in ex P ired air ) >' or ' 265 ( con ' stant factor) X 79 - 5 21.07.f The amount of O 2 actually absorbed by the animal is therefore: 21.0716.504.57%. and the amount of CO 2 excreted is: 4.00 - 0.03 (CO 2 in inspired air) 3.97 % CO 2 . The respiratory quotient, or ratio of CO 2 excreted to O 2 absorbed is therefore: 3.97 _ =0 .87. Total Gas Exchange. The volume of air expired in 15 minutes into the Tissot spirometer was found to be 100 liters measured at 20 C. and 747 mm. Hg. (brass-scale barometer). This volume of gas must be corrected so as to give the volume of dry *This is the constant Oa percentage in air. tThis calculation can be simplified by using an abbreviated table (page 597) giving the O 2 figure j corresponding to the various percentages of N in the expired air. METHOD FOR DETERMINING RESPIRATORY EXCHANGE IN MAN 597 air at and 760 mm. Hg. To do this two things must be taken into account. (1) Since the expired air is saturated with water, the pressure due to water vapor must be subtracted from the observed barometric pressure to obtain the true pressure. The vapor tension of water for various temperatures is given in Table II on page 598. (2) Since the barometer tube lengthens or contracts with heat or cold, the baro- metric readings must be corrected. The corrections for ordinary barometric read- ings are found in Table III, page 598. The figure corresponding to the temperatures is subtracted from the barometric reading in order to obtain correct barometric pres- sure. In the above experiment, the correction for the barometer is 2.41 mm. (see Table III, page 598), and that for vapor tension at 20 C. is 17.4 (see Table II, page 598). Actual Barometric Pressure, 747 (17.4 + 2.4) = 727.2 mm. The coefficient of expansion of gases is taken as 0.003665) or 1/273; therefore the volume at equals the volume at 1 divided by 1 + 0.003665 t; and hence "\r Vo = , when Vo Volume at and V Volume at t. 1 + 0.003665 t ' VT* The volume of gas varies inversely as the pressure, Vo = , where V = volume at 760 P pressure; therefore working both corrections together, we have 760 (1 + 0.003665 t) This formula applied to the present problem reads: Vo = 100X727.2 =89.14 liters. 760 (1 + 0.003665x20) The latter calculation can be considerably simplified by using standard tables which give constants for corrections of gas volumes. These are easily obtainable and are given in part in Table IV. According to these tables for 20 C. and 727.21 mm. Hg. B.F., the factor is 0.89124; therefore the total volume of air breathed in 15 min. was: 0.89124 x 100 = 89.124 liters, at 0C. and 760 mm. Hg. and the absorbed O 2 , 0.89124 x 4.57 = 40.7 liters of O 2 , or 16.28 L. per hour. The Caloric Value Calculated from the Gas Exchange (Indirect Calorimetry). This can be done by using Table V but it is more accurate to know the nonprotein R.Q. which is given in the first column of the table. This latter is obtained by deducting from the total CO 2 eliminated, the CO 2 derived from protein (found by multiplying the urinary N by 9.35) and by deducting from the total O 2 absorbed the O 2 required to oxidise protein (found by multiplying urinary N" by 8.45). Suppose, for example, that 14.4 gm. N is excreted in the 24 hrs. urine; i.e., 0.6 gm. per hr. Then, 9.35x0.6 = 5.610 gm. CO 2 or, (since 1 gm. CO 2 = 0.5087 L) 2.85 L must be subtracted from 14.152 L giving 11.302 L and similarly 8.45 x .6 = 5.070 gm. O 2 or (since 1 gm. O 2 = 0.7 L) 3.5490 must be subtracted from 16.28 giving 12.73. The nonprotein R.Q. is therefore 11 302 ' =.88. Referring to Table V we see that at R.Q. 0.88 one liter of O, equals 1^.730 4.900 C. in 1 hr. 4.9 x 16.28 79.77 C were expended. As a matter of fact for most purposes it is sufficiently accurate to use the uncorrected R.Q. for Table V. 598 METABOLISM TABLE I THE PERCENTAGE OF OXYGEN WHICH is EQUIVALENT TO THE NITROGEN FOUND IN THE EXPIRED AIR To obtain the nitrogen in the expired air, add the percentage of CO 2 and O 2 found and subtract the sum from 100. The table gives the percentage for O 2 corresponding to this figure : %N a 78.7 78J3 78^9 79^0 79O 79^2 79^3 79^4 79J5 79^6 79/7 79.8 %0 2 20.86 20.88 20.90 20.93 20.96 20.98 21.01 21.04 21.07 21.10 21.12 21.14 79.9 80.0 80.1 80.2 80.3 80.4 80.5 80.6 21.16 21.19 21.22 21.25 21.28 21.31 21.35 21.38 TABLE II TENSION OF AQUEOUS VAPOR IN MILLIMETERS OF MERCURY To obtain the dry barometer pressure, subtract the mm. Hg corresponding to the temperature of the air from the barometer pressure at the time of the experiment: Temp. 15 16* 17 18" 19 20 21 22 23 24 25" Mm. 12.7 13.5 14.4 15.4 16.3 17.4 18.5 19.7 20.9 22.2 23.5 TABLE III TEMPERATURE CORRECTIONS TO REDUCE READINGS OF A MERCURIAL BAROMETER WITH A BRASS SCALE TO 0C. Subtract the appropriate quantity as found in table from the height of the barometer. The table is for a barometer with a brass scale, and the values are a little lower (about .2 mm.) than for the glass scale. The corrections for intermediate temperatures can be approximated by interpolation. Temp. 700 mm. 710 mm. 720 mm. 730 mm. 740 mm. 750 mm. 760 mm. 770 mm. 15 20 25 1.69 2.26 2.83 1.72 2.22 2.87 1.74 2.32 2.91 1.77 2.36 2.95 1.79 2.39 2.99 1.81 2.42 3.03 1.84 2.45 3.07 1.86 2.48 3.11 TABLE IV TABLE FOR REDUCING GASEOUS VOLUMES TO NORMAL TEMPERATURE AND PRESSURE The observed volume, when multiplied by the factor corresponding to the temperature and corrected pressure, will give the volume of the expired air reduced to and 760 mm. Mm. 15 16 17 18 19 20 21 22 23 24 25 720 730 740 750 760 770 .898 .910 .922 .935 .947 .960 .894 .907 .919 .932 .944 .957 .891 .904 .916 .928 .941 .953 .888 .901 .913 .925 .938 .950 .885 .897 .910 .922 .934 .948 .882 .894 .907 .919 .931 .945 .880 .891 .904 .916 .928 .940 .877 .888 .901 .913 .925 .936 .873 .885 .897 .910 .922 .933 .870 .882 .894 .907 .919 .930 .867 .879 .891 .904 .916 .927 METHOD FOR DETERMINING RESPIRATORY EXCHANGE IN MAN 599 TABLE V R. Q. CALORIES FOR 1 LITER O a RELATIVE CALORIES CONSUMED AS Number Carbohydrate Fat per cent per cent 0.707 4.686 100 0.71 4.690 1.4 98.6 0.72 4.702 4.8 95.2 0.73 4.714 8.2 91.8 0.74 4.727 11.6 88.4 0.75 4.739 15.0 85.0 0.76 4.752 18.4 81.6 0.77 4.764 21.8 78.2 0.78 4.776 25.2 74.8 0.79 4.789 28.6 71.4 0.80 4.801 32.0 68.0 0.81 4.813 35.4 64.6 0.82 4.825 38.8 61.2 0.83 4.838 42.2 57.8 0.84 4.850 45.6 54.4 0.85 4.863 49.0 51.0 0.86 4.875 52.4 47.6 0.87 4.887 55.8 44.2 0.88 4.900 59.2 40.8 0.89 4.912 62.6 37.4 0.90 4.924 66.0 34.0 0.91 4.936 69.4 30.6 0.92 4.948 72.8 27.2 0.93 4.960 76.2 23.8 0.94 4.973 79.6 20.4 0.95 4.985 83.0 17.0 0.96 4.997 86.4 13.6 0.97 5.010 89.8 10.2 0.98 5.022 93.2 6.8 0.99 5.034 96.6 3.4 1.00 5.047 100.0 0.0 (From Lusk.) CHAPTER LXV STARVATION In order to provide a standard with which we may compare other conditions, we shall first of all study the metabolism during starva- tion. A valuable chart compiled from observations made in the Carne- gie Institution of Washington on a man who fasted for thirty-one days is produced in Fig. 182. The Excretion of Nitrogen. When an animal is starved, it must live on its own tissues, but in doing so it saves its protein, so that the excretion of nitrogen falls after a few days to a low level, the energy requirements being meanwhile supplied, so far as possible, from stored carbohydrate and fat. Although always small in comparison with fat, the stores of carbohydrate vary considerably in different animals. They are much larger in man and the herbivora than in the carnivora. Dur- ing the first few days of starvation it is common, in the herbivora, to find that the excretion of nitrogen is actually greater than it was before starvation, because the custom has become established in the metabolism of these animals of using carbohydrates as the main fuel material, so that when carbohydrates are withheld, as in starvation, proteins are used more than before and the nitrogen excretion becomes greater. We may say that the herbivorous animal has become carnivorous. The same thing may occur in man when the previous diet was largely carbohy- drate; thus, almost invariably in man the nitrogen output is larger on the third and fourth days of starvation than on the first and second. Another factor influencing the nitrogen excretion during the early days of the fast is the amount of previous intake of nitrogen; the greater this has been, the greater the excretion. By the seventh day, however, a uniform output of nitrogen will usually be reached irrespective of the individual's protein intake. During the greater part of starvation, most of the energy required to maintain life is derived from fat, as little as possible being derived from protein. This type of metabolism lasts until all the available resources of fat have become exhausted, when a more extensive metabolism of protein sets in, with the consequence that the nitrogen excretion rises. This is really the harbinger of death it is often called the premortal rise in nitrogen excretion. It indicates that all the ordinary fuel of the animal economy has been used up, and that it has 600 STARVATION 601 [NUTRITION LABORATORY OF THE CARNEGJE INSTITUTION OF WASHINGTON. BOSTON. MASSACHUSETTS] METABOLISM CHART OF A MAN FASTING 31 DAYS APRIL 14 -MAY 15. 1912 OXYGEN AND CARBON DIOXIDE. c.c. ALVEOLAR CO, TENSION, mm. a. 3<6 38.0 3.50 DIOOO PRESSURE.. f I 23456789 10 II 12 1314 15 16 17 18 192021 22 23 24 25 26 27 28 29 30 3f XYGENKR KILO. PER MINUTE 36.0 3.40 34.0 3.30 32.0 3.20 30.0 3.10 8.0 3.00 2.90 128 2.80 124 2.70 120 2.60 I 16 2.50 I 12 2.40 108 I7"00 104 1650 100 1600 96 1550 36.8 1500 36.4 1450 36.0 1400 2 1350 V -* RB ON WOXI E PER K La PER MINUTE/ PHOSPHORUS (P,0s). CMS. Sihi 3 4 5 6 7 8 9 1.0 I.I 12 13 14 15 1.6 17 18 I? 20 21 2.Z 23 24 25 26 2.7 2 29 30 3 CARBON IN URINE B-OXYBUTYRIC ACID. URICACID-N.GM. TOTAL SULPHUR (S),GM. AMMONIA-N. CMS. Fig. 182. Curve constructed from data obtained from a man who fasted for thirty-one days. The days of the fast are given along the abscissae, and the various measurements along the or- Oinates. (From F. G. Benedict.) ' 602 METABOLISM become necessary to burn the very tissues themselves in order to obtain sufficient energy to maintain life. Working capital being all exhausted, an attempt is made to keep things going for a little longer time by liq- uidation of permanent assets. But these assets, being represented largely by protein, are of little real value in yielding the desired energy because, as we have seen, only 4.1 calories are available against 9.3, obtainable from fats. These facts explain why during starvation a fat man excretes daily less nitrogen than a lean man, and why the fat man can stand the starva- tion for a longer time. The premortal rise is, however, not prevented by feeding oil, which would seem to indicate that death may be due not so much to the absence of fuel as to serious nutritional disturbance of es- sential organs; e. g., there may be no available material to supply the glands of internal secretion with the building stones they must have (see page 614). Not only is there this general saving of protein during starvation, but there is also a discriminate utilization of what has to be used by the different organs, according to their relative activities. This is very clearly shown by comparison of the loss of weight which each organ un- dergoes during starvation. The heart and brain, which must be active if life is to be maintained, lose only about 3 per cent of their original weight, whereas the voluntary muscles, the liver and the spleen lose 31, 54 and 67 per cent, respectively. No doubt some of this loss is to be accounted for as due to the disappearance of fat, but a sufficient remainder represents protein to make it plain that there must have been a mobilization of this substance from tissues where it was not absolutely necessary, such as the liver and voluntary muscles, to organs, such as the heart, in which energy transformation is sine qua non of life. The vital organs live at the expense of those whose functions are accessory. The energy output per square meter of body surface steadily declines. In the man examined by Benedict, it was 958 C. per square meter of surface at the end of the first twenty-four hours, but only 737 on the thirty-first day of the starvation period. The oxygen intake and carbon- dioxide output correspondingly diminish. The behavior of the nitrogenous metabolites in the urine is of par- ticular interest, the following facts being of significance: Urea nitrogen relatively falls and NH 3 nitrogen rises. For example, on the last day of feed- ing the percentage output of NH 3 nitrogen in relation to total nitrogen was 3.16 ; on the eighth day of the fast it was 14.88 (Cathcart) . 2 Acidosis is the cause. The total amount of creatinine and creatine shows only a slight fall, but creatinine relatively decreases and creatine increases (Cathcart). Since creatine is a substance peculiar to muscle tissue, it is possible by STARVATION 603 comparing the creatine and creatinine output with that of nitrogen to determine whether all of the nitrogen liberated by the breakdown of muscle has been excreted, or whether some has been retained either for resynthesis in' the muscle itself or for use elsewhere. As a matter of fact the muscle breakdown as calculated from the creatine-creatinine output is greater than that calculated from the nitrogen, indicating that synthesis of the noncreatine remainder must be occurring. That transference of nitrogenous substances from place to place in the body in starvation is proved (1) by the constant presence of amino ni- trogen in the blood and tissues (Van Slyke) ; and (2) by the effect of copious water drinking. The latter causes a decided increase in the out*- put of nitrogen, because of the excretion of some of the nitrogenous substances. It is probable, however, that in such cases there is also a subsequent increase in endogenous protein metabolism, since the washed- out free nitrogen would have to be replaced. Excretion of Purines. Although at first they fall somewhat, the total amount increases as the fast progresses. Perhaps the first decline is due to general using up of hypoxanthine of muscle and the later rise to the breakdown of nuclei (page 671). Excretion of Sulphur. It is important to compare the excretion of sulphur and nitrogen. In the early days of starvation a ratio of 17 N : 1 S has been found, but later one of 14.5: 1, which is practically the same as that in muscle (i.e., 14; 1), indicating that late in fasting the main source of protein supply is muscle. Several of the changes observed during starvation can be attributed to the condition of acidosis which supervenes. The acids are derived from incomplete combustion of fat (see page 715), and are represented by /?-oxybutyric, the amount being sometimes considerable (10-15 grams a day), especially in obese individuals. The large ammonia excretion (sometimes 2 grams a day) is evidently for the purpose of neutralizing the excess of acid. Another consequence of the acidosis is the decline in the alveolar tension of C0 2 (page 379), and it is possible that some of the circulatory changes shown in the chart may also be dependent on it. For reducing obesity the method of repeated fasting is quite safe provided the acidosis is carefully watched and the diet which is given contains accessory food factors (page 618). Many secondary changes also occur in the starving organism. Thus, the mobilization of fat is often responsible for a pronounced increase in the fat content of the blood (see page 698). The amino nitrogen of the blood is not perceptibly reduced by starva- tion (page 613) but early in the condition the blood sugar becomes much lower than normal, after which it remains steady. This is significant 604 METABOLISM when we remember that after two or three days of starvation all of the available glycogen has been used up. It indicates that carbohydrate must be essential for life, and that it is produced in starvation from proteins (see page 699). The glycogen content of the skeletal muscles becomes reduced but not that of the heart. Starvation ends in death in an adult man in somewhat over four weeks but much sooner in children, because of their more active metab- olism. At the time of death the body weight may be reduced by 50 per cent. The body temperature does not change until within a few days of death, when it begins to fall, and it is undoubtedly true that if means are taken to prevent cooling of the animal at this stage, life will be prolonged. Death from starvation must be due either to a general failure of all the cells or to injury of certain organs that are essential for life. Since the loss of protein from the body as a whole only amounts to between 20 and 50 per cent at the time of death by starvation, it is unlikely that gen- eral failure can be the cause. If it were so, death would always occur when some fixed loss of protein had occurred. Certain organs evidently cease to perform their function, either because they are deprived of raw material for the elaboration of some substance (hormone) necessary for life, or because they themselves wear out from want of nourishment. NORMAL METABOLISM Apart from the practical importance of knowing something about the behavior of an- animal during starvation, such knowledge is of great value in furnishing a standard with which to compare the metabolism of animals under normal conditions. Taking again the nitrogen balance as indicating the extent of protein wear and tear in the body, let us consider first of all the conditions under which equilibrium may be .re- gained. It would be quite natural to suppose that, if an amount of pro- tein containing the same amount of nitrogen as is excreted during starvation were given to a starving animal, the intake and output of nitrogen would balance. We are led to make this assumption because we know that the balance sheet of a business concern showing an excess of expenditure over income could be adjusted in this way. But it is a very different matter with the nitrogen balance sheet of the body; for, if we give the starving animal just enough protein but no other foodstuffs to cover the nitrogen loss, we shall cause the excretion to rise to a total which is practically equal to the starvation amount plus all that we have given as food ; and although by daily giving this amount of protein there may be a slight decline in the excretion, it will never become the same as STARVATION 605 that of the intake. The only effect of such feeding will be to prolong life for a few days. Nitrogenous Equilibrium. To attain equilibrium we must give an amount of protein the nitrogen of which is at least two and one-half times that excreted during the starvation level. For a few days follow- ing the establishment of this pure protein diet, the nitrogen excretion will be far in excess of the intake, but it will gradually decline until the two practically correspond. Having once gained an equilibrium, we may raise the level at which it stands by gradually increasing the protein in- take. During this progressive raising of the ingested protein, it will be found, at least in the carnivora (cat and dog), that a certain amount of nitrogen is retained by the body for a day or so immediately following each increase in protein intake. The excretion of nitrogen, in other words, does not immediately follow the dietetic increase. The amount of nitrogen thus retained is too great to be accounted as a retention of dis- integration products of protein; it must therefore be due to an actual building up of new protein tissue that is, growth of muscles. Nitrogenous equilibrium on a protein diet alone is readily attainable in the cat, and less readily, in the dog. But in man and the herbivorous animals, it is impossible to give a sufficiency of protein alone to maintain equilibrium; there will always be an excess of excretion over intake. Indeed it scarcely requires any experiment to prove this, for it is self- evident when we consider that there are less than 1000 (X in a pound of uncooked lean meat, and that there are few who could eat over three pounds a day, an amount, however, which would scarcely furnish all of the required calories. A person fed exclusively on flesh is therefore being partly starved, even though he may think that he is eating abundantly >and is quite comfortable and active. This fact has a practical application in the so-called Banting cure for obesity. Protein Sparers. Very different results are obtained when carbohy- drates or fats are freely given with the protein to the starving animal. Nitrogen equilibrium can then be regained on very much less protein, so that we speak of fats and carbohydrates as being "protein sparers." Carbohydrates are much better protein sparers than fats; indeed they are so efficient in this regard that it is now commonly believed that car- bohydrates are essential for life, and that when the food contains no trace of carbohydrates, a part of the carbon of protein has to be con- verted into carbohydrate. This important truth is supported by evi- dence derived from other types of investigation (e. g., the behavior of diabetic patients, in whom the power to use carbohydrates is greatly depressed). The marked protein-sparing action of carbohydrates is il- lustrated in another way namely, by the fact that we can greatly 606 METABOLISM diminish the protein breakdown during starvation by giving carbo- hydrates. By using protein sparers we can indeed reduce the daily nitro- gen excretion to about one-third its amount in complete starvation. Re- moval of carbohydrate from the diet is said to entail a failure of the muscles to use again in their metabolism certain of the products (e. g., creatine) which result from their disintegration. At any rate it has been found that creatine is excreted in the urine under these conditions. As to the nature of the processes occurring in the body during protein sparing, two possibilities have to be borne in mind: either the body pro- tein is catabolized less rapidly or the protein sparer unites with certain of the breakdown products of protein to form new protein. Recent work by Davis, Hall and WTiipple 68 affords strong support to the latter view. These workers investigated the rate of repair of the liver and the curve of urinary nitrogen excretion in dogs after causing destruction of a large part of the liver tissue by chloroform administration. In animals in which about one half of each lobule had been destroyed, only about 50 per cent was found to be repaired in nine days, and the urinary nitrogen was higher than the normal starvation level although no food was given ; to other animals to which sugar was given the repair of the liver was complete in nine days and the curve of nitrogen decidedly below that of starvation. These observations open up a new field for the investiga- tion of problems of the growth of new tissue in the adult animal and their prosecution should afford aid in determining the influence of various dietetic conditions on such growth. Davis and Whipple 69 have already published some important observations in this direction and among other things have found that a diet of bread and milk, or one of cooked liver or kidney, causes more rapid repair than one of cooked skeletal muscle. Fats do not accelerate the repair process. The Irreducible Protein Minimum. In the case of a man living on an average diet, although the daily nitrogen excretion is about 15 grams, it can be lowered to about 6 grams provided that in place of the protein that has been removed from the diet enough carbohydrate is given to bring the total calories up to the normal daily requirement. If an excess of carbohydrate over the energy requirements is given, the protein may be still further reduced without disturbing the equilibrium. It has been found that it is not the amount of carbohydrate alone that determines the ease with which the irreducible protein minimum can be reached ; the kind of protein itself makes a very great difference. This has been very clearly shown by one investigator, who first of all determined his nitro- gen excretion while living exclusively on starch and sugar, and who then proceeded to see how little of different kinds of protein he had to take in order to bring himself into nitrogenous equilibrium. He. found that STARVATION , 607 he had to take the following amounts : 30 gm. meat protein, 31 gm. milk protein, 34 gm. rice protein, 38 gm. potato protein, 54 gm. bean protein, 76 gm. bread protein, and 102 gm. Indian-corn protein. The organism is evidently able to satisfy its protein demands much more readily with meat than with vegetable proteins. This variability in the food value of different proteins depends on their ultimate structure that is, on the proportion and manner of linkage of the various amino acids that go to build up the molecule. In no two proteins are these building stones, as they are called, present in exactly the same proportions, some proteins having a preponderance of one or more and an absence of others, just as in a row of houses there may be no two that are exactly alike, although for all of them the same build- ing materials were available. Albumin and globulin are the most im- portant proteins of blood and tissues, so that the food must contain the necessary units for their construction. If it fails in this regard, even to the extent of lacking only one of the units, the organism will either be unable to construct that protein, and will therefore suffer from partial starvation, or it will have to construct for itself this missing unit. It is therefore apparent that the most valuable proteins will be those that contain an array of units that can be reunited to form all the varieties of protein entering into the structure of the body proteins. Naturally, the protein which most nearly meets the requirements is meat protein, so that we are not surprised to find that less of this than of any other protein has to be taken to gain nitrogenous equilibrium. The most exact information regarding the "food value " of different proteins has been secured by observations on the rate of growth of young animals. This method yields more reliable information than can be secured by studies on the nitrogenous balance, because it is not usually possible to keep up the latter observations for a sufficient period of time, or to secure an adequate number of data. During growth the building-up processes are in excess of the breaking-down, so that the effect is an increase in bulk of the tissues, thus permitting us, by the sim- ple expedient of observing the body weight, to draw conclusions as to the influence of various foodstuffs on tissue construction. CHAPTER LXVI NUTRITION AND GROWTH In the growth of animal tissues two factors are concerned, one being the property of the cell to grow, the growth factor; and the other, the availability of suitable material to grow upon, the food factor. Concern- ing the growth factor little is known; its variability in different species of animal, its irregularity despite proper adjustment of the food factors, its abnormality leading to tumor formation, etc., are all well-known but apparently inexplicable facts (Mendel 8 ). The growth factor is very sen- sitive towards the activity of certain of the ductless glands such as the anterior lobe of the pituitary (page 808), and towards substances of unknown chemical nature contained in various foods. These are called accessory food factors or vitamines (page 618). THE FOOD FACTOR OF GROWTH Our knowledge is constantly increasing concerning the food factor of growth, and many facts of extreme practical importance have been ac- cumulated in recent years. In seeking for the relationship of food to growth, we must first of all consider whether this process entails a greater expenditure of energy than is necessary for mere maintenance in adult life. Important results bearing on this question have been se- cured by observations on the basal metabolism of young children. In computing the energy supply of fasting adult animals of different sizes, it will be remembered that the smaller the animal, the greater is the energy exchange in relationship to the body weight, although when computed in relationship to body surface tolerably constant values are obtained. When the calorie output per square meter is determined in growing children, there is, as we have already seen, clear evidence of greater energy expenditure (see page 577), particularly marked in boys just before puberty. An increased energy metabolism has also been de- scribed in the case of infants, but the uncontrollable muscular activity, the psychic disturbances, etc., may explain the result. Even after dis- counting these factors, however, it is possible that there may be a cer- tain influence, depending probably on the active mass of growing proto- plasmic tissue, which stimulates the energy expenditure. The question is not yet finally settled. 608 NUTRITION AND GROWTH 609 The Relationship of Proteins to Growth and Maintenance of Life. Since protein constitutes the fundamental chemical basis of the cell, it is natural to devote attention in the first place to this food principle. In the pioneer investigations, studies on the nitrogen balance in young animals yielded results from which it was concluded that the conditions for the disintegration of protein are less developed in young animals than in adults, so that the growing organs rapidly withdraw circulating protein and build it into tissue protein. In consideration of the accumulation of data extending over several decades, Rubner denied these conclusions, and showed that the diet of the growing infant is by no means relatively rich in protein. He con- cluded that ' ' growth is not proportional to the quantity of protein in the diet. " Important though this pioneer work may have been in the de- velopment of our present-day conception, the viewpoint of the men who carried it out was very much narrowed on account of the paucity of knowledge concerning the structure of the protein molecule. No allow- ance was made for the fact, which has recently been firmly established, that the protein molecule may vary extremely in regard to the units of which it is composed, and that the growing tissues may demand, not so much an abundance of protein as such, but rather a proper supply of all the building stones which are required for growth (Mendel). QUANTITATIVE COMPARISON OF AMINO ACIDS OBTAINED BY HYDROLYSIS OF PROTEINS* (Compiled by T. B. Osborne, 1914) f CASEIN OVAL- BUMIN GLIADIN ZEIN EDESTIN LEGUMIN ox MUSCLE Glycocoll 0.00 0.00 0.00 0.00 3.80 0.38 4.0 Alanine 1.50 2.22 2.00 13.39 3.60 2.08 8.1 Valine 7.20 2.50 3.34 1.88 6.20 ? 2.0 sLeucine 9.35 10.71 6.62 19.55 14.50 8.00 14.3 Proline 6.70 3.56 13.22 9.04 4.10 3.22 8.0 Phenylalanine 3.20 5.07 2.35 6.55 3.09 3.75 4.5 Glutaminic acid 15.55 9.10 43.66 26.17 18.74 13.80 10.6 Aspartic acid 1.39 2.20 0.58 1.71 4.50 5.30 22.3 Serine 0.50 ? 0.13 1.02 0.33 0.53 ? ^Tyrosine 4.50 1.77 1.61 3.55 2.13 3.55 4.4 XJystine ? ? 0.45 ? 1.00 ? ? Histidine 2.50 1.71 1.84 0.82 2.19 2.42 4.5 Arginine 3.81 4.91 2.84 1.55 14.17 10.12 11.5 Lysine 5.95 3.76 0.93 0.00 1.65 4.29 7.6 Tryptophane, about 1.50 present 1.00 0.00 present present present Ammonia 1.61 1.34 5.22 3.64 3.28 1.99 1.07 65.49 48.85 85.68 88.87 82.28 57.43 102.87 *These analyses are combinations of what appear to be the best determinations of various chemists. fThe figures for the more recent analyses of gliadin are inserted. From the accompanying table giving the percentage of the various ammo acids, etc., present in certain proteins, it will be evident that there 610 METABOLISM are very marked variations in the units of which different proteins are composed. If any one of these units should be essential for growth and the organism be unable to manufacture the missing unit for itself, it is clear that growth could not proceed however much protein not contain- ing the necessary unit we might feed to the animal. It is an application of the law of the minimum, and is analogous with the failure of growth which has long been known to ensue when certain inorganic substances are withheld from the growing animal. A diet might be perfectly bal- anced as judged by comparison of the nitrogen intake and output, and yet if it should fail to contain even one of the essential units and the organism should be incapable of supplying this unit, then would the diet be inadequate for growth. These important facts are the outcome of modern work, and they Days Each divi$ion=&0days. Fig. 183. Curves of growth of rats on basal rations plus the various proteins indicated. The normal curve may be taken as that with casein (I). (Adapted from Lafayette B. Mendel and T. B. Osborne.) have been established by observations on the growth of young animals fed with a " basal ration " to which were added mixtures of amino acids or various proteins which differ considerably from one another in the nature of the units entering into their make-up. In such experiments the periods during which growth is observed must be prolonged, since a transient increase in weight might depend merely on repair processes occurring in tissues which had previously for some reason been brought below par. Among the most important observations have been those of F. Gow- land Hopkins, Lafayette B. Mendel and T. B. Osborne 8 and of McCollum 9 and his collaborators. The animals chosen for Mendel and Osborne 's experiments were young white rats. Large batches of these animals NUTRITION AND GROWTH 611 were fed on a basal ration consisting of protein-free milk (containing the inorganic salts, the sugars, traces of protein, and unknown substances having an important influence on growth vitamines), to which were added more carbohydrate, purified fat, and the protein whose influence on growth it was desired to study. The same diet was fed at regular in- tervals to a given batch of rats, and the weight of each rat was period- ically taken, the observation being prolonged until the animals grew to maturity and produced young, and these again grew to maturity, repro- duced, and so on. By plotting the results in curves, with the time peri- ods along the abscissae and the average weight of the rats of each batch along the ordinates, the extent of the influence of a given diet on the curve of growth was obtained. A normal curve of growth is shown in Days Each division - 80 days Days Each division -20 days Fig. 184. Curves of growth of rats on basal rations plus the proteins indicated. In curve III the effect of the addition of zein to an inadequate allowance of the perfect protein, lactalbumin, is shown; and in IV the effect of the addition of cystine to a deficient casein allowance. (From Lafayette B. Mendel and T. B. Osborne.) . No. 1 of Fig. 183. It was obtained from results secured by adding liberal amounts of casein to the basal diet. Similar curves were obtained with lactalbumin of milk and ovalbumin and ovovitellin of egg. Perhaps the most interesting substances capable of producing the normal curve of growth are certain of the proteins that T. B. Osborne has succeeded in separating in crystalline form from vegetable foodstuffs. These are edestin (hempseed), globulin (squash seed), excelsin (Brazil nut), glu- telin (maize), globulin (cottonseed), glutein (wheat), glycinin (soy bean), cannabin (hempseed). That growth proceeds normally with any one of these proteins when it is fed abundantly does not, however, necessarily indicate that each con- 612 METABOLISM tains in adequate proportion all of the necessary units to meet the pro- tein demands of growing tissues. In the case of casein, for example, one of the units, namely, glycocoll, which is the simplest of all the amino acids, is entirely missing, and another, cystine, which is a sul- phur-containing amino acid, is present only in small amount. The ab- sence of glycocoll, however, is not of importance, because the organism can manufacture it for itself (see page 663). In the case of cystine, which the tissues can not manufacture themselves, the deficiency has to be made up for by feeding an excess of casein so as to cover the needs of the tissues for this amino acid. By so doing a superabundance of most of the other units will be ingested, and this superabundance will entail the destruction and excretion of the useless amino acids, a process, however, which is conducted in such a way as to permit of the utilization, by the organism, of a part of the energy which the cast-off amino acids contain (see page 699). It is, therefore, not entirely a wasteful process. When the supply of casein is limited, on the other hand, the curve of growth becomes subnormal, because an insufficient supply of cystine is thereby offered (Fig. 184). Similar results have been obtained in the case of edestin, a protein from hempseed. This contains an insufficiency of the diamino acid, lysine. Fed in abundance, edestin gave a normal curve of growth, but when fed in insufficient amount the curve failed to ascend properly (which, however, it could be made to do by adding some lysine to the edestin). There is a large group of proteins which fail to permit of any growth no matter in what amounts they may be added to the basal ration. These include: legumelin (soy bean), vignin (vetch), gliadin (wheat or rye), legumin (pea), legumin (vetch), hordein (barley), conglutin (lupine), gelatine (horn), zein (maize), phaseolin (kidney bean). The adequacy to maintain growth of any of these pure proteins varies according to the deficiency in their amino acids. In the case of gliadin of wheat or rye, glycocoll is -lacking, and lysine is present only in small amount (see table). The absence of glycocoll can not, however, as we have already seen in the case of casein, explain the inadequacy of gliadin as a foodstuff for growth (Curve II in Fig. 183) . It must be the lysine that is at fault. A still more deficient protein is the zein of maize. With this as the only protein added to the basal diet, the curve of growth actually descends (Curve III of Fig. 183), thus indicating that the animal is starving and must soon succumb. The missing units in this protein are glycocoll, lysine and tryptophane (see table on page 609), and it is very signifi- cant that if the latter two amino acids are supplied along with zein, an almost normal curve of growth will result. Some improvement can even be brought about by giving tryptophane alone ; that is to say, the NUTRITION AND GROWTH 613 curve assumes a horizontal line instead of descending, indicating that, although inadequate for growth, the diet is now sufficient for the main- tenance of life. An important fact demonstrated by these experiments, is that cer- Fig. 185. Photographs of rats of same brood on perfect diet (uppermost picture) ; on a main- tenance diet but inadequate for growth (middle picture) ; and on a diet that was inadequate both for maintenance and growth. (From Mendel and Osborne.) tain diets are adequate for the maintenance of life although they are inadequate for growth. In conformity with this conclusion, it was found when young white rats were fed with gliadin alone for periods of time ex- ceeding those in which they should have become full grown, that they remained in an ungrown stunted condition. The capacity to grow 614 METABOLISM had not, however, been lost, for when the gliadin was replaced by milk, the animals resumed growth at a very great rate. The capacity to grow had only been inhibited by the inadequate diet, and there was nothing really abnormal about the stunted animals. For example, the reproduc- tive function developed normally, as was shown in the case of a young female rat which, after being fed with gliadin as the sole protein sup- ply for 154 days, was mated and produced four young. Although the mother was still maintained on the gliadin diet, the young rats pre- sented normal growth, for they were living on the milk supplied by the mother, and this milk, because it contained either casein or some other necessary accessory factor (vide infra), was an adequate food. After removal from the mother, three of these rats were fed on an arti- ficial diet of casein, edestin and the basal ration, and continued the nor- mal course of growth, but when one of them was placed on the gliadin food mixture it immediately failed to grow properly. It would appear from these experiments that, of the two amino acids that are missing or deficient in gliadin namely, glycocoll and lysine it must be the lysine that is essential for growth. This very important conclusion was fully corroborated by finding that, in young rats stunted by previous gliadin feeding, growth immediately started when lysine was added to the diet and ceased again when the lysine was removed, and so on, the experi- ments being often repeated in various modifications. Mendel and Os- borne call attention to the relatively high percentage of lysine in all those proteins that are concerned in nature with the growth of young animals; thus, it is present in large amounts in casein, lactalbumin and egg vitellin. It is particularly in protein of vegetable origin that indispensable units are likely to be missing, the best known of these units being the aromatic amino acids, tyrosiiie and tryptophane; the diamino acid, lysine; and the sulphur-containing acid, cystine. Some animal proteins, such as gelatine, also fail to contain aromatic groups, and are therefore utterly inadequate as foods. That the absence of one or two units should render a protein in- capable of maintaining life suggests that a specific role may be taken by certain amino acids in the maintenance of nutritional rhythm; thus, they may be necessary for the elaboration of some hormone or other in- ternal secretion essential to life, such as epinephrine, the active principle of the suprarenal gland. This is an aromatic substance not far removed in its chemical structure from tyrosine (see page 773). It is therefore natural to suppose that the absence of the tryptophane unit in zein is the reason that this protein is incapable of maintaining the initial body weight. NUTRITION AND GROWTH 615 In attacking the problem from this viewpoint, Hopkins and Willcock 10 made observations on the survival period of young mice; that is, the period during which the animals survived when fed on a basal diet mixed either with zein alone or with zein plus small quantities of tryp- tophane. It was found that, with zein alone, the mice were unable to maintain growth; they lost in weight and died in from about a week to about a month. Other mice fed on the same amount of basal diet and zein, but to which was also added some tryptophane, although they did not grow, were capable of maintaining their body weight and lived in some instances for nearly a month and a half. There were other indica- tions of the difference in the efficiency of the two diets. The mice fed on the zein alone were very inactive, and remained for a considerable period of the time in a condition of torpor. The hair was ruffled, the eyes w^ere half closed, and the ears, feet and tail were cold. The ani- mals, however, gave evidence of having a good appetite. On the other hand, the mice to which tryptophane was also given manifested a strik- ingly different behavior, being active and more or less normal until just before death. That both groups of animals failed to live more than forty-four or forty-eight days is probably to be accounted for by the absence in the zein of the other unit, lysine. Had this been added along with the tryptophane it is probable, in the light of Mendel and Osborne's observations, that the animals would have survived much longer. To supply the missing unit, besides using the pure amino acid, we may employ other proteins which contain the required amino acid (Curve III of Fig. 184). That mixtures of protein foodstuffs are desirable has long been apparent to those who have studied practical dietetics. We must com- bine the unsuitable protein with others which, although in themselves perhaps also unsuitable, yet furnish us with a mixture which contains all the essential units both for maintenance and growth. As Mendel points out, these considerations suggest that we may be able to utilize certain of the low priced protein by-products of the cereal, meat and milk in- dustries. The test of the adequacy of the corrected diet must, however, be determined by experiments of the type which we have just described. It is probably in stock-raising rather than in connection with human nu- trition that these facts will prove of practical value ; for, not only is the diet of man more varied, but it contains animal proteins in which the deficien- cies are not so common. Most important work of this character is being conducted by McCol- lum and his collaborators. 12 It would take us beyond the confines of this book to discuss the results in detail, but it may be mentioned that they have shown that, since the adequacy of the diet depends on a multiplicity of factors besides the ammo-acid make-up of proteins, some of which we shall discuss immediately, very extensive observa- 616 METABOLISM tions with various food mixtures must be conducted over long periods of time. The nutritive values of the common cereals added to a stand- ard diet that had brought the animals (rats) to the threshold of death, were found to be as follows: With cornmeal there was immediate recov- ery and rapid growth, both of which were also secured in considerable degree by wheat embryo and entire wheat kernel ; with rye and oats, on the other hand, there was little if any improvement. Much work is, of course, yet to be done before we can determine the exact role which each unit plays in the physiological development of young animals. To sum up what we already know, it may be said that glycocoll is not essential, since it can be manufactured by the animal itself; that tryptophane is essential for maintenance, probably because it is required for the production of certain essential hormones, for the make-up of which in its absence other tissues must become disintegrated, leading therefore to a diminution in body weight; and that lysine ap- pears to be essential for growth. Tissues can be maintained without lysine, but they can not grow. That the young rats in the experiments of Mendel grew normally while living on milk supplied by the stunted mother indicates that the requisite lysine must have been produced in the mother's body. In the application of the foregoing principles to human dietetics, it is undoubtedly safe to follow Bayliss's advice to take care of the calo- |ries and allow the proteins to take care of themselves. 11 For example, in the case of milk the deficiency of cystine in its chief protein, casein, is corrected by the presence of lactalbumin, which, though present in only small amounts, contains sufficient quantities of this amino acid to meet the demands of the growing tissue. These observations on maintenance and growth suggest very interest- ing applications in connection with the growth of tumors. Is it possible that we might retard the growth of tumors by a diet that was insufficient for growth while sufficient for maintenance. In an experiment devised to test this proposition mice were fed on a diet of starch, lard, lactose and gluten on which they could merely maintain existence but failed to grow. Some of these rats were inoculated with a rapidly growing tumor at the same time as another batch of mice kept on normal diet, and it was found that the tumor grew much more slowly in the stunted mice than in the others. One mouse, for example, on the restricted diet had a scarcely visible tumor 52 days after the inoculation. When this mouse, however, was placed on a normal diet of bread, milk, etc., the tumor immediately began to grow at a very great rate. 13 Too much importance should not be placed on this experiment. We shall now pass on to consider some of the factors besides the pro- tein content which have an important bearing on dietetic efficiency. CHAPTER LXVII NUTRITION AND GROWTH (Cont'd) THE RELATIONSHIP OF OTHER FACTORS THAN PROTEINS The Relationship of Carbohydrates, As we have seen elsewhere, car- bohydrates are almost certainly essential for normal metabolism. If they are not given with the food, they must be manufactured out of protein by the organism itself. It is not surprising, therefore, that their absence from the diet of growing animals should lead to abnormality in the rate of growth. Pediatrists have not infrequently insisted that one form of carbohydrate is more advantageous for growth than another. This no doubt in the main is true, but the whole question of adequacy probably depends on the digestibility of the carbohydrate and not upon its essential chemical nature. It is likely that the only carbohydrate required by the tissues is glucose. The readiness with which the car- bohydrate of the food becomes converted into this monosaccharide is probably the only determinant of its efficiency as food material. The Relationship of Fats. Although fats are an invariable constit- uent of practically every diet, it is yet a debatable question as to whether they are essential to the maintenance of a healthy normal organism. Difficulties standing in the way of a solution of this problem are that it is not only technically very difficult to remove fat entirely from the common foodstuffs, but also that the simple fats are usually associated with substances having similar solubilities and physical properties: namely, the lipoids, phosphatides, cholesterol, pigments, etc. Since these substances are present in practically every cell, it is almost certain that they can be manufactured by living protoplasm. Indeed, experimental evidence is not wanting to show that this is actually the case. Although the cell can manufacture lipoids, a young animal can apparently not grow when these substances, as well as simple fat, are entirely absent from the diet. This has been shown by feeding young mice on a diet from which all traces of fat and lipoids had been removed by extraction with alcohol and ether (Stepp) 14 . On such a diet the mice lived only a few weeks. They could be kept alive much longer when some of the alcohol-ether extract was mixed with the diet, but not so when neutral fat instead of the alcohol-ether extract was added. The 617 618 METABOLISM addition of the ash of the lipoid extract failed to maintain the mice, so that the lacking substance could not be inorganic in nature. As we shall see immediately, these results are dependent upon the presence in fats of so-called accessory food factors or vitamines. The Relationship of Inorganic Salts. Inorganic salts are also an es- sential ingredient of the diet. McCollum found that young animals soon ceased to grow when fed on a diet of corn and purified casein, but that rapid growth returned when a suitable salt mixture was added. Oats, wheat, and beans have also been shown to require some adjustment of their ash content to make them adequate for growth. Most of the animal foods contain in themselves sufficient inorganic material, as is evidenced among other things by the adequacy of milk alone as diet for growing animals and the abhorrence of salt that is shown by strictly carnivorous animals. In the usual mixed diet of man there is almost always enough inorganic material, the salt which he adds being largely for seasoning purposes. When a preponderance of vegetable food is taken, however, the salt comes to have a real dietetic value. ACCESSORY FOOD FACTORS, VITAMINS Even when the requirements of the animal body for calories and protein building stones are fully met, the diet will fail to maintain health unless it also contains substances of an unknown chemical nature called "accessory food factors" or "vitamins." These are entirely of plant origin, and require to be taken only in very small quantities to display their beneficial action. They do not become rapidly destroyed in metabolism, but may remain attached to the tissues sufficiently long so that carnivorous animals obtain them indirectly. Great advancement in our knowledge of vitamins had been made in recent years, particu- larly through the work of F. Gowland Hopkins and Harriette Chick, 70 Osborne and Mendel, 71 Funk, 15 McCollum, 12 and McCarrison. 75 Serious and prolonged absence of certain vitamins from the dietary may hinder the growth of young animals or may be the cause of various serious diseases in adults. The human diseases which are known definitely to be due to the absence of one or other of the vitamins are beriberi, scurvy and rickets, and accordingly three vitamins are distinguished: 1. Antiberiberi or antineuritic vitamin (also called water soluble "B" growth factor). 2. Antirachitic vitamin (also called fat-soluble A growth factor). 3. Antiscorbutic vitamin. It will be observed that the vitamins also differ from one another in their solubilities. NUTRITION AND GROWTH 619 Investigation of the distribution of the vitamins among the various foodstuffs, and their degree of stability towards heating, etc., has been very materially facilitated by the fact that certain of the lower animals suffer diseases like those seen in man when vitamins are absent from the diet. This renders it possible to prosecute the investigations in- tensively and under scientifically controlled conditions, thus affording knowledge which enables us to alleviate human suffering. The Antiberiberi or Antineuritic Vitamin. Beriberi is a disease char- acterized by wasting, anesthesia and paralysis, and sometimes by ex- cessive edema. Pathologically, it is a form of severe neuritis. It is common in rice-eating communities, and the first clue to its chief cause was afforded by the observation that it does not occur among people who take unmilled rice, and that it disappears in those who take "pol- ished" rice when the millings or a watery extract of them (pericarp and germ) are added to the diet. It was observed by Eijkman that the poul- try of a prison where beriberi was prevalent exhibited symptoms very like those of the human disease, and further investigation showed ex- tensive nerve degeneration to exist in the affected animals. Pigeons fed on polished rice develop exactly the same symptoms so that experi- mental investigation soon rendered it possible to determine with accu- racy which foodstuffs prevent beriberi. Meanwhile McCollum and Davis discovered that the absence of the same water-soluble vitamin interfered seriously with the growth of young animals. This is shown in Curve II of Pig. 186, from the observations of Hopkins and Chick, and is con- structed on the same principles as those of Fig. 184. It will be observed that the withdrawal of the vitamin caused an immediate cessation of growth followed by a period during which the body weight remained more or less constant, but ultimately declined. The fact discovered by McCarrison 75 that atrophy of the gastrointestinal tract also results from deficiency in this vitamin, may be responsible in part at least for the failure of growth. During this stage muscular incoordination is a prominent symptom, and death ultimately occurs. The curves show that this vitamin must soon disappear from the organism when it is with- drawn from the food and that the animal cannot synthetize it. It will be noted in the table on page 623 that vitamin B is present in abundance in the seeds of plants and the eggs of animals. It is very plentiful in yeast and in yeast extracts, which may therefore be added to the diet when there is risk of its deficiency. It is ab- sent from bread made with white wheat flour, but beriberi is rare in people living on this food, since other foodstuffs containing the vitamin are usually also taken. Beriberi is unknown where rye bread is the staple food. The Antirachitic Vitamin (Fat-Soluble A Factor). The first inkling 620 METABOLISM of the existence of this factor we owe to Stepp who found that mice could not live for long on animal foods from which all fatty substances had been thoroughly extracted by alcohol and ether. If the extract was restored to the extracted food, this again became adequate. Hopkins then showed in carefully controlled work that animals (rats) not only failed to grow, but declined and died when they were fed on artificial diet composed of the purified constituents of milk, although they might eat voraciously. The addition of a few drops of milk, insufficient to raise the energy or protein value appreciably, invariably caused normal 6RAM5 ISO loo 150 100 50 50 IS of the ' I u HI / / // jlf ---^ * X / / / ^ \ ^' FACTOR X / . /"- \ '&' F ACTOJ? ABSENT -1 MONTH - ABSENT S U V/ VD AFTER 5 -1 WEEK -' ^ REMOVING <_ (rtOiiTH) ? A MCTOTt ^ yCK/NG. FED Pi" FA BY MOTHER ABSENT 1 ?,^' Curves of growth of rats as influenced by the accessory food factors. I. The effect / fat - s ? lubl . e > factor ' Note that the curve d es not immediately descend after removal of ... -_ *B" (water-soluble) factor. Note that growth ceases ** 1S withdrawn. III. The time during which animals survive after removal of the A tactor at different ages, the uppermost curve being that obtained for rats that were nearly mature before the factor was removed from the diet and the lower ones for less mature animals. it u i C( ?. ntmuous c r ve is for young fed by mothers receiving "A" factor int their diets; the broken-line curve is for young fed by mothers receiving none of this factor. (From Hopkins and Chick.) growth to return. Osborne and Mendel also found that although the rats in their experiments already referred to on page 609 grew for about two months upon a diet containing protein, protein-free milk, starch and lard, they ultimately declined, but that this could be avoided by substi- tuting butter for the lard, and that the active substance was concen- trated in the butter-fat portion of the butter. Later work by various investigators showed that most animal fats, but not those of plants, contain this essential, and it was hence called fat-soluble A factor or NUTRITION AND GROWTH 621 vitamin. Lard does not contain it, so that young animals fed with this as the only fat of an otherwise perfect diet fail to grow as shown in Curve I, Fig. 186. An important difference will be observed in this curve from that of II in which the factor B (antiberiberi vitamin) is alone deficient, namely, that a small amount of growth continues for a time after the removal of the proper diet. This indicates that there must be some reserve of the fat-soluble vitamin in the body, and it is only after this is exhausted that growth entirely ceases and decline then sets in. Further evidence of tissue storage of this vitamin is afforded in Curve IV in which the upper curve represents the normal growth of young rats, the lower, of those nursed by a mother receiving a diet that was deficient in the vitamin. When the reserves of the fat-soluble vitamin are exhausted, not only do the young animals fail to grow, but they become highly susceptible to bacterial disease, one symptom of which is a very characteristic eye in- fection (xerophthalmia) which begins with a swelling of the lids, and later develops into a purulent conjunctivitis, often leading to blindness. Administration of some fat-soluble vitamin in the diet dispels the eye symptoms usually within a few days. When the dietary of adult ani- mals contains none of this vitamin, the eye symptoms also develop, the general condition greatly deteriorates and the animals become ex- tremely susceptible to bacterial infections, particularly those affecting the lungs, and from which they readily succumb. Adults are, however, much less susceptible to the absence of the fat-soluble vitamin than growing animals. This may be because of great storage capacity for it and is shown in Curve III of Fig. 186. At the same time it is worthy of note that there is reason to believe that the condition known as war edema is due to a deficiency of this vitamin. It may be stated here that when both the A and B factors are absent from the diet young animals immediately cease to grow and develop the nervous symptoms due to the absence of the B factor, from which they usually succumb before the symptoms due to the absence of the A factor have had time to develop. It is believed that it is with the metabolism within the cells rather than with that of fat itself that the fat-soluble vitamin is concerned. A most important relationship probably exists between this vitamin and the occurrence of rickets. Thus when the puppies of large dogs are fed with separated milk, bread, linseed oil, yeast and orange juice, they grow at a normal rate, but in about six weeks develop undoubted symp- toms of rickets (bones defectively calcified so that the long bones bend, swelling at the epiphyses, a rosary at the costochondral junctions, the ligaments loose, general lethargy and loss of muscular tone). In this diet both the water-soluble and the antiscorbutic factors are present in 622 METABOLISM abundance (the yeast and the orange juice), but the fat-soluble factor is very low. Judged by its effect upon the growth curve of rats this factor would appear to be absent from linseed oil. The latter, however, does con- tain a trace which is sufficient to allow of an abnormal form of growth in puppies. When animal fats, such as cod-liver oil or butter, are sub- stituted for the linseed oil in the above, or a similar diet, rickets does not occur. It will be seen in reference to the table on page 623 that there are two main sources for the fat-soluble vitamin, (1) certain animal fats, and (2) green leaves. It is particularly abundant in cream, butter, beef fat, fish oils (particularly cod-liver oil and whale oil) and egg yolk. It is absent or present only in traces in most vegetable oils, such as lin- seed, olive, cotton-seed, but in some of them such as peanut oil it is present in larger amounts. Its presence in green leaves stands out in contrast to its absence from root vegetables. It is also present in certain cereals and pulses. The Antiscorbutic Vitamine. That scurvy is definitely due to the absence from the dietary of some vitamin has been known in a general way for a long time. It used to be very common among the crews in the days of sailing ships, and nautical history records many interesting observations, by captains and ship's surgeons, showing that it could be prevented by adding certain fruits or the juices of fruits or vegetables to the daily ration. Indeed lime juice became a regular part of the mariner's ration. Great progress was made in the investigation of the precise distribution and behavior of the antiscorbutic vitamin by the discovery that guinea pigs develop the disease when all green stuff is removed from the diet and the animals are fed on grains and water or autoclaved milk. The symptoms appear in about 20 days, up to which time if young animals are used, the growth curve continues. The symp- toms and the postmortem findings are similar to those seen in man, (ten- derness and swelling in the joints, swelling of the gums, loosening of the teeth, tendency to hemorrhages and fractures of the bones). The curve of growth declines, and the animal dies usually between the 30th and 40th days. Important work is being done in the Lister Institute in London to determine the minimal amounts of various foodstuffs that are required in the scurvy diet to prevent the occurrence of the disease. Regarding distribution it may be said that this vitamin is found in nature in all tissues which are actively undergoing metabolic change. It is abundant in growing green leaves, in fruits and in germinating seeds. But it is absent, or present only in traces, in dormant seeds, or in plant tissues that have been dried. It is also present, though less abundant in fresh animal tissues and in milk. Although potatoes do not contain much of this vitamin, a diet composed mainly of them is NUTRITION AND GROWTH 623 seldom scorbutic because of the large quantities that are usually con- sumed. Canned vegetables and meats only contain traces of it because it is destroyed in the heating process. Canned fruits, however, contain more of it because it is preserved by the acid. The practical application of the results of these observations to the nutrition of man, and particularly to the dietetic treatment of disease, is undoubtedly very great. This is especially so in infants and growing children, in whom the correction of some slight inadequacy in the diet may have the most pronounced results, not only on growth and nourish- ment, but also on the power of resistance against disease and infection. The beneficial influence of cod-liver oil, for example, may depend on some fat-soluble accessory food factors, while the miraculous benefit which scorbutic children derive from the addition of the juice of limes, lemons, etc., to the food is undoubtedly due to such influences. The accumu- lating mass of evidence as to the faulty nutrition in animals fed on certain kinds of food that fail to contain food factors emphasizes the necessity in the dietetic treatment of such diseases as diabetes, nephritis, etc., of seeing to it that the diet is sound, not only in calories, protein content, and palatability, but also with regard to the presence of these factors. In the accompanying table the relative amounts of the various vita- mines in different foodstuffs is indicated by the use of plus signs. THE DISTRIBUTION OF THE THREE ACCESSORY FACTORS IN THE COMMONER FOODSTUFFS WATER-SOLUBLE B CLASSES OF FOODSTUFFS FAT-SOLUBLE A OR OR ANTINEURITIC ANTISCORBUTIC ANTIRACHITIC (ANTIBERIBERl) FACTOR FACTOR FACTOR Fats and oils. Butter + + + Cream + + Cod-liver oil + + + Mutton or beef fat or suet + + P'ea-nut or arachis oil + Lard Olive or cottonseed oil Linseed oil Fish oil, whale oil, herring oil, etc. + + Hardened fats, animal or vegetable origin Margarine from vegetable fats or lard Nut butters + Meat, fish, etc. Lean meat (beef, mutton, etc.) + + + Liver ++ + + + Kidney and Heart + -f + Brain and sweetbreads + + + Fish, white very slight, if any 624 METABOLISM THE DISTRIBUTION OF THE THREE ACCESSORY FACTORS IN THE COMMONER FOODSTUFFS (CONT'D.) CLASSES OP FOODSTUFFS FAT-SOLUBLE A WATER-SOLUBLE B OR OR ANTINEURITIC ANTISCORBUTIC ANTIRACHITIC (ANTIBERIBERl) FACTOR FACTOR FACTOR Fish, fat (salmon, herring, etc.) Fish, roe Tinned meats Milk, cheese, etc. Milk, cow's whole, raw Milk, skim, raw Milk, dried whole Milk boiled whole Milk condensed, sweetened Cheese, whole milk Cheese, skim Eggs Fresh or dried Cereals, pulses, etc. Wheat, maize, rice, whole grain Wheat, germ Wheat, maise, bran White wheaten flour, pure corn-flour, polished rice, etc. Custard powders, egg sub- stitutes prepared from cereal products Dried peas, lentils, etc. Soy beans, haricot beans Germinated pulses or cereals Vegetables and fruits Cabbage, fresh Cabbage, fresh cooked Swede, raw expressed juice Lettuce, Spinach (dried) Carrots, fresh raw Potatoes, cooked Beans, fresh, scarlet runners, raw Onions, cooked Lemon juice, fresh Lemon juice, preserved Lime juice, fresh Lime juice, preserved Orange juice, fresh Raspberries Apples Bananas Tomatoes (canned) Nuts Miscellaneous. Yeast, dried Yeast, extract and autolysed Meat extract Malt extract Beer very slight, if any very slight less than + + undetermined less than -I- less than + less than + to 4- (at least) very slight -f + + 'in some specimens very slight + T I CHAPTER LXVIII DIETETICS THE CALORIE REQUIREMENT In the application of the important facts that have been reviewed in the preceding chapters to the science of dietetics, the question arises as to how we may determine with scientific accuracy just exactly how much food should le taken under varying conditions of bodily activity. In a general way, we know that the amount of food that we require to take is proportional to the nature and amount of bodily exercise that is being performed at the time ; and that, if the food supply is inadequate, the work before long will fall off not only in quantity but in quality as well. "Horses (also men) work best when they are well fed, and feed best when they are well worked, " is an old adage and one the truth of which can not be overestimated in the consideration of all questions of dietary requirements. An ill-fed beggar will rather suffer from the pain and misery of starvation than attempt to perform a piece of work that the well-meaning housewife bargains should be done before she gives him a meal. The spirit may be willing but the flesh is weak. If he could be trusted, he should be fed first and worked afterwards. Besides the amount of work, two other factors are well known to influence the demand for food namely, growth and climate. A young, growing boy will often demand as much if not more food than would appear to be his proper share, from a comparison of his body weight with that of his seniors; and, other things being equal, it is well known that we are inclined to eat much more heartily of food during the cold days of winter than during the sultry days of July and August. That we know these facts in a general way, indicates that the first step to take in the exact determination of dietetic requirements is to find out how much energy the body expends under varying conditions of activity. This, as we have seen, may be done by having the person live for some time in a respiration calorimeter, so that we may measure the calorie output. To the conclusions drawn from results of observa- tions made under such artificial and unusual conditions of living, the objection might, however, quite justly be raised that they need not apply to persons going about their ordinary routine of life. To meet 625 626 METABOLISM this objection another method, which we may call the statistical, is avail- able. It consists in taking the average diet of a large number of indi- viduals and comparing the calorie value with the average amount and type of work that they are meanwhile called upon to perform, and can best be used where the diet is accurately known, as in public institu- tions, the army, the navy, etc. The total food supplied is then divided by the number of individuals, this giving the per capita consumption. Obviously some get more than others, but when a sufficient number of individuals is included, such errors become eliminated by the law of averages. The reliability of this method is testified to by the remarkable corre- spondence in the calorie value of the food consumed by farmers in widely different communities: Calories Farmers in Connecticut 3,410 " " Vermont 3,635 " " New York 3,785 " " Italy 3,565 " " Finland 3,474 Average 3,551* *Lusk: The Fundamental Basis of Nutrition. The average inhabitant of various cities: London 2,665 Paris 2,903 Munich 3,014 Konigsberg 2,394** **Rubner. Individuals in different callings: Farmers' families (U.S.A.) 3.560 Mechanics' families (U.S.A.) 3,605 Professional men's families (U.S.A.) 3,530 Army (U.S.A.) 3,851 Navy (U.S.A.) 4,998t tAtwater. In general, it is usually computed that a man --^weighing 70 kg. requires in calories: ^,500 for a sedentary life, 8,000 for light muscular work, 3,500 for medium muscular work, 4,000 and upwards for very hard toil.J JMcKillop. These figures apply to the average man, but in calculating the calorie requirements of a family or a community we must make allowance for the lesser requirements of women and children. Several dietitians have compiled tables showing how many calories are expended according to age and sex, and from the figures have calculated a generalized mean, which shows in comparison with men the percentage that should be al- lowed for women and children. The mean values are as follows : DIETETICS 627 Man 100 _ Woman S3 Boy over 16 92 Boy 14-16 81 Girl 14-16 74 Child 10-13 64 Child 6-9 49 Child 2-5 36 Child under 2 . . 23 In calculating the calorie requirement of the population as a whole, the necessity of making allowance for the varying needs of men, women, and children would obviously make the calculations far too complicated for practical purposes. It is necessary to have a factor by which we may multiply the total population in order to determine its ' ' man value. ' ' This factor is based on the relative proportion of men to women and children, and it amounts very nearly to 0.75, i. e., three-quarters of the total population gives "the man value." Knowing the total population, say, of a city, we must therefore multiply this by 0.75 in order to ascer- tain for how many men doing moderate muscular work (3000 C.) food has to be provided. THE PROTEIN REQUIREMENT The facts considered in the previous two chapters lead to the question: To what extent may the proportion of protein in the diet be reduced with safety? It is evident that there must be a minimum below which every one of the necessary building materials of protein could not be supplied in adequate amount to reconstruct the worn-out tissue protein. The extent to which the protein content of the diet of man can be lowered with safety depends on several factors, of which the most im- >ortant are: first, the nature of the protein; second, the number of non- protein calories ; and third, the extent of tissue activity. Where so many factors must be taken into consideration, the only method by which the actual minimum can be determined consists in what may be called "cut and try experiments." Of the many investigations of such a nature, probably the best one for us to consider, is that recently published from the Nutrition Laboratory of Copenhagen. The subject, an intelligent laboratory servant, lived a perfectly normal and active life for a period of five months on a diet of potatoes cooked with margarine and a little onion, and containing 4000 C., with a total protein content of 29 grams. During another period he did outdoor work as a mason and laborer, and took 5000 C. daily, and 35 grams of protein. It is important to contrast these results with the following based on municipal statistics of gross consumption. 628 METABOLISM MUNICIPAL FOOD STATISTICS PROTEIN FAT CARBOHYDRATES CALORIES Konigsberg Munich Paris London gm. 84 96 98 98 gm. 31 65 64 60 gm. 414 492 465 416 2394 3014 2903 2665 1 It is certain that man can lead a normal existence and remain in good uiealth on very much less protein than the 100 grams which statistical studies show to be the amount he actually takes. This discrepancy be- tween the amount which experiment demonstrates to be adequate and that which habit and custom demand, raises the question as to whether, after all, our instincts may not have erred and so made us unnecessarily extravagant in our protein intake. It has been suggested that such pro- tein extravagance will in various ways have a deleterious effect on the organism; thus, that the excretory organs, such as the kidneys, will be overtaxed in eliminating the unused amino acids, that the constant pres- ence of these bodies in excess in the blood will cause degeneration and sluggish metabolism, and that the excess protein in the intestine will lead to the production of poisonous decomposition products, the subse- quent absorption of which into the blood will cause toxemic symptoms. Important support to such views appeared to be supplied some dozen years ago by Chittenden, who was able to show that he himself and many other persons doing different kinds of work could be supported on daily amounts of protein that were not more than from one-third to one-half of the amount usually taken. Not only so, but it was averred that dis- tinct improvement was experienced in the general sense of well-being and of mental efficiency as a result of the lesser protein consumption. Taking these results as a whole, it is quite clear that man can get along under ordinary conditions with much less protein than he usually takes; but that really proves nothing, for the question is not can he, but should he, so deprive himself? Are instinct and custom wrong and is Chittenden right ? That is the question. To answer it many studies have been made of the condition of peoples who for economic or other rea- sons are compelled to live on less protein than the average. Are these people healthier, less prone to infections and degenerative diseases, and more efficient mentally than others? In such studies great care must be exercised to see that conditions other than diet, such as climate, exercise, etc., are properly allowed for. It would not be fair, for example, to compare the mental and bodily condition of peoples living in the tropics and who take comparatively little protein, with those living in temperate zones, who consume much more. After discounting all of these other DIETETICS 629 f, factors, it has been quite clearly shown that, when the protein allowance , is materially reduced, the people as a whole are less robust, mentally in- ferior, and, instead of being less prone to the very diseases which are [usually supposed to be due to overloading of the organism with useless excretory products, are more liable to suffer from them. That a decided reduction in protein weakens the defense of the organ- ism against infection is probably due to the fact that the fluids of the body normally contain a great variety of so-called antibodies that is, of highly complex substances that are largely protein in nature. When bacteria, or the poisons produced by them, enter the body, they are met by one or more of these defense substances and destroyed or neutralized. Now it is clear that there should always be a surplus of protein-building materials from which the antibodies may be constructed. Such an excess will constitute a "factor of safety" against disease. And there are fac- tors of safety of another nature to be provided for. For example there must always be an adequate supply of tryptophane, of lysine, and of cystine, not only to meet the bare necessities of the protein constructive processes that go on under normal conditions, but also to make good the larger amount of protein wear and tear that greater degrees of tissue activity will entail. Although moderate muscular exercise does not ap- pear to cause any immediate consumption of protein (carbohydrate and, later, fat being the fuel material that is used), yet it does throw a greater strain on the tissues thus causing a greater wear and tear of the ma- chinery, and hence a demand for more protein-building material. There are also certain of the internal secretions of the body, such as epineph- rine (adrenaline), that are essential for life, and as crude materials for the manufacture of which certain amino acids are essential. Tyro- sine is one of these, and since proteins, as we have seen, differ from one another quite considerably in the amount of this amino acid which they contain, it is advisable to provide an excess, so that an adequate supply of tyrosine may always be available. The answer to one of the most important practical questions in die- tetics namely, "What proportion of protein should the diet contain? " depends on these scientific principles. The source of the protein is the important thing. With animal protein there is no doubt that we could get along with perfect safety by taking daily not more than 50 or 60 grams, which is about half of what we actually consume. If the protein is of vegetable origin and part of it of the first quality, as, jjvheat and Indian corn preparations, more should be taken so as to allow for the deficiency of certain amino acids. When vegetable proteins of the sec- ond quality, such as those of peas, beans, lentils, etc., are alone available, much larger amounts are necessary. Such proteins are inadequate in the C>30 METABOLISM case of growing children at least, and even in adults it is undoubtedly advisable that other proteins should supplement them. To insure safety, therefore, it is almost imperative that the diet should contain proteins of various sources. If for economic reasons the main source must be proteins of vegetable origin, then some animal protein, such as is contained in milk or meat or eggs, should be added to at least one of the daily meals. When^peas' and beans are mainly depended on for the protein supply, they should be taken either with milk or one of its prep- arations, or with a thick gravy or sauce made from meat and containing the finely minced meat. This must not be strained off, for if it is, the sauce will contain only the meat extractives but not any of the protein, which is coagulated by the boiling water. Meat extract, in other words, contains no proteins; it is not a food but merely a condiment of no greater dietetic value than tea or coffee. ACCESSORY FOOD FACTORS The practical point to be remembered is that three accessory fac- tors or vitamins are known. There is little danger of the diet being inadequate with regard to food factors if it contains some fruits or green vegetables or unheated fresh milk. Certain of the food factors are destroyed by prolonged cooking. It is during times of food scarcity that the restricted diet may require to be scrutinized to see that it con- tains the essential vitamins. The reader is referred to page 618, where this subject is more fully dealt with. DIGESTIBILITY AND PALATABILITY We have seen that practical dietetics depends on several factors, the exact relative importance of which cannot perhaps be gauged in every case, but preparation of the food so as to make it appetizing must un- doubtedly rank high. The importance of good cooking will now be ap- parent. It is the act of making food appetizing and therefore digestible. It is really the first stage in digestion, the stage that we can control, and one therefore to which much attention must be given, especially when it becomes necessary to make attractive, articles of diet that are ordinarily considered common and cheap. Most people can cook a lamb chop so as to make it reasonably appetizing, but few can take the cheaper cuts of meat and con- vert them into cooked dishes that are as popular and attractive. And there are still fewer who can take the left-overs and trimmings and convert them in the same way. This is the real art of cooking, and too much encourage- ment cannot be given to the effort which our cooking experts are making DIETETICS 631 to show people how these things can be done. The waste of good food in a large city is truly appalling. Cooking has other advantages than making the food appetizing. The jheat loosens the muscle fibers of the meat so that it is more readily masticated; it destroys microorganisms and parasites in the meat; it de- stroys antibodies which, might interfere with the action of the digestive ferments. Thus, untreated raw white of egg is not digested in the stom- ach because it contains one of the antibodies which prevent the pepsin from acting on it; but boiled egg white, if properly chewed, can be di- gested, and whipping the egg white into a foam partly destroys the in- hibiting substance. Before concluding, something should be said about the laxative quali- ties of food, for it is often in this particular alone that one food is more satisfactory than another. A diet of meat, milk, eggs, and white bread is apt to be unphysiological because there is nothing in it to act as what has been called intestinal ballast; that is, a material which will keep the intestines sufficiently filled to stimulate their muscular movements. This ballast is best furnished in the shape of cellulose, the most important constituent of green food. Peas, beans, cabbage, salad, and many fruits, 'j especially apples, should always occupy a place in the daily menu. An- i other food which is valuable because it yields this ballast is the outer grain of wheat, oats, etc. So much must not be taken as to produce a constant intestinal irritation, and each person must determine for him- self where this limit lies. The difference among various breads depends partly on the degree to which they supply ballast. It must not be lost sight of that many of these foods of plant origin are most important be- cause of the valuable vitamines they contain. The all-important subject of food economies can receive no attention here, except to point out that it is one which must be most carefully con- sidered in the solution of all problems of dietetics. An admirable ac- count of the subject will be found in Graham Lusk's "Science of Nutri- tion" (third edition) and .in McKillop's "Food Values." 16 CHAPTER LXIX THE METABOLISM OF PROTEIN Introductory. The older physiologists believed that the protein taken with the food was brought into a soluble condition by the digestive en- zymes, and that it was then absorbed into the blood and directly incor- porated with the tissues. The discovery of the enzymes trypsin and erepsin and of free amino acids in the gastrointestinal contents clearly showed that this simple theory of Liebig could not be correct. It was, furthermore, found that when an excess of proteins such as egg albumin gains entry to the blood, part of the protein appears in an unchanged condition in the urine ; and that enzymes capable of digesting this pro- tein, but not other varieties, make their appearance in the blood. After the injection of foreign proteins into the blood, symptoms of varying severity often develop, from the almost instantaneous death produced by snake venom to the slowly developing anaphylactic reac- tions which follow the injection into the blood of many proteins chemi- cally indistinguishable from those of the blood serum itself. When pro- tein is taken in the usual amounts by mouth, these poisonous reactions do not supervene, even snake venom is harmless when swallowed, nor is it possible during digestion of a protein meal to detect food protein in the blood by means of the precipitin reaction. Finally it was discovered that the very slow intravenous injection of completely digested flesh did not produce on the part of the body any of the reactions that injected protein Itself produces, indicating that perfect assimilation had occurred. From these and similar observations it soon became clear that protein 61 not be absorbed as such from the alimentary canal, but must first of be completely 'broken down into the amino acids, which are then rebuilt o the protein of the organism. The direct evidence for this important change in belief concerning protein metabolism has been gained by the discoveries that: (1) nitrogen equilibrium can be maintained in animals fed with completely digested protein mixtures; and (2) amino acids can be isolated from the blood. The experiments of the first group consist, in principle, in breaking down protein until there is no longer the characteristic biuret test and then feeding this digestion mixture to animals and observing them from day to day, using as criteria of their nutritional condition the body weight and the nitrogen equilibrium. (Page 605.) It has been shown that success in maintaining nutritional efficiency depends partly on the 632 THE METABOLISM OF PROTEIN 633 nature of the process used for digesting the protein, and partly on the presence or ab- sence of carbohydrate in the digestion mixture. It was found that the products of hydrolysis by acid failed to maintain equilibrium, and it was believed that this was owing to the fact that the acid had more completely disrupted the protein molecule, and had left no polypeptides, which, it was imagined, remained intact during enzyme action and were essential for proper protein metabolism. This view has now been consider- ably altered, since it has been shown that the acid actually destroys certain amino acids which the enzyme leaves/ intact. The amino acid particularly concerned is tryptophane. Thus, when different groups of animals were fed with diets, consisting of (1) fully digested casein, (2) fully digested casein from which the tryptophane had been removed, it was found that nitrogen equilibrium could not be maintained on the second diet, whereas it was maintained on the first. When the protein was only partly digested by acid that is, not digested enough so as to break up all the trypto- phane or when tryptophane was added to the second diet, nitrogen equilibrium could be satisfactorily maintained. These results obtained in different classes of animals have also been confirmed for the hum'an subject. For example, nitrogen retention has been observed in a case of a boy suffering from a stricture of the esophagus, when he was fed by rectum for fifteen days with digestion products resulting from the action of trypsin and erepsin on flesh. Concerning the second type of evidence, many investigators attempted to separate the amino acids themselves from the blood, particularly during the digestion of a large amount of protein, but the results were at first entirely negative because of the lack of methods that were sufficiently delicate to make it possible to detect the slight increase that could be expected even when a maximum absorption of nitrogen had occurred. The very large flow of blood through the portal vein causes such extensive dilution of any substances added to it that the concentration of the substance in an isolated sample of the blood can be only trivial. This brief historical survey of the subject brings us to a position where we may proceed to discuss the present-day teaching regarding protein metabolism. Briefly stated, this teaching is to the effect that the protein molecule is broken down into its ultimate building stones, the amino acids, ~by the digestive enzymes of the gastrointestinal tract. These amino acids are absorbed into the blood, by which they are carried ta the various organs and tissues, which sift out the amino acids and use 'those of them which they require for the reconstruction of their broken-down protein. The amino acids not required for the process, along with those which may 'be liberated in the tissues themselves by disintegration of tissue proteins, are then split into two portions, one represented by ammonia and the other by the remainder of the OMiwLJicitLjnolecide. The former is excreted as urea and the latter is oxidized to produce energy. elusions depend, it will be necessary to consider some of the most important CHEMISTRY OF PROTEIN Before proceeding to discuss the evidence upon which the above con- facts concerning the chemistry of the protein molecule. We shall require this information not only to understand the history of protein in the 634 METABOLISM animal body, but also to follow intelligently the important work that has already been discussed concerning the relative value of different proteins as food. A knowledge of protein chemistry has come to be essential in practically all branches of medical science. Proteins, like starches, are composed of numerous smaller molecules. In the case of starch these molecules are the various monosaccharides glucose (dextrose), levulose and galactose; in the case of proteins they are the amino acids. The breaking apart of the links that hold the mole- cules together is effected in both cases by the process of hydrolysis, so called because of the fact that the reaction consists in the taking up of a molecule of water at each of the places where the chain falls apart. This hydrolysis may be effected either by the action of mineral acids or alka- lies, or by enzymes, the only difference in the action of these reagents being that in the former case the breaking apart takes place more or less indiscriminately, whereas in the latter it proceeds according to a definite plan, which varies somewhat with the type of enzyme employed. Just as a chemical knowledge of the -structure of sugar or monosac- charides is the basis of carbohydrate chemistry, so is that of the amino acids the basis of protein chemistry. Amino Acids. There are, so far as known, eighteen different amino acids concerned in the constitution of protein, but they are all alike in their characteristic structure. The most striking characteristic depends on the presence in the molecule of: (1) an amino group with a basicity comparable to that of ammonia, and (2) an acid group with an acidity comparable to that of acetic acid. Let us take in illustration one of the simplest fatty acids namely, acetic. It has the formula CH 3 COOH. The COOH group is called carloxyl, and on it depend the acid properties of the compound. The CH 3 group is known as methyl, and the amino group (NH 2 ) is attached to it in place of one of the hydrogen atoms, thus giving the formula CH 2 NH 2 COOH, which is aminoacetic acid or gly- cocoll. If we take the next higher acid of the fatty acid series, having the name propionic and the formula CH 3 CH 2 COOH, its amino acid, called alanine, has the formula CH 3 CHNH 2 COOH. Now let us place the formu- las of these two acids side by side in the following manner: H CHs I I NH 2 - C - COOH NH 2 - C - COOH (amino group) H (acid group) (amino group) H (acid group) Aminoacetic acid Aminopropionic acid (glycocoll) (alanine) It will be observed that the only difference between the two acids is dependent upon a change in the group that is attached to the upper verti- THE METABOLISM OF PROTEIN 635 cal valency bond of the central carbon atom, which therefore must be considered as the center of the entire molecule. The various amino acids entering into the structure of protein differ from one another solely with regard to the chemical nature of the group that is attached to this ver- tical valency bond. Evidently, then, the reactions that amino acids pos- sess in common must depend on the end groups containing the carboxyl and amino radicles, whereas the characteristic reaction of each of the eight- een amino acids must depend upon the differences in the radicles attached to the upper vertical bond. This may be represented thus : Any radicle NH 2 -C-COOH H (Any amino acid) The end groups endow the amino acids with the power to combine with both acids and bases. With acids they behave like substituted ammonias to form salts, which can here ionize into the amino acid, as the cation, and the acid group, as the anion. With bases the carboxyl group reacts to form' salts, which yield amino acid as the anion. A most important reaction consists in the condensation of aldehydes with the amino group. This occurs particularly readily with formaldehyde, water being eliminated in the re- action, and the basic nature of the amino acid thus destroyed. Upon this reaction de- pends the method of Sorensen for determining the amount of amino acid in a mixture (see page 641). The titration is performed by rendering the solution of amino acids neutral, then adding formaldehyde and titrating with standardized acid, using phe- nolphthalein as the indicator. This tells us to what degree the acidity of the mixture has become increased as a result of adding the formaldehyde, and since this increase in acidity must depend upon the number of amino groups, we are furnished with an indirect estimate of the concentration of the amino acids. The reaction is illustrated by the equation : radicle H radicle NH 2 -C-COOH + H-Cz=O = CH 2 = N - - COOH + H 2 O (amino acid) (formaldehyde) Another reaction of amino acid of physiological interest is that known as the car- b amino reaction, consisting in a union of the amino acid with calcium and carbonic acid. Finally, it is important to note that the amino group is very firmly attached; it remains intact in acids and alkalies and is removable only by a process of oxidation. This can be accomplished by treating the amino acid with such reagents as hydrogen peroxide or with potassium permanganate, when the amino group is displaced and a so-called ketonic acid formed. The reaction will be evident from the accompanying equation : CH 3 CH 3 I I O + NH,-C-COOH ^ O = C-COOH+ NH 3 H (alanine) (pyruvic acid) 636 METABOLISM To illustrate this reaction we have chosen aminopropionie acid or alanine, because the substance formed by its oxidation and known as pyruvic acid is of very great im- portance in intermediary metabolism. It serves as the common substance from which proteins, carbohydrates or fats may be formed, and therefore as the intermediary sub- stance through which one of them may pass on being transformed into another (page 698). The use of two arrows pointing in opposite directions in the above equation in- dicates that the reaction may proceed readily in either direction. The ammonia set free from amino acids may be oxidized to free nitrogen by using nitrous acid according to -the general equation: NH 3 + HONO=2H 2 O + N 2 . Upon this reaction depends another extremely important quantitative metttod for measuring the number of amino groups prese'nt in protein (Van Slyke). To make the estimation, nitrous acid is allowed to act on the amino acids, and the volume of nitrogen gas set free by the reaction is measured, the principle being similar to that used for the de- termination of urea by the hypobromite method. The apparatus employed for decomposing the substance and collecting and measuring the evolved nitrogen consists essentially of a mixing bulb, connected below through stop- cocks with two small burettes, one containing a solution of sodium nitrite and glacial acetic acid, and the other a solution of the substance to be investigated. The upper end of the mixing bulb is connected through a three-way cock with a graduated gas burette and with another bulb containing potassium permanganate solution. By allowing some nitrite and acid solution to run into it and shaking, the mixing bulb is first of all filled to a certain mark with nitrous oxide gas. A measured quantity of the amino solution is then allowed to mix with the nitrite ; the apparatus is shaken for five minutes at 15 to 20 C., and the evolved nitrogen and nitric oxide are driven over into the permanganate, which absorbs the nitric oxide, leaving the nitrogen, which is then measured in the burette. The apparatus has now been so perfected that numerous analyses may be made with it in a very short time and with a degree of accuracy that is scarcely surpassed in any other biochemical estimation. Protein Synthesis. From the point of view of protein chemistry, the most significant reaction of the amino acids is their ability to link to- gether to form compounds called peptides. This linking occurs between the amino group of one amino acid and the carboxyl group of the next. When alanine and glycocoll, with which we are familiar, are thus linked together, the reaction takes place according to the equation: H CH 3 H CH, | | | /IH + HO; OC-C-NH 2 = HOOC-C-NH-CO-C-NH 2 HOOC-C-N I ! : I \H H H H (alanine) (glycocoll) (alanyl - glycocoll) In this manner, then, a so-called dipeptide is formed, in which it will be observed there still remain free carboxyl and amino groups, thus per- mitting the linking on of other amino-acid groups to form tripeptides or tetrapeptides or other polypeptides. Indeed, this process of condensa- tion may go on practically indefinitely, a polypeptide containing eighteen amino-acid groups namely, three leucine and fifteen glycocoll groups hav- THE METABOLISM OF PROTEIN 637 os.5't If o o - o o s; o 55 JS a a " * ,11 s i-S > 3 a - 8 | o 1 1 mi 638 METABOLISM ing actually been synthesized. The resulting polypeptides have the proper- ties of derived proteins like the proteoses; thus, they give the biuret and other reactions characteristic of proteins and are precipitated by such reagents as mercuric chloride and phosphotungstic acid. Some are also digested by trypsin and erepsin. They have the same optical activities as proteins. One of them has been prepared which produces a typical anaphylactic reaction. So far a polypeptide that can be coagulated by heat or is in other ways identical with the naturally occurring proteins, has not been synthetized; but there is no doubt that it is only a matter of time before this will be accomplished. Eighteen distinctly different amino acids have been isolated from pro- tein, and it may assist in the conception of our problem if we place these amino acids side by side and link them together in the manner described above. This is done in the accompanying chart compiled by D. D. Van Slyke, in which also various other important facts concerning the chem- istry of the amino acids are incidentally added. At the lower part of each formula will be seen the characteristic car- boxyl and amino groups of neighboring acids linking together the ter- minal carbon atoms. The upper vertical bond of this carbon atom is con- nected with the characteristic group of the amino acid, which may be very simple and represented only by hydrogen, as in glycocoll, or highly com- plex and including a ring formation, as in tryptophane. It will further be observed that there may "be other amino groups connected in various positions in this radicle. This is particularly the case in the first three of the amino acids in the table namely, the basic amino acids. In lysine the extra amino group reacts with nitrous acid, liberating free nitrogen by the Van Slyke method; but in other cases, as in arginine, it fails to give this and the other characteristic reactions of the amino group. It will further be observed that the amino acids are arranged in three main groups: one basic, another neutral, and the third acid. The basic amino acids are three in number and have an alkalinity similar to that of ammonia. They have been called the hexone bases, because each contains six carbon atoms. They are alone present in certain forms of pro- tein called protamines. The neutral amino acids contain one amino group and one carboxyl group, which exactly neutralize each other. This is the largest group of amino acids, and is further subdivided into three: one containing aromatic or benzene rings and including the very im- portant amino acids, tyrosine and tryptophane; another containing the so-called pyrrolidine ring; and the third, the largest of all, containing the so-called aliphatic chains; that is, the chains characteristic of the fatty acids and which may be either straight or branched. When the chains are branched, the substance is called an isosubstance, as in isoleucine. The acid amino acids, including glutamic acid and aspartic acid, are THE METABOLISM OF PROTEIN 639 characterized by containing two carboxyl groups and only one amino group. They therefore resemble acetic acid in acidity. It may be of assistance to some if we restate these chemical facts from a slightly different standpoint as follows: Glycine, or glycocoll, is aminoacetic acid, CH 2 NH 2 COOH Alanine is glycine plus a methyl group, CH 3 CH ; it is therefore amino- COOH OH propionic acid and is closely related to lactic acid, which is CH 3 CH . Many of COOH the other amino acids may be considered as derivatives of alanine, thus: 1. Serine is alanine with an "OH" (hydroxyl) group in place of one of the "H" NH 2 atoms of the methyl group, CH 2 OH - CH COOH 2. Cysteine is alanine with an "SH" (thio) group in this position, CH 2 SH - CH COOH Two cysteine molecules united at the ' ' S " groups give cystine. NH 2 1 CH 2 S - CH COOH NH 2 3. Phenylalanine has a C 6 H 4 (phenyl) group, CH 2 C 6 H 5 -CH COOH NH 2 4. Tyrosine has a C 6 H 4 OH (phenol) group, CH 2 C 6 H 4 OH - CH COOH C 5. Tryptophane has a C 6 H 4 CH (indole) group : NH C CH 2 - CH - NH 2 - COOH. C 6 H 4 CH NH CH N NH 6. Histidine has a CH C - (imidazole) group: 640 METABOLISM CH N NH CH = C.CH 2 . CH. NH 2 -COOH. The last two are also called heterocyclic compounds, of which there is another, viz.; Proline (and oxyproline), which is a-pyrrolidine carboxylic acid: CH a CH 3 CH 2 CH.COOH \ Other ami/no acids are: CH S CH 3 CH 3 CH 8 CH 3 C 2 I \ / \ / \ / CH CH CH (1) Valine Leucine CH.NH 2 ok CH.NH. Isoleuciue I 1 I thus: COOH CH.NH 2 1 COOH (valine) COOH (isoleucine) (leucine) (2) The amino dibasic acids: Aspartic, which is aminosuccinic acid, CH 2 COOH I CHNH 2 COOH; and Glutaminic, which is aminoglutaric acid, CH 2 CH 2 - COOH CHNH, COOH. Lastly there are the diamino acids, in which two groups exist : Lysine a e-diaminocaproic acid. NH 2 NH 2 CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH Arginine a-animo 5-guanidine-valerianic acid. NH 2 HN = C COOH. NH 2 NH.CH 2 - CH 2 - CH 2 - CH COOH The guanidine group in this acid is of interest because of its close relationship to NH 2 urea, which is O=C NH 2 CHAPTER LXX THE METABOLISM OF PROTEIN (Cont'd) AMINO ACIDS IN THE BLOOD AND TISSUES In the Blood. Furnished with the general facts concerning the chem- istry of protein, we may now proceed to consider the more precise knowledge recently acquired concerning the history of this substance in the animal economy. Although no one has succeeded in separating amino acids in pure condition from drawn blood even during the height of digestion, it has nevertheless been possible to do so from circulating blood by a method of dialysis, known as vividiffusion, elaborated by Abel 33 and his pupils. The method consists in connecting a long tube of collodion with the two ends of a cut artery in an anesthetized animal. (Fig. 187.) The tube, coiled many times, is then immersed in a solution containing approximately the same salt content as the blood plasma of the animal. The diffusible constituents of the blood plasma dialyze into the saline solution ; or any one of them may be prevented from dialyzing by add- ing that particular substance to the saline in such amounts as will make its concentration in plasma and saline alike. It has been possible in this way to isolate several of the amino acids and other ammonia-yielding substances from blood. Thus, alanine and valine have been obtained as crystalline salts, and histidine and creatine have been (see page 656) shown to be present by their reactions. All of the amino substances, however, do not dialyze, and these exceptions are further characterized by the fact that they do not readily give up their ammonia on the ad- dition of sodium carbonate, as do the diffusible substances (Rohde). Although amino acids can thus be separated in a pure state from cir- culating blood, their concentration in a drawn specimen is too low to ike direct quantitative estimation possible. By the methods of Van ttyke and Sorensen, already described, however, it has been shown imong other things that the blood always contains a certain concentra- ;ion of amino acids ; thus, in that of fasting animals from 3 to 5 mg. per LOO c.c. of blood are usually found present. During the absorption of a )rotein meal, the amino content of the blood undergoes a marked in- crease, becoming doubled or more; and a similar result has been ob- tained by placing pure amino acids in the small intestine. After 10 grams of alanine, for example, the amino nitrogen of the mesenteric blood rose from 3.7 to 6.3 mg. per cent.* "This is a convenient way of stating per 100 c.c. of blood. 641 642 METABOLISM In the Tissues. After entering the circulation, the excess of ammo acids very quickly disappears from it again. This has been demonstrated by observing the amount of amino acids in the blood after the intravenous injection of a solution of amino acid into an anesthetized animal. After injecting 12 gm. of alanine into the vein of a dog, 90 per cent was found to have disappeared from the circulation within five minutes. The ques- tion is, What becomes of the amino acids that rapidly disappear? Are they decomposed in the blood, or do they become absorbed by the tis- sues? This problem has been attacked by analyzing portions of various organs and tissues removed before and some time after the injection Fig. 187. Vividiffusion apparatus of J. J. Abel. into an animal of solutions of amino acids. In the case of the muscles it has been found that the amino-acid content increases until from 60 to 80 mg. per cent of amino acid has accumulated. Beyond this point, however, the muscles do not seem to be able to take up any more amino acid (Fig. 188). The capacity of the abdominal organs, however, is more elastic; for example, the amino nitrogen of the liver has been observed to become increased to 125 or 150 per cent of the original amount. Al- though this absorption of amino acids by the tissues is extremely rapid, it never proceeds to such a point that the blood becomes entirely free of them, for even after many days' starvation the blood contains its normal quota of from 3 to 10 mg. per cent (Fig. 189). This indicates that under these THE METABOLISM OF PROTEIN 643 conditions a certain equilibrium must become established between the ammo-acid content of the blood and that of the tissues, the concentration in the tissues being approximately from five to ten times greater than in the blood. The absorbed amino acids are very loosely combined with the tissues, for they can be extracted by such feeble reagents as water or dilute al- cohol. Their presence can not, however, be merely due to diffusion; for if it were, the concentration could not become greater in the tis- sues than in the blood. The further fate of the amino acids is difficult 150 100 50 Injectio Muscle NH 1 2 Hours Fig. 188. Curves showing the amount of amino nitrogen taken up by different tissues after the intravenous injection of amino acids. The lowermost curve shows the urea concentration of the blood. (From D. D. Van Slyke.) to follow. We know that they do not remain in the body for a long time, because most of the protein nitrogen in the food is excreted as urea within twenty-four hours after ingestion; and when single amino acids I are fed, they quickly reappear in the urine as urea. The tissues can therefore be only a stopping-place for the amino icids. When the latter are determined in blood collected from different 3arts while absorption of protein from the intestine is in progress, it las been found, as shown in Fig. 189, that during the passage of the )lood through the liver there is a greater fall in the concentration of 644 METABOLISM amino acids than during its passage through the entire remainder oi' the body. It will be seen that the above conclusions are drawn from estimations made on blood taken from the vena cava, the portal vein, and the hepatic artery, the upper curve in the chart being from animals during digestion and the lower, from fasting animals. The results show that the liver must be particularly greedy of amino acids, which, however, must rapidly be- come transformed into other substances, since no conspicuous varia- tion has been found to occur in the amino-acid content of the tissues Fig. 189. Curves showing the concentration of amino-acid nitrogen in the blood during fasting and protein digestion. (From D. D. Van Slyke.) according to whether the animal is fasting or is digesting protein food. This result, it is to be noted, is quite different from that which is ob- tained after the intravenous injection of amino acids, and the results of the two experiments taken together, indicate that the amino acids after their absorption can not remain in the tissues, in a free condition for a long time. It means that the amino acids during natural digestion must be disposed of at a rate which is practically the same as that at which ab- sorption is proceeding. THE METABOLISM OF PROTEIN 645 THE FATE OF THE AMINO ACIDS To follow the metabolism of the amino acids further we must deter- mine the end product into which they are converted. This is urea, the estimation of which can nowadays be made with considerable accuracy on account of the discovery, by Marshall, of the action of urease in con- verting its nitrogen into ammonia, which can then be estimated by com- paratively simple methods (Folin). When the viscera are compared before and at various periods after the intravenous injection of amino acids, the immediate increase in amino nitrogen remains undiminished in all of them except the liver, in which a very rapid reduction is observed to occur. At the same time the percentage of urea in the blood steadily rises. These facts are illus- trated in Fig. 188. The simplest interpretation of these results is that the liver converts the amino acids into urea and discharges this urea into the blood. This conclusion, however, it must be observed, is not inevitable; for it is pos- sible that the amino acids may be condensed into polypeptides in the liver, just as sugar is condensed by this organ into glycogen, and that the increase in urea in the blood is merely coincident (Fiske). It must not be imagined that the conversion of the amino acids into urea is exclusively a function of the liver. On the contrary, it is well known that this process may occur in animals from which the liver has' been entirely removed. It is probably safe to conclude, however, that the liver is the most active center for amino-acid transformation and urea formation. When urea is estimated in samples of blood removed at short inter- vals of time after the ingestion of a large amount of protein, it is found that the increase becomes very early established. In one experiment, before the food was taken the concentration of urea nitrogen in the blood was a little over 10 mg. per cent; one hour after taking 500 grams of meat, it had risen to about 18, and in two hours to nearly 25. Evidently the increase had occurred about the same time as the passage of food from the stomach into the duodenum. These facts indicate that urea formation in the liver becomes stimulated long before the other tissues, such as the muscles, have had time to take up their full quota of amino acids. During digestion of protein the liver does not appear to wait until the other tissues have become saturated with amino acids before it begins to destroy the unnecessary excess by conversion into urea; on the contrary, this process sets in with the very first installment of amino acid that reaches the liver by the portal blood. This conclusion is in harmony with the well-established fact that, when protein is given to a 646 METABOLISM starving animal, the greater part of its nitrogen is soon excreted as urea, leaving only a small fraction to be used for rebuilding the wasted tissues. The amino acids that are absorbed by the extrahepatic tissues become very quickly converted into formed protein, as is evident from the fact that the concentration of free amino acids in the tissues of an animal during absorption of protein is not perceptibly greater than in those of a fasting animal, and the question remains to be considered, What be- comes of the protein thus formed? The answer is, that it is gradually /used up in the metabolic processes, so as to liberate again the amino iacids, which add themselves to those absorbed from the intestine and be- y?ome used again or excreted, according to the demands of the tissues at the time for amino acid. This process of liberation of ammo acid from the breakdown of body protein goes on of course irrespective of absorption of amino acid from the intestine. It goes on, for example, during starvation; indeed, in this condition the percentage of free amino acids in the muscles is, if anything, somewhat higher than that observed in an ordinarily fed an- imal. In starvation also the migration of amino acid is going on among the various organs, of which those whose activity is essential to the maintenance of life, such as the heart and the respiratory muscles, are supplied with amino acids from tissues that are less vital, such as the skeletal muscles (see page 602). These experiments further show that- free amino acids can not serve to any significant extent as food reserves in the same way as glycogen and fat. If amino acids were of value as food reserves, we should expect the store of them to be depleted by starvation. As to how long a period of time elapses between the incorporation of the absorbed amino acids into tissue protein and their subsequent liberation again by autolysis, we are entirely ignorant. The researches which we have just been considering do not throw any light on the relative value of different proteins in tissue metabolism. They do not inform us as to which of the amino acids must be absorbed ready-made from the digested food, and which of them may be dispensed with since the organism can manufacture them for itself. We know that the higher animals can synthetize some amino acids, such as glycocoll, but not others, such as tryptophane ; but which amino acids belong to the glycocoll and which to the tryptophane groups, can not as yet be definitely stated. The investigation of this problem has to be under- taken by experiments of an entirely different type namely, by observing the welfare and growth of animals fed on proteins of varying amino- acid composition. A full discussion of these experiments is given in the chapters on Nutrition and Growth. CHAPTER LXXI THE METABOLISM OF PROTEIN (Cont'd) THE END PRODUCTS OF PROTEIN METABOLISM Introductory. So far we have approached the problem of protein metabolism by studying the behavior of the absorbed products of pro- tein breakdown, and we have seen that these become gradually assimilated by the tissues and used by them in their metabolic processes. We have been unable, however, to offer any facts, regarding the exact chemical changes which each amino acid undergoes during this process of tissue metabolism. At first sight it might appear an easy matter to collect such information by direct examination of the tissues themselves, either by searching in them for amino derivatives which might be derived from absorbed amino acids, or by studying the changes which occur when the amino acids are subjected to the action of the isolated tissue en- zymes that must be responsible for the change. Such methods of in- vestigation are, however, fraught with technical difficulties so great that very little can be learned from them, and for the present at least we must be content to piece our information together from facts derived by less direct methods. Such a method is offered by investigating the behavior of the en^. products of protein metabolism. The main end product is urea along with traces of its precursor am- monia, but these are not the only ones, for some amino acids after being incorporated with the tissue proteins break down into products that are no longer members of the amino-acid series, although they may be closely related to certain amino acids. Such substances are creatine and its anhydrid creavtmne. A part of the amino acids during their pres- ence in a free state in the blood may also be excreted unchanged by the kidney. Our list so Jar therefore includes urea, ammonia, creatine, creath^ijie, and amino nitrogen, of which the last is usually included in metabolism investigations in the fraction designated undetermined nitrogen. Another group of closely related substances coming, not from the general protein metabolism of the tissues, but from the metabolism which is peculiar to the nuclei, consists of the so-calleds^pt/rme bodies. Furthermore, so as to serve as a check on results obtained by examining these nitrogenous metabolites, it is important to observe the manner of 647 648 METABOLISM excretion of the sulphur moiety of the protein molecule, for it will be remembered that it is in protein alone that sulphur is usually taken into the animal body. .The excretion of sulphur therefore runs more or less parallel with the intensity of protein metabolism. After selecting the end products that are most likely to be of signif- icance, the first question concerns the amount of each of them excreted during twenty-four hours on diets that are either rich or poor in pro- tein. The possibility of conducting such investigations obviously de- pends on the use of quick and yet reliable methods for the estimation of the nitrogenous metabolites. Such methods have been furnished by the painstaking and careful work of Folin, an example of whose results is given in the accompanying table. NITROGEN-RICH DIET NITKOGEN-POOR DIET Volume of urine 117U c.c. 385 C.C. -^^^ Total nitrogen 16.8 grams 3.60 grams ^SJrea nitrogen 14.7 grams 87.5% 2.20 grams = 61.7% ^Ammonia nitrogen ^HJric-acid nitrogen Crea,tinine nitrogen 0.49 gram 3.0% 0.18 gram 1.1% 0.58 gram 3.6% 0.42 gram 11.3% 0.09 gram 2.5% 0.60 gram =17.2% Undetermined nitrogen 0.85 gram 4.9% 0.27 gram 7.3% Total S0 3 3.64 grams 0.76 gram Inorganic SO 3 Ethereal SO, ' 3.27 grams 90.0% 0.19 gram 5.2% 0.46 gram 60.5% 0.10 gram 13.2% MeutraT"SO 3 0.18 gram 4.8% 0.20 gram 26.3% (Folin.) The general conclusions which may be drawn from these results are s follows: 1. With a protein-rich diet much more urine is excreted in twenty- four hours than with one that is protein-poor. Evidently the nitrogenous metabolites act as diuretics. 2. The total or absolute amounts of nitrogen and of all the other nitrogenous metabolites, save creatinme, become diminished during the starvation period. The same is true of the^sulphur derivatives, except in the case of the neutral sulphur, which behaves like creatinine. 3. The decrease in nitrogen is not borne proportionately by all of the metabolites. This is seen by examination of the percentage figures which are obtained by calculating the nitrogen of each substance as a percentage of the total nitrogen. SThe urea decreases relatively much more than the total nitrogen. The inorganic sulphate behaves in a manner similar to the urea that is, the percentage of total sulphate excreted in the inorganic form becomes much less during starvation. 4. The relative amount of all the other nitrogenous metabolites, as well as that of the ethereal and neutral sulphates, becomes increased during starvation. The most striking results of the above investigation are that creatinme remains unchanged. during starvation, but that urea becomes relatively THE METABOLISM OF PROTEIN 649 increased. The former must be derived from metabolic processes going on in the tissues independently of the supply of foodstuff carried to them, whereas the latter must depend, if not entirely, yet very largely, on the protein content of the food. Creatinine may therefore be called an end product of endogenous metabolism, and urea an end product of exogenous metabolism. Other metabolites namely, ammonia, uric acid and the undetermined nitrogen, as well as the ethereal sulphates must represent processes of metabolism that are partly exogenous and partly endogenous. Having made ourselves acquainted with the general nature of the changes that occur in the nitrogenous metabolites when protein metab- olism is stimulated by the taking of food or is depressed by starvation, we may now proceed to a study of the source and origin in the animal body of each of the metabolites. UREA AND AMMONIA For various reasons it is important to consider these two metabolites together. During the intermediary metabolism of the majority of the amino acids, the amino group becomes broken off as ammonia, which immediately combines with the available acids to form neutral ammonium salts. The most available acid for this purpose is carbonic acid; there- fore ammonium carbonate is formed in large amounts. A small propor- tion of the ammonia may combine with other acid radicles, such as chlorine, to form ammonium chloride. The fate of these two types of salt is very different. The ammonium carbonate becomes quickly trans- formed into urea, whereas the ammonium chloride is excreted in the urine. The process of urea formation may therefore be considered as having the function of preventing the accumulation of ammonium car- bonate in the animal body. It is the means by which a harmful substance is converted into an innocuous substance a detoxication process, in other words. Regarding the nature of the chemical process involved in this trans- formation of ammonium carbonate into urea, reference to the following formulae will show that the ammonium carbonate that is formed by the union of carbonic acid with ammonia, by losing one molecule of water becomes ammonium carbamate, which by repetition of the process be- comes transformed into urea : OH ONH 4 ONH 4 NH, / \] / / / CO + 2NH 3 <= CO -H O^CO - H.,O = CO \ \ \ \ OH ONH, NH, NIT, (carbonic (ammo- (ammonium (ammonium (urea) acid) nia) carbonate) cnrbnmate) 650 METABOLISM Some of the urea may come from metabolic processes of an entirely different type. One of these at least is known ; namely, the splitting-off of urea from arginine, which it will be remembered is guanidine-amino- valerianic acid (see page 640). An enzyme called arginase, having this action, has been isolated from various organs and tissues. The diamino- valerianic acid, or ornithine, which remains after the urea is split off, may be further used in protein metabolism. The reaction is shown in the following equation: NH 2 - C - NH - CH 2 - CH 2 - CH 2 - CHNH 2 - COOH + H 2 O NH (arginine) NH 2 -CO I 4- NH 2 - CH 2 - CH 2 - CH 2 - CHNH 2 - COOH NH 2 (urea) (ornithine) On an ordinary diet, as we have seen, a man excretes somewhat more than 90 per cent of his total nitrogen as urea and about 3 per cent as ammonia, the remainder of the nitrogen appearing in the other nitrog- enous metabolites. Influence of Acidosis on Ammonia-Urea Ratio. It sometimes happens that a large proportion of the ammonia is used for the purpose of neu- tralizing abnormal acids present in the organism. The ammonia is appar- ently produced in the kidney (page 563). When mineral acids are given to an animal, or when acids are produced in the organism itself by some faulty type of metabolism, the ammonia excretion by the urine immedi- ately rises. In diabetes, for example, where considerable quantities of /?-oxybutyric acid are produced (see page 715), a decided increase in the ammonia excretion is observed. A milder type of acidosis may also be in- duced in normal persons by withholding carbohydrates from the diet, and here again the ammonia excretion is relatively increased. In such cases the ammonia is to be regarded as an alkaline re- serve in the sense that it carries the excess of acids out of the body and so saves fixed alkalies. It does not appear, however, that all types of acidosis entail the utilization of ammonia as reserve alkali, and an increase in the relative amount of ammonia in the urine does not necessarily indicate a condition of acidosis. In the pernicious vomiting of pregnancy, for example, a relatively high excretion of am- monia has been found associated with no greater a degree of acidosis than in normal cases of pregnancy, as determined by the power of the plasma to absorb carbonic acid. When there is a relative excess of al- kali in the blood (alkalosis) the ammonia excretion becomes depressed, as is the case after taking alkali with the food, or in the alkalosis pro- duced by forced breathing (page 382). THE METABOLISM OF PROTEIN 651 Influence of Liver on Ammonia-Urea Ratio. Experimental Observa- tions: (1) REMOVAL OF LIVER. There are several facts which indicate that other causes than acid-production may interfere with the conversion of am- monia into urea. What are these causes? Since, as we have seen, the liver is the organ which most actively converts amino acids into urea, it would be natural to expect that, when the functions of this organ were interfered with, relatively more of the nitrogen excre- tion would occur as ammonia and relatively less as urea. In order to determine the exact significance of the liver as a urea-forming organ, two types of investigation have been used; namely, (1) observation of the changes produced in the ammonia-urea ratio in the urine by partial or total removal of the liver; and (2) observation of the urea-forming power of a liver perfused outside the body. To remove the liver from the circulation the portal vein is brought in apposition with the vena cava, the two are sewed together, and a passage opened between them, after which the portal vein is ligated above the anastomosis (forming the so-called Eck fistula). The portal blood then passes directly into the vena cava, and the liver is now supplied only by the hepatic artery. The animals live for a considerable time after the operation, and the urine frequently contains relatively less urea and more ammonia than normal. The results are, however, not nearly so striking as would be expected if the liver were the main seat of urea formation. The experiments have nevertheless brought to light a fact of considerable clinical interest namely, although the animals may thrive if kept on a diet not containing an excess of flesh, they im- mediately begin to develop peculiar symptoms, not unlike those of ec- lampsia or uremia, when they are fed with large amounts of flesh food. Most of the symptoms can be referred to abnormal stimulation of the central nervous system, and examination of the urine has shown a large increase in the excretion of ammonia and a change from the normal acid reaction to an alkaline one. At one time it was assumed that the toxic symptoms were caused by the presence in the blood of ammonium carbamate, since large quantities of the calcium salt of this substance could be separated from the urine. It is now known, however, that the ammonium carbamate is present only because of the excess of ammonium carbonate, the two salts existing to- gether in solution according to the laws of mass action. That the intox- ication is not due to ammonium carbamate does not exclude the pos- sibility that it may be due to ammonia itself, although it is more likely that other nitrogenous metabolites, produced when excess of flesh food is taken, are the responsible agents. If the liver is entirely removed by ligating the hepatic arteries in an 652 METABOLISM animal with an Eck fistula, a more pronounced decrease in urea and increase in ammonia occur during the short period of time that the animal survives the operation. The percentage of blood sugar falls steadily after the removal of the liver, and when it reaches about 0.05, the toxic symptoms appear, soon' followed by death. If glucose solutions be now given intravenously, the symptoms are immediately relieved and the life of the animal prolonged (Mann). In corroboration of these observations on mammals, it may be of in- terest to note that when the liver is removed from birds, which is a com- paratively simple operation on account of a natural anastomosis between the portal and renal veins, there is a marked decrease in the excretion of uric acid and a corresponding increase in the excretion of ammonia during the twelve hours or so that the birds survive. In birds and reptiles urea is excreted as uric acid, being produced by a synthetic process in the liver (see page 677). The changes in this experiment are of considerable magnitude; thus, before the operation the amount of ammonia nitrogen relative to total nitrogen has been found to vary be- tween 10 and 18 per cent; after the operation it may be increased to between 45 and 60 per cent. The uric-acid nitrogen normally varies be- tween 60 and 70 per cent of the total nitrogen; after the operation it may fall to between 3 and 6 per cent. In animals with an Eck fistula and with the hepatic artery ligated, an increase in the urea output occurs when amino acids are injected under the skin. This result corroborates the conclusion that the liver can not alone be responsible for the conversion of ammonia into urea. (2) PERFUSION OP ORGANS. This method consists in removing the or- gan into a warm chamber or bath and perfusing it, through cannulae inserted in its main artery and vein, with a solution of defibrinated blood or of defibrinated blood mixed with saline solution. The perfusion liquid is kept at body temperature and is saturated with oxygen. By means of a pump it is made to circulate in a pulsatile flow, and the total amount of urea or other metabolite in the circulating fluid is determined before and after the fluid has been circulated several times through the organ. When the liver is perfused, urea gradually accumulates in the fluid, particularly after the addition of one of its known precursors for example, ammonium carbonate. When other organs or viscera are perfused, no urea is formed. The evidence shows that the liver is an important seat of urea formation, but not necessarily that other organs are unable to form it in the intact animal, for there are many sources of inaccuracy in perfusion experiments, for although we exercise the THE METABOLISM OF PROTEIN 653 greatest care, we can not hope to maintain the organ in other than a slowly dying condition. It is certainly far removed from the normal- state, as is revealed not only by histological examination, but by the fact that edema almost invariably sets in and the blood vessels become ex- tremely constricted, thus necessitating a gradual increase in the per- fusion pressure as the perfusion goes on. Furthermore, the organ being isolated- from the nervous system, there can be no control of the rela- tive blood supply of different parts. In the intact animal the circula- tion is more or less distributed according to the particular needs of the different viscera, and such conditions obviously can not be simulated in a perfusion experiment. Another objection to perfusion experiments in general depends on the fact that the well-being of the organs in the intact animal is largely dependent on hormones conveyed to them from other organs. Such hormones are frequently quite labile in nature, and soon disappear from the perfusion fluid. Notwithstanding these objections, there can be no doubt that many of the functions of an organ are retained much longer than they would be if the organ were not perfused ; for example, the contractility of the muscle or the power of forming urea in the liver. Perfusion experiments are of value therefore when they yield positive results. Negative re- sults may indicate either that the organ does not perform the particular function that is being investigated or that it has lost this function as a result of partial death. That a perfused muscle retains its power of contraction does not necessarily indicate that it maintains all of its metabolic functions; neither does the fact that the liver forms urea prove that it is capable of performing its other functions. It is easy to show that the liver dies piecemeal; some functions, such as glycogen- formation, die early, while others, such as urea-formation, remain for a long time intact. The use of perfusion experiments in the investigation of problems of metabolism should always be very carefully controlled and the results should never constitute the only evidence upon which impor- tant conclusions are based. (3) Before leaving this subject it may be well to point out that the method which at first sight might appear to be the simplest for throwing light on such problems as that under consideration namely, the examina- tion of the inflowing and outflowing blood of different parts or organs is not applicable in most cases. This is because of the extremely small changes in concentration which may occur even although large amounts of the particular substance in question are being absorbed or produced. As we shall see later, this criticism is particularly applicable in the case of sugar. Even during the injection of considerable quantities of sugar into the portal vein, no difference in percentage can be demonstrated 654 METABOLISM between the blood of the two sides of the liver, although we know that sugar is being retained to form glycogen. For the same reasons, differ- ences in the percentage amounts of ammo acids or of urea are often dif- ficult to demonstrate in the blood entering and leaving the liver even when we know that large quantities of them are being added to or re- moved from it. Clinical. Since the liver is an important seat of urea formation, the question arises as to whether the relative percentage of urea and am- monia in the urine will become altered by disease of the liver. Many observations with this point in view have been undertaken, but it can not be said that the results are very striking. In extreme destruction, such as that produced by phosphorus poisoning, there may indeed be a great increase in the relative amount of ammonia and a decrease in that of urea. The same is true in acute yellow atrophy of the liver, in which disease the nitrogen excreted as ammonia may amount to as much as 70 per cent of that excreted as urea. In milder forms of liver dis- turbance, however, such as cirrhosis, the figures are much less striking. When an increased ammonia excretion is observed in such cases, we must be cautious in drawing the conclusion that it is due primarily to interference with the hepatic function. It may just as well be caused by the development of acids in the organism that require the ammonia for their neutralization. It is significant, for example, that considerable quantities of acids are produced in phosphorus poisoning. Although the urea and ammonia excretions become altered by exten- sive destruction of liver tissue, it is a remarkable fact that very little if any change occurs in the amino nitrogen, either of the urine or of the blood. In experimental necrosis of the liver caused by chloroform or by phosphorus, it is only in the latest stages of the condition and when it is of the very severest type that the amino acids have been found to increase in the blood and urine. The conditions seem to be some- what different in man, abnormally high amounts of amino nitrogen hav- ing been observed in the blood in a considerable proportion of patients with impaired liver function. In very severe cases of diabetes, for ex- ample, figures that are distinctly higher than normal have been observed (VanSlyke, etc.). In eclampsia the marked pathological changes in the liver might be expected to be associated with an upset in the metabo- lism of amino acids. Losee and Van Slyke 35 have, however, recently shown by the most accurate methods that neither in the blood nor in the urine is any excess of amino acids to be found in this condition, although in cases of pernicious vomiting of pregnancy, there was a relative in- crease in the ammonia excretion. We have already seen that this THE METABOLISM OF PROTEIN 655 increase did not bear any relationship to the acid-absorbing power of the blood plasma (see page 650). The importance of the kidneys in removing the urea from the blood is readily seen from the change in the percentage of urea in this fluid after the partial or complete removal of the kidneys. Animals sur- vive nephrectomy for about three days, and during this time urea rapidly accumulates in the blood and begins to make its appearance in the saliva and the intestinal secretions. In man also where the kidneys are extensively diseased, a similar accumulation of urea occurs in the blood, some of the excess being got rid of through the sweat and to a certain extent through the intestine. The importance of encouraging perspiration and a free movement of the bowels in cases of nephritis is thus indicated. It must not be concluded that the accumulation of urea in the organism is the direct cause of the symptoms. Urea itself is comparatively inert, and it is generally believed that other metabolic products with which the urea runs parallel in amount are the toxic agents. Hewlett has found, however, that very large injections of urea cause certain symptoms. 34 CHAPTER LXXII THE METABOLISM OF PROTEIN (Cont'd) CREATINE AND CREATININE Creatine and creatinine are very largely products of endogenous metab- olism; they are mainly derived from chemical processes occurring in the tissues although some of the creatine and creatinine present in the food may appear as creatine in the urine. Essential Chemical Facts Before we proceed to discuss the metabolism of these important substances, it will be necessary to refer briefly to some points in their chemistry. The simpler of the two bodies is creatine, which is methyl-guanidine-aeetic acid; creatinine is its anhydrid, being formed from creatine by the removal of a molecule of water, so that the NH 2 groups become joined together in the same way as they do in the formation of pep- tides from ammo acids (page 636). The relationships are illustrated in the following formulas : CH 3 -N-CH-CO (methyl) , CH 3 N / \ / CH..COOH NH = C NH C - H 2 = \ \ (acetic acid) \ \ \ i (guanidine) -NH., NH (creatine) (creatinine) It should be noted that guanidine is closely related to urea, 0:=C(NH 2 ) 2 , and that when creatinine is formed from creatine a ring formation occurs, giving what may be regarded as an imidazole derivative (see page 639). Creatine is also related to one of the important diamino acids, arginine, since both contain guanidine radicles, NH= 0(NH 2 ) 2 , and to histidine and the purines (see page 669), both of which contain the imidazole ring. The close relationship which creatine bears to urea is illustrated by the fact that urea is formed when creatine is subjected to the action of boiling barium hydrate. When it is oxidized by means of potassium permanganate, urea is also formed, the remainder of the molecule, more % or less intact, being split off as methyl- "NTTT f^TT amino-acetic acid (CH < ',-r 3 ), also known as sarcosine. The conversion of creatine to creatinine goes on slowly in aqueous solutions, but is much accelerated by heating with acid. Heated in an autoclave at a temperature of 117 C. for thirty minutes, with half normal hydrochloric acid, the creatine goes over almost quantitatively into creatinine. It will be noted that the creatinine ring is partly oxidized. This renders it unstable, so that creatinine in the presence of alkalies 656 THE METABOLISM OF PROTEIN 657 has the power of reducing metallic oxides. Like glucose it can reduce alkaline solu- tions of copper, silver and mercuric salts; it also reduces picric acid in weakly alkaline solution to picramic acid, which, being red, furnishes us with a solution the strength of which can be estimated colorimetrically. Quantitative Estimation. Although the presence of creatinine in the urine has been known for many years, there being from 1 to 2 grams of it in the twenty-four-hour urine, little progress was made in the study of its metabolism because of the absence of a reliable method for its estimation. The elaboration by Folin of a colorimetric quantitative method for creatinine, depending on the reduction of picric acid, has furnished the starting point for the modern work which has been done. To estimate the creatine by this method, it is usual to proceed as follows: The creatinine content is first of all determined, another portion of urine being then heated with acid in the autoclave until all of its creatine has been converted into creatinine. A second de- termination of creatinine is then made, and the difference between the two is calculated as creatine. It should be pointed out that, since the creatine is estimated by an indirect method, there are considerable chances for inaccuracy. Indeed, it has been shown that errors may have been incurred in some of the recent work on account of the fact that when aeetoacetic is present in the urine it prevents the creatinine from developing its full reducing power on picric acid in the cold, so that when subsequently the urine is heated with acid for the purpose of converting the creatine into creatinine, the destruction of aeetoacetic acid allows the reducing power of the creatinine to develop to full intensity. It is obvious that this would make it appear as if creatine had been converted into creatinine. It is particularly in the urine of diabetic patients, in which aeetoacetic acid is present that mistakes are likely to be made. Metabolism When we come to consider the metabolism of creatine and creatinine, we find that there are remarkably few facts definitely known concerning it. The average amount excreted daily, expressed as the number of milli- grams of creatinine in twenty-four hours per kilogram body weight, is known as the creatinine coefficient (Shaffer). 36 For a lean person this is about 25 mg. ; for a corpulent person, about 20 mg., the difference in- dicating that muscle mass, and not body weight, is the important factor determining the coefficient. Further evidence that this relationship ex- ists is furnished by the fact that in the muscular atrophies creatine ex- cretion is distinctly below normal. It must be the mass of the muscles rather than their activities that is the determining factor, for the creatine excretion does not become increased by muscular exercise. Influence of Food, Age, and Sex. Although creatine and creatinine are endogenous metabolites, it must be remembered that, under ordinary dietetic conditions, a part of each is derived from these substances pres- ent in the food. It is important therefore to consider the conditions under which the creatine and creatinine in the food appear in the urine. Regarding creatinine, it is pretty well established that practically all that is taken with the food reappears as creatinine in the urine. Shaffer I 658 METABOLISM has, for example, succeeded in recovering 76 per cent of ingested creat- inine in the urine excreted during twenty-one hours following the in- gestion of 0.7 gm. creatinine. The conditions for the excretion of creatine are more complex. It is present in the urine of children in considerable amount, but in that of adults, only as traces. In the first years of life the creatine in boys' urine may amount to one-half of the total creatine and creatinine, but it becomes gradually less and practically disappears at about seven years of age. Girls, on the other hand, continue to excrete creatine until about puberty, after which, although ordinarily absent, it reappears in the urine at each monthly sexual cycle, and is present during pregnancy fend for some days after delivery. Feeding creatine to children causes it to appear in the urine, accompanied usually by a slight increase in the creatinine. The same results can be observed in women during the monthly periods, when as much as 0.1 gm. may be present, and during pregnancy. Creatine is also present in the urine of most if not all of the other mammalia. Some of these facts are shown in the following table: AGE CREATININE-N CREATINE-N EXCRETED IN 24-HR. URINE 2 0.025 0.023 3 0.057 0.022 Boys 5 8 0.112 0.163 0.025 0.0 11 0.157 0.0 15 0.378 0.0 5 0.069 0.005 6 0.032 0.003 Girls 7 0.157 0.066 10 0.147 0.020 12 0.201 0.011 (From Mathews.) When creatine is given to an animal that has been kept in a starved condition, most of it seems to disappear. It can not be recovered in the urine either as creatine or as any other nitrogenous metabolite. It seems to functionate more as a food than as a useless substance. The possi- bility that some of it can be destroyed by the intestinal bacteria being admitted, there is nevertheless some justification for the view that the creatine finds a useful function in the anabolic process of the muscles. Influence of Complete and Partial Starvation. Although, as we have seen, the creatinine excretion remains constant when the amount of pro- tein in the diet is greatly reduced, yet it does not remain constant during complete fasting or when carbohydrates are entirely withheld from the diet. In fasting it has been found that creatine appears in place of the creatinine which has disappeared, so that if both creatine and creatinine THE METABOLISM OF PROTEIN 659 are determined, very little if any diminution will be found to have oc- curred. Fasting, therefore, causes the creatine and creatinine metabol- ism of the adult to become like that of the juvenile metabolism. As pointed out by Mathews, it would be interesting in the light of this ob- servation to see whether other substances, passed in the urine of young animals but absent in that of the adult, would reappear in the urine when the animals were made to fast. A similar replacement of some of the creatinine by creatine appears when carbohydrate is entirely withheld from the diet, or in diabetic animals, either in the disease diabetes mellitus in man or in the experi- mental condition induced in animals by giving phlorhizin. Unfortu- nately, in a considerable part of the work that has been done on this phase of the subject a method of estimation was employed which did not take sufficiently into account the influence of acetoacetic acid on the creatine estimation; but even after allowing for this possible source of error, there can be no doubt that creatine appears in the urine when carbohydrates are improperly metabolized. If carbohydrates are given to a starving animal, for example, the creatine is replaced in its urine by creatinine, although this will not occur when either protein or fat is fed. The general conclusion which may be drawn from these observations is that carbohydrates in some way are required for the proper conversion of creatine into creatinine in the animal body (Cathcart) 37 . Origin of Creatine and Creatinine Notwithstanding the large amount of excellent work that has recently been done on the metabolism of creatine' and creatinine, we know very little indeed regarding the origin of these bodies in the animal organism. It would be profitless to discuss this problem to any great extent, but a few of the most important facts so far established may be of interest and of value. The first step in attacking such a problem is to compare the amounts present in the various organs and tissues, in the blood, and in the excreta. Of the approximately 120 grams of creatine and creatinine in the body of an average adult, a very large proportion is in the muscles, the voluntary muscles containing the largest percentage, the heart con- taining a medium percentage, and the involuntary (intestinal) muscles containing relatively a small amount (Mye'rs and Fine) 38 . Next to the skeletal muscles, but containing more than the involuntary muscles, come the testes and brain. The liver, pancreas, thyroid, kidneys, spleen, etc., contain traces. The blood (human) contains about 1 mg. creatinine per 100 c.c. and about 3 mg. creatine, the former being equally distrib- uted between plasma and corpuscles, whereas the latter is contained mainly in the corpuscles. (Hunter and Campbell, 58 ) METABOLISM In all these places by far the greatest proportion of the total creatine- creatinine exists as creatine, which is exactly the reverse of the condi- tion obtaining in the urine of adults, where practically all is excreted as creatinine. The close chemical relationship between creatine and creat- inine, considered along with the above facts regarding their quantitative distribution in the body, indicates that the creatinine of the urine is de- rived from the creatine of the tissues. The question is, How does the creatine come to be converted into creatinine f Such a transformation is probably effected by many of the tissues of the body and certainly by the blood, the active agency in all cases being no doubt an enzyme. That the blood contains such an enzyme is indicated by the fact that creatine is transformed to creatinine by blood serum more quickly than it is when merely dissolved in water. Even heated blood serum possesses some of this power. The liver also probably brings about the transfor- mation, as has been shown by perfusion experiments, and by the fact that in cases of phosphorus or hydrazine poisoning creatine displaces creatinine in the urine. The problem therefore narrows itself down to the question of the origin of creatine. In the light of chemical knowledge there are several precursors from which creatine might be formed. One, for example, is arginine, which it will be remembered is guanidine-amino-valerianic acid (see page 640). By oxidation this might become changed into guani- dine-amino-acetic acid, which by methylation would then be changed into creatine. That such a process of methylation may actually occur in the animal body is definitely known, for it happens when such substances as pyridine or naphthalene are given with the food. They appear in the urine as methyl derivatives. The possibility of the derivation of creatine by methylation of arginine is suggested by the result of the injection into ducks of arginine, combined with such substances as paraformalde- hyde (Thompson 59 ). Even in this case however the results are not very convincing. The closely related substance, guanidine-acetic acid, when fed to animals (rabbits) also causes a slight increase in the excretion of creatine (Jaffe), and, it is said, an increase in the creatine content of the muscle. Even in this case, however, by far the largest proportion of the administered guanidine-acetic acid is excreted in the urine unchanged. The large percentage of creatine in muscle tissue leads one to expect that some relationship must exist between muscular metabolism and the amount of creatine present either as such in the muscles or as creatinine in the urine. Regarding the latter point it is definitely established that muscular exercise leads to no increase in the creatinine excretion, al- though it is said that such an increase occurs following a state of tonic muscular contraction. In the light of the fact already stated that there THE METABOLISM OF PROTEIN 661 is creatine in other organs than the muscles, it seems probable that the substance has really little to do with muscular contraction as such, but rather is concerned in some way in the formative metabolism of the cell, with its general growth or maintenance. Indeed, it is a question whether creatine is an actual constituent of the living tissue. It may rather, as has been suggested by Folin, be a postmortem product, represented dur- ing life by creatinine. Creatine appears in the urine in phosphorus poisoning, in carcinoma of the liver and during postpartum involution of the uterus. It is not de- rived from the disappearing uterine muscle, however, for creatinuria also occurs after cesarean section with removal of the uterus. Creatine elimination is not an index of cellular destruction. Muscular fatigue also leaves the creatine content of muscle unchanged. In late stages of nephritis, creatinine accumulates in the blood and serves as an index of the gravity of the condition (page 683). CHAPTER LXXIII THE METABOLISM OF PROTEIN (Cont'd) UNDETERMINED NITROGEN AND DETOXICATION COMPOUNDS In the present chapter we shall refer briefly to the groups of urinary substances styled undetermined nitrogenous compounds and to the com- pounds that are excreted in the urine as the result of the combination in the body of certain toxic bodies with chemical substances that render them harmless (detoxication compounds). Undetermined Nitrogen Included under undetermined nitrogen are amino acids, peptides and basic substances. The amount of amino acids and peptides in normal urine is very small but may become considerable in disease, especially of the liver, when leucine and tyrosine may appear. The presence of traces of amino acid and peptone in normal urine is to be expected, for although the actual concentration of amino acids in the blood is never very great, a certain leakage of amino acids must occur into the urine. The peptide is sometimes known as oxyproteic acid. It becomes dis- tinctly increased in phosphorus poisoning and in such conditions as are accompanied by excessive protein metabolism. The basic constituents include such substances as trimethylamine, ethylamine, putrescine and cadaverine (page 536), and there are probably many more of a similar nature. Many of these substances are similar to the so-called ptomaines found in meat, etc., and they have been called the ptomaines of urine, from which they can be isolated by rendering the urine alkaline and shaking out with ether. It is probably to the presence of these sub- stances that urine mainly owes its toxic action. The Detoxication Compounds Certain nocuous substances are produced in the intestine during the digestive process (see page 535), and others may result from the meta- lic processes in the tissues. To guard against the harmful action of these substances on the organism, they become detoxicated in various 662 THE METABOLISM OF PROTEIN 663 ways, mainly by forming inert compounds with other substances, par- ticularly with glycocoll, sulphuric acid or glycuronic acid. The com- pound thus formed is then excreted in the urine. Hippuric Acid, Glycocoll is used mainly to detoxicate the benzoic acid which results from the oxidation of the aromatic substances pres- ent in large quantities in vegetable food and fruit (particularly in cran- berries). Some benzoic acid may also be produced by the breakdown of the aromatic group of the protein molecule; phenylalanine, for ex- ample, gives rise to benzoic acid by bacterial decomposition. The com- pound formed is hippuric acid, this name indicating that it is present in large quantities in the urine of the horse, as it is also in the urine of all herbivorous animals. Hippuric acid is benzoyl-glycine (C 6 H 5 .CO.NH.CH 2 COOH), and it can readily be produced in the laboratory by bringing together benzoyl chloride with glycocoll, thus: C 6 H 5 .CO : Cl + H HN . CH 2 COOH = C 6 H 5 CO . NH . CH 2 COOH + HC1. (benzoyl chloride) (glycocoll) (hippuric acid) Under ordinary dietetic conditions only a trace of hippuric acid is present in the urine of man, but much larger quantities, 2 grams a day for example, may appear when the diet contains a large proportion of fruit or vegetables. It is not known to undergo any characteristic varia- tions in disease. The benzoic acid which is contained in certain canned foods as preservative also combines in the body with glycocoll, so that any toxic effect which it might produce is practically negligible. There is certainly no very evident reason why canned foods containing benzoic acid should be tabooed, for in so far as the benzoic acid is concerned, they can be no more toxic than a diet composed largely of vegetables and fruit. This detoxication of benzoic acid requires the presence in the organ- ism of a constant supply of glycocoll, which, it will be recalled, is the lowest in the series of amino acids, being aminoacetic acid (CH 2 NH 2 COOH). It is present in greatest amount in the protein of the connective tissues. It is said, however, that not more than from 2 to 3.5 per cent of glycocoll is available in the proteins of the body. Al- though this amount of glycocoll would amply suffice to detoxicate the benzoic acid produced by the metabolism of the food in carnivora, it is quite inadequate for this purpose in the case of herbivora, and the question naturally presents itself as to where the glycocoll in these animals comes from. It is said, for example, that of the total nitrogen excretion in herbivora 50 per cent may appear as glycocoll under cer- tain conditions. These facts along with those gained by observations on 664 METABOLISM the growth curves (see page 610) indicate that the organism is capable of producing new glycocoll for itself, and it is interesting to consider how this glycocoll may be derived. A very probable source is by synthesis between ammonia and glyoxylic acid (CHO. COOH). That glyoxylic acid or its aldehyde, glyoxal, is readily produced during metabolism from car- bohydrates and that ammonia is always available would seem to lend some support to this view (see page 698). The synthesis of glycocoll from glyoxal and ammonia occurs thus: H.COCHO + NH 3 CH,NH 2 COOH. (glyoxal) (glycocoll) The linking up of glycocoll with benzoic acid occurs in various organs, particularly the kidneys and the liver. An isolated perfused preparation of the kidney produces hippuric acid provided benzoic acid is added to the perfusion fluid, and the latter also contains an abundance of oxy- gen, which is best secured by using defibrinated arterialized blood in- stead of artificial serum (Locke's solution). The necessity of a plentiful supply of oxygen is further shown by the fact that, if the hemoglobin of the blood is rendered incapable of carrying 2 by bubbling carbon mon- oxide gas through it, no synthesis of hippuric acid will result from per- fusing the blood through the kidney. The kidneys are not the only site of this synthesis, since hippuric acid is still formed after nephrectomy, in both carnivorous and herbivorous animals. It has been isolated from the liver of nephrectomised dogs after injection of glycocoll and benzoic acid (Kingsbury and Bell 60 ) ; and after damaging the liver cells by poi- soning with hydrazin, in dogs, it has been found that the excretion of hippuric acid falls decidedly. Hydrazin does not act on the kidney cells. 61 The actual chemical process by which the synthesis occurs (de- hydration) is similar to that by which polypeptides are formed by the union of amino acids, or creatinine from creatine. (C 6 H 5 co ;OH+H; HNCH 2 coon). Glycocoll may be used for detoxicating other substances than benzoic acid, particularly cholic acid, forming the glycocholic acid of the bile (see page 528) and phenylacetic acid. In birds the benzoic acid be- comes combined with diamino-valerianic acid or orni thine (NH 2 - CH 2 - CH 2 -CH 2 -CH-NH 2 -COOH) in place of glycocoll, so that in the urine of these animals in place of hippuric acid a compound called ornithuric acid occurs. It is of importance to point out here that this pairing of aromatic toxic substances with certain of the metabolic products of the organism has THE METABOLISM OF PROTEIN 665 frequently been found an excellent experimental method for demon- strating the presence of intermediary metabolic substances that other- wise would not have appeared in the excreta. These substances are thus diverted from their normal course in metabolism so as to form neutralization or detoxication compounds. Glycuronic acid is an example. Ethereal Sulphates and Glycuronates. The other substances used for detoxication purposes are sulphuric and glycuronic acids. Phenol, and its derivative cresol, after being absorbed from the intestine, in the contents of which they are produced by the bacterial decomposition of protein (see page 535) become combined in the body, probably in the liver, with sulphuric acid or with glycuronic acid to form the sulphate or glycuronate. The aromatic sulphate further combines with potassium to form the so-called ethereal sulphates, as which the substance is excreted in the urine. A small amount of phenol may however appear in the urine unchanged. As we have already seen, the sources of the phenol in the intestine are tyrosine and phenylalanine (see page 564), and since these ammo acids are also present in the tissues, it might be sup- posed that some of the phenol sulphate of potassium present in the urine could come from the tissues. It is usually assumed, however, that derivation from the tissues does not occur. Another ethereal sulphate is indoxyl sulphate of potassium, which re- sults from the absorption into the blood of the indole and skatole pro- duced by intestinal putrefaction from tryptophane (see page 536). Immediately after absorption indole is oxidized to indoxyl, which then combines with sulphuric acid and with potassium to form indoxyl sul- phate of potassium, which is the well-known indican of the urine. As in the case of phenol sulphate of potassium, none of the urinary indican seems to come from the normal metabolism (of the tryptophane) of the tissue proteins. It is a much more reliable indicator of the extent of intes- tinal putrefaction than is phenol sulphate of potassium, but it also becomes increased in amount during putrefaction in the body itself, as for example in abscess formation. The amount of indican in the urine may be roughly gauged by oxi- dizing the urine by means of hypochlorite and then shaking out with phloroform. If the resulting extract is more than light blue in color, it indicates excessive putrefaction. A negative test does not neces- sarily mean that intestinal putrefaction is absent, but a markedly positive test always indicates that it is occurring. Skatole, the methyl deriva- tive of indole, may undergo similar processes and appear in the urine during excessive intestinal putrefaction. Its presence in the blood some- times confers on the breath a distinctly fecal odor, for this body, as its name indicates, is that to which the odor of the feces is due. 666 METABOLISM Glycuronic acid, the other substance used for detoxication processes, is of the nature of a dextrose molecule with the one end-group oxidized to carboxyl (CHO- (CHOH) 4 - COOH). It is probably produced under normal processes of metabolism in the animal body, but is destroyed except when such poisonous substances as camphor, chloral hydrate or certain aromatic alcohols are given, when it is used for the purpose of detoxicating them. The resulting glycuronates have reducing powers and may be confused with glucose when present in large amount. Gly- curonates may be distinguished from glucose in the urine (1) because they are levorotatory, and (2) because they do not ferment. The free acid itself, however, is dextrorotatory. CHAPTER LXXIV URIC ACID AND THE PURINE BODIES Introductory. The participation by highly trained organic chemists in the investigation of biochemical problems has brought our knowledge of the history of the purine substances in the animal body from a state of chaos and guesswork to one of system and scientific accuracy. The peculiar solubility reactions of uric acid and its salts and the discovery of urates in gouty deposits served to make uric acid metabolism one of the earliest research problems in both the medical clinic and the bio- chemical laboratory, but the earlier results were practically valueless, partly because they were inaccurate and partly because their interpretation was impossible in the absence of even the most elementary facts concerning the chemistry of uric acid. Before any real progress was possible, a clean sweep had to be made of all the old speculations and hypotheses, such as that dignified by the high-sounding name of " uric-acid diathesis," and a foundation of ac- curate chemical knowledge established. This foundation is now wonder- fully complete, and a superstructure of biochemical fact is already beginning to grow upon it. In the present chapter we shall examine some of the most important contributions that have made this progress possible, As in the study of any other problem of metabolism, we must, however, make ourselves familiar with the main facts concerning the chemistry of the purine bodies and of the tissue constituents into the composition of which they enter, before proceeding to the more strictly biological aspect of the subject. The Chemical Nature of the Purines By an examination of the empirical formulas of the purines of biochemical interest, it will be observed that they are all derivatives of a substance purine, which although in itself of no importance is interesting, since it serves as the basic substance from which the others are derived. The list is as follows: Purine . C 6 H 4 N 4 Hypoxanthine C 5 H 4 N 4 O Monoxypurine f Adenine . C 5 H 3 N 4 .NH 2 Ammo-purine I Purine Xanthine . C 5 H 4 N 4 O 2 Dioxypurine 1 bases. Guanine . C 5 H 3 N 4 O.NH 2 Amino-oxypurine 1 ' Uric acid . C 5 H 4 N 4 O 3 Trioxypurine The first oxidation product of purine is hypoxanthine, which has long been known as a constituent of meat extract. Adenine, the amino derivative qf hypoxanthine, 667 668 METABOLISM occurs iii combination with other substances in the nuclear material. The second oxi- dation product is xanthine and its amino derivative, guanine. They occur in the same places as hypoxanthine and adenine. The highest oxidation product of all is the well- known urinary constituent, uric acid, which may therefore be chemically designated as trioxypurine. In addition to the purines of animal origin, there are also certain ones of vegetable origin the methyl purines, which exist as the alkaloids of tea and coffee namely, caffeine, theobromine, and theine. To understand the chemical structure of this group of substances, it is perhaps simplest to start with that of uric acid. This consists essentially of two urea molecules linked together by a central chain of three carbon atoms, as will be evident from the accompanying structural formula : HN-CO -NH I I! \ CO HN-C-NH (urea) (urea) (central chain) This structure can be shown by methods both of decomposition and of synthesis. When uric acid is decomposed by oxidizing it with nitric acid, it yields urea and a residue called alloxan; or it can be synthetized from urea and trichlorlactamide, a derivative of lactic acid, w^hich it will be remembered contains three carbon atoms. The changes involved in this synthesis will be made clear by examination of the ac- companying structural formula, in which the manner of production of the by-products of the reaction (NH , H o O and HC1) are shown by dotted lines: NH. ! H NH.. : - C = O CO \ \ \ \ (urea) NH. H Cl I -C | OH H NH \ CO C- ; Cl H NH (urea) (trichlorlactamide) By milder oxidation by means of potassium permanganate in the cold, uric acid be- comes quantitatively converted to allantoine: C S H 4 N 4 3 + H 2 + O :=C 4 H 6 N 4 3 + CO 2 (uric acid) (allantoine) The importance of this transformation lies in the fact that in most animals, man and the higher apes being exceptions, uric acid is thus decomposed in the animal body. The structural formulas for the other purine bodies in relationship with those of pnrine and uric acid are given below. URIC ACID AND THE PURINE BODIES Purine itself has the following structural formula: H2 - H S - NH? \ 1- C8-H ( For convenience of description the atoms in purine are numbered as shown.) HN-C=O I I H-C C-NH HN-C=O 0=0 C-NH \ / C-H N-C- N (hypoxanthine) (6-oxypurine) N = C-NH 2 I HN-C- (xanthine) \ / C-H (2-6-oxypurine) I I H-C C-NH I HN-C=0 I I H,N = C-NH C-H N-C - X / N-C - N \ / C-H (adenine) (6-amino-purine) (guanine) (2-amino-6-oxypurine) HN-CO 00 I C-NH \ CO HN- C -NH Uric acid (2-6-8-trioxypurine) The substances with which the purine bases are most closely related are the pyrir inidine bases. Three of these are known: thymine (NH-CO CO C.CH 8 I !! NH-CH cytosine ( N = C-NH 2 I CO CH I II- NH-CH) ; and uracil (NH-CO I I CO .CH NH-CH). From an examination of the structural formulas, it will be seen that they are more or less related to purine (having one of the urea radicles omitted), although it can scarcely be doubted that they exist as separate constituents of the nucleic acid group in the animal body, and are not derived from purine. They are primary products. The Chemical Nature of the Substances in Which Purine and Pyrimidine Bases Exist in the Animal Body. In general it may be said that the amino purines adenine and guanine together with the pyrimidine bases thymine and cytosine occur combined with phos- phoric acid and a carbohydrate in the various nucleic acids, each of which 670 METABOLISM is again combined with some simple protein to form nuclein, the essen- tial constituent of the chromatin of the nucleus. One of the oxypurines, hypoxanthine, may also exist combined with phosphoric acid and carbo- hydrate to form a substance present in muscle and known as inosinic acid. The simplest form of nucleic acid is that known as guanylic, which is found in certain organs (liver, pancreas, etc.) side by side with the more complex variety. It consists of phosphoric acid, a pentose (5 C- atom sugar) and the amino purine, guanine. The pentose which can be detected in these organs is apparently derived solely from guanylic acid. The more complex form of nucleic acid, and probably that present in all nuclei, is composed of phosphoric acid, a hexose (in animal cells) or a pentose (in vegetable cells), the two amino purines, adenine and guanine, and the two pyrimidine bases, cytosine and thymine. I. Nucleic acid may therefore be considered as a compound of polyphos- Vbhoric acid, containing carbohydrate groups, which serve to link the phos- phoric acid molecules to those of purine and pyrimidine. It has been found necessary to introduce certain terms to designate the different parts of the nucleic acid molecule ; thus, the whole molecule is called a tetranucleotide, composed of four mononucleotide molecules, each of which consists of a phosphoric acid molecule plus a micleoside y which again is composed of a purine or pyrimidine nucleus attached to pentose or hexose. The nucleoside is so named because it is similar in structure to a glucoside. Apart from differences in the carbohydrate group, it appears that there is a close similarity in the structures of nucleic acids from dif- ferent cells. This would indicate a common function for them all, which may be either of a structural or of a physiological nature ; that is, nucleic acid may have to do with the sustentacular material that builds the nucleus, or it may have to do with some physiological function common to all cells, such as irritability, or growth, or respiration. If nucleic acid is merely a sustentacular material, then the study of the behavior of chromosomes and chromatine in cells can not have the significance that it would have were nucleic acid concerned in the more vital activ- ities of the nucleus. All the so-called nuclear stains owe their specific staining properties to the fact that they are of a basic nature and com- bine with nucleic acid. Until we know more definitely what the exact function of nucleic acid may be, it is unwise to place too much weight on the behavior of the chromosomes in cytologic researches. By studying the behavior of cells (ameba) from which the nucleus was removed, it has been found that the process of reproduction and growth alone are affected. The other functions proceed normally. In URIC ACID AND THE PURINE BODIES 671 this connection it is interesting to note that much evidence is accumulat- ing to show that the respiratory functions of cells are linked up with the presence in the cytoplasm of bodies called mitochondria composed of phospholipin and protein. (Lynch. 61 ) The History of Nucleic Acid in the Animal Body. We shall first of all study the manner in which nucleic acid may be broken down. As is to be expected from its complex structure, various types of enzymes are concerned in this process. The first to act are known as the nucle- ases. They split the tetranucleotide molecule into two dinucleotides, which immediately afterward split further into mononucleotides. Four nucleotides, two of purine and two of pyrimidine, are thus formed from each molecule of nucleic acid. Each nucleotide molecule may now un- dergo decomposition in one of two w^ays: (1) either by the splitting off of phosphoric acid, leaving a nucleoside (guanosine or adenosine), or (2) by the splitting off of both phosphoric acid and carbohydrate, leaving free purine bases. Nucleases have been found which specifically effect either of these decompositions, and they have been called phospho- nucleases* (1), and purine-nucleases (2), respectively. In the decompo- sition of nucleic acid all of the four purine compounds guanine, guano- sine, adenosine and adenine may be formed. This is illustrated in the accompanying schema, in which the nucleic acid is represented as a purine nucleotide: NudeicAcid (without the pyrimidine group) W^ *(2) ases) \ \ \ denosine 8 > Adeni (1) (Action of nucteases) Guanine 4-(7) Guanosine Adenosine (8) > Adenine (4) (5) (6) (Action of deaminiaing enzymes) >Jr ^ Xanthosine Inosine (9) (Action of hydrolyzing enzymes) (10) "*i . Uric Acid*- ( 11 ) Xanthine < - ( 11 ) - - -+ Hypoxanthine (Action of xanthine oxidase) (Jones.) The next step in the disintegration process is that the amino group is removed and the corresponding oxypurine is produced. To bring this | about, there exists a specific deaminizing enzyme for each of the above amino compounds, and each enzyme is named according to the exact amino purine upon which it acts; thus, guanase (3), guanosine-deaminase (4), adenosine-deaminase (5), and adenase (6) have all been identified. *The numbers refer to the enzymes indicated in the schema. 672 METABOLISM The free base may then be split off from the nucleosides by specific hydrolyzing enzymes (7) (8) (9 1 ) (10). The joint action of these enzymes leads to the formation of oxypurines, xanthine and hypoxanthine, which are oxidized to uric acid by xanthine- oxidase (11). In man and the anthropoid apes uric acid is the end product of the above changes, but in other mammals most of the uric acid is further oxidized into allantoine. It has also been found, except in man and the chimpanzee, that extracts of organs such as the liver, are capable of decomposing uric acid into allantoine. The identification of these specific enzymes is sought by a determination of the free amino-purine bases and the phosphoric acid produced by allowing an aqueous extract of the tissue in question to act on nucleic acid (of yeast)* at body tempera- ture. Another portion of the digested mixture is then hydrolyzed by means of boiling sulphuric acid and the constituents again determined. From the results it is often possible to draw conclusions as to the exact nature of the enzymes present. The most remarkable outcome of this work has been to show that the distribution of the enzymes is not the same in the tissues and organs of different animals. Very briefly, some of the most important results that have so far been obtained are as follows: Gastric and pancreatic juices do not contain a trace of any of the enzymes. Intestinal juice, on the other hand, contains a nuclease capable of splitting the poly- nucleotides into mononucleotides. The two pyrimidiiie nucleotides split off do not undergo further change, but the purine nucleotides are con- verted into nucleosides (the enzyme being designated "nucleotidase"). Extract of the intestinal mucosa, besides having the same action as the intestinal juice, can also decompose the purine, but not the pyrimidine nucleosides, into carbohydrate and purine groups (specific action of "nucleosidase"). A similar action is produced by extracts of kidney, heart muscle, and liver. Blood serum, hemolyzed blood, and extract of pancreas, on the other hand, are capable of carrying the decomposition only as far as the mononucleotides. Regarding the other enzymes mentioned in the above list, it is im- portant to note that they appear at different stages in embryonic develop- ment, and that their distribution varies considerably in different species of adult animal, the spleen, liver, thymus, and pancreas containing them most abundantly. The distribution of enzymes in the organs of the monkey resembles that in the lower animals considerably more than it does that in man. Some remarkable facts have come to light regarding guanase and adenase, particularly that guanase is deficient in the organs *Yeast nucleic acid is used because it is less resistant to disintegration than thymic nucleic acid. URIC ACID AND THE PURINE BODIES 673 of the pig, in the urine of which animal it has also been found that the purine bases are in excess of the uric acid. This absence of guanase no doubt accounts for the fact that deposits of guanine may occur in the muscles, and that these may be so large as to constitute the condition known as guanine gout found in this animal. Adenase, on the other hand, is absent from the organs of the rat, which again corresponds with the fact that, when adenine is injected subcutaneously into these ani- mals, it undergoes oxidation without the removal of its amino group. In the human organism, adenase appears to be absent from all of the organs, whereas guanase is present in the kidney, lung and liver, but not in the pancreas or spleen. Xanthine-oxidase exists only in the liver. The distribution of uricase is perhaps the most interesting. It is pres- ent in most of the lower animals. On account of its presence extracts of the liver, spleen, etc., in all breeds of dogs, with the exception of Dalmatians, rapidly destroy uric acid; and practically no uric acid when injected subcutaneously can be recovered unchanged in the urine, but appears as allantoine. Uricase, however, is absent in man. This has been demonstrated by finding (1) that when uric acid is injected sub- cutaneously, nearly all of it reappears in the urine, and (2) that uric acid is not destroyed when extracts of the organs are incubated with uric acid or its precursors at body temperature. It must of course be kept in mind that, although the uric acid is thus shown not to be destroyed in vitro, it may nevertheless be destroyed in the living animal. The importance of the above described results rests in the fact that from them we may hope to be able, ultimately, to state exactly in what rgans and tissues the intermediary metabolic processes concerned in ucleic acid metabolism occur. The work at the present time is of spe- cial significance, since it represents one type of evidence which we must have before we can trace exactly every step in the metabolism of any ther biochemical substance. The absence of uricase from the tissues of man places him in a unique osition with regard to the metabolism of nucleic acid, and renders the vestigation of the problem particularly difficult, since investigations n the usual laboratory animals are useless. Recently, however, S. R. enedict has discovered that the Dalmatian breed of dog also known as the carriage dog, and having a spotted or mottled skin has a purine metabolism like that of man. 4 When fed on food containing no purine substances, a dog of this breed excretes large quantities of uric acid, and when the latter substance is injected subcutaneously, it is eliminated quantitatively as such in the urine. We shall see later how experiments on this animal have been made use of in the investigations of problems of purine metabolism as applied to man. In all other animals most of the 674 METABOLISM uric acid is oxidized to allantoine before being excreted. The degree to which this occurs varies between 79 and 98 per cent of the uric acid in different species. This has been called the uricolytic index (Hunter and Givens 42 ) . The Balance between Intake and Output of Purine Substances under Various Physiological and Pathological Conditions. The main purine ex- cretory product in man is uric acid, but there is also a certain amount of purine bases. The presence of uric acid in urine has attracted at- tention for decades in medical investigation, because of the relative ease with which it can approximately be determined quantitatively, and because of the well-known fact that it may be responsible for certain diseases, such as gout, when it accumulates in the tissues in an insoluble form. On a diet containing meat, or more particularly on one con- taining glandular substances, the total daily excretion of uric acid is very considerably greater than w r hen the diet contains no such food stuffs. The conclusion which Burian and Schur 43 drew from this ob- servation is that purine. must be partly of exogenous and partly of endogenous origin. In other words, some of it is derived more or less directly from preformed purine substances in the food, and the remain- der from the purine constituents of the animal's own tissues. Endogenous Purines. It was thought that a definite proportion of each of the administered purines could be invariably recovered from the urine. Although this has not been found to be exactly true, there is nevertheless a certain constancy in the proportion of administered purine that is excreted. Thus, Mendel and Lyman have found recently ithat about 60 per cent of injected hypoxanthine, 50 per cent of xan- thine, 19-30 per cent of guanosine, and 30-37 per cent of adenine are eliminated in the urine as uric acid. When combined purines i. e., nu- clear material are given, only a small proportion of the purine thus reappears. There is, therefore, a general parallelism between the purine content of the food and that of the urine, w r hich indicates that purine- rich food ought to be eliminated from the diet of patients who are suffering from deposition of insoluble urate in the tissues, as in gout. The fate of the purine that disappears in the body is unknown; some of it may be decomposed in the intestine, but why so much of the remainder should disappear, after absorption by the blood, is a mystery, since no uricase can be discovered in any of the organs or tissues. The destroyed purines can not be shown to influence any of the other well-known nitrogenous metabolites of the urine. The following table of experiments by Taylor and Eose 45 may serve /to illustrate these points. The subject was placed on a purine-free diet consisting of milk, eggs, starch and sugar, for three days. After this URIC ACID AND THE PURINE BODIES 675 period a part of the total nitrogen (3 grams) was supplied as sweet- breads thynms gland, etc. containing a high percentage (0.482) of purine nitrogen; for another period of four days still more of the nitro- gen (6 grams) was replaced by sweetbread nitrogen; and this was fol- lowed by a final period in which the original diet of milk, etc., without purine substances, was restored. The following table gives the results: 1ST PERIOD PURINE-FREE DIET 2ND PERIOD 3RD PERIOD 4TH PERIOD PURINE-FREE DIET Total urinary 1ST Urea N and NH 2 Creatinine Purine N (total) Uric acid N Eemainder N 8.9 7.3 0.58 0.11 0.09 0.91 8.7 7.1 0.55 0.17 0.14 0.88 9.1 7.1 0.56 0.26 0.24 1.18 8.8 7.05 0.47 0.10 0.07 1.18 The increase of uric acid accounted for less than half of the purine nitrogen ingested. This appeared as uric acid, the excretion of purine bases being practically unchanged. CHAPTER LXXV URIC ACID AND THE PURINE BODIES (Cont'd) SOURCE OF ENDOGENOUS PURINES Even after the entire elimination of all purine substances from the food in the case of man, purine continues to be excreted in the urine as uric acid. This, as above remarked, is called endogenous excretion. At first it was thought by Burian and Schur that the total nitrogen of the purine-free diet could be considerably varied without causing any alteration in the amount of the endogenous purine excretion, but a rep- etition of the work has shown that, when these changes are of consider- able magnitude, the endogenous moiety does not remain constant. This has already been demonstrated in the table on Folin's results (see page 648), and is still better illustrated in the accompanying table, which shows the excretion of uric acid and coincidently of urea from hour to hour in the urine after taking food which is free from nuclein or purine substances. After a fast of six hours, a diet consisting of bread and potatoes was taken at 1:30, and the urea and uric acid measured in the urine each hour thereafter. TIME UREA GM. URIC ACID MG. AMOUNT OF URINE C.C. 10-11 1.07 26 175 11-12 1.13 27 118 12-1 P.M. 1.07 24 164 1-2 (meal) 0.64 21 60 2-3 1.12 22 43 3-4 1.16 38 41 4-5 0.84 40 53 5-6 1.16 56 59 6-7 1.20 39 56 7-8 1.37 30 95 8-9 1.47 33 183 9-10 1.33 24 155 10-11 1.33 23 180 (Hopkins and Hope.) 46 A postprandial increase of endogenous purine excretion is very dis- tinct, and it indicates that during the process of assimilation something must be occurring in the organism which entails the production of purine from the organism itself. As to what this may be, it is impossible to say. It may be associated with the work of the gastric and intestinal glands, which recalls the interesting suggestion, originally made by 676 URIC ACID AND THE PURINE BODIES 677 Horbaczewski, that ingested substances increase the excretion of uric acid by causing a leucocytosis, the purine being derived from the nucleic acid set free when the leucocytes become broken down. That this is not the correct explanation, however, is indicated by the fact that in- gested substances that give rise to an increased number of leucocytes affect the excretion of uric acid during the period the leucocytes are present in the blood, and not after they have disappeared, which would be expected to be the case were the uric acid a product of purine substances liberated by their breakdown. This would indicate that the purine sub- stance is a metabolic product of the living leucocytes and not a break- down product of those that are dead. It should be noted that the increase in the postprandial uric-acid excretion occurs earlier than that of urea. The most pressing question concerns the origin of the endogenous purines. Uric acid is the purine with which we are most concerned in the case of man, and chemistry shows us that it may be produced either by a synthesis of two urea molecules with a carbon residue containing three carbon atoms, or by the oxidation of the lower purines namely, of those which are the constituent parts of the nucleic-acid molecule. There are consequently two sources from which the endogenous purine excretion in man may be derived : (1) synthesis of two urea molecules, and (2) oxidation of the lower purines. We will consider first the possibility of synthesis. In birds and reptiles practically all the nitrogen is excreted in the form of uric acid, and it is easy to show that this has been produced in the organism mainly in the liver by the synthesis of urea with carbon-rich resi- dues. Minkowski found that by removing the liver from geese, which is a comparatively simple operation on account of an anastomotic vein between the portal and the renal veins, the uric acid in the urine became very markedly decreased and ammonium lactate took its place (page 651). Since we know that ammonium in the animal body is ordinarily converted into urea, we may conclude from this observation that some- thing has occurred to prevent the synthesis of urea into uric acid. In confirmation of this conclusion it was subsequently found that, if am- monium lactate was added to the blood perfused through the isolated liver of the goose, uric acid accumulated in the perfusion fluid. Fur- thermore, when birds and reptiles are fed with ammonium salts or with the degradation products of protein, there is an increase in the ex- cretion of uric acid instead of urea. Everything which in a mammal tends to cause an increase in urea excretion causes in birds and reptiles a similar increase in the excretion of uric acid.* *The reason for the formation of this relatively insoluble metabolite in place of the soluble urea is connected in some way with the fact that birds and reptiles do not take such large quantities of water with their food as other animals. 678 METABOLISM In the early days of research in the uric-acid problem, not inconsid- erable mistakes were made on account of failure to recognize the essen- tial difference in the metabolism of uric acid in birds and mammals, and the tendency for some time after the exact state of affairs was discovered was to consider that in mammals none of this synthetic proc- ess occurs. The latter view, however, is surely incorrect, for a cer- tain amount not only of uric acid itself but of the lower purine bodies can be produced by synthesis in the mammalian body. Thus, Ascoli and Izar 47 discovered that uric acid could be made either to disappear or to be formed when a minced preparation of liver was incubated, the ex- act result depending upon whether the incubation was conducted in the presence of oxygen or of carbon dioxide. With oxygen uric acid disap- peared, whereas with carbon dioxide uric acid accumulated, indicating that in the presence of this gas the destroyed uric acid became reformed from the disintegration products of the oxygenation process. As similar results were obtained from the livers of birds, it is clear that no essential difference exists between the purine metabolic processes occurring in the livers of birds and of mammals. The difference is a quantitative not a qualitative one. Eegarding the chemical nature of the product into which uric acid is broken down and from which it may be resynthetized, it has been pos- sible so far to identify but one substance namely, dialuric acid. This is a perplexing result, for from all other investigations it would appear that in mammals, with the exception of man and the anthropoid apes, uricase splits uric acid into allantoine (see page 673), which substance, however, when added to liver extract does not cause any uric acid to be formed; nor do any of the other known decomposition products of uric acid have such a result. The chemical reaction involved in the produc- tion of uric acid from dialuric acid and urea is indicated as follows: NH C = / \ O = C C I H.OH H ! NH \ 4 = NH c; = o H i NH (dialuric acid ) ( urea ) The synthesis of uric acid is brought about by the combined action of a thermolabile enzyme in the blood and a thermostable body in the tissues. An aqueous extract of blood-free liver of the dog can destroy uric acid only in the presence of oxygen; it can not reform it even in URIC ACID AND THE PURINE BODIES 679 the presence of carbon dioxide. On the other hand, blood serum can not reform uric acid, whereas a mixture of the bloodless liver extract and blood serum produces uric acid readily under suitable conditions. Boiling of the liver extract does not affect the result, but boiling of the blood serum renders it incapable of exerting its joint action with the bloodless liver extract. These experiments with dog's liver serve only as circumstantial evi- dence that uric-acid synthesis occurs in mammals as well as in birds. More direct proof that purine synthesis occurs in mammals is as follows: (1) It was discovered long ago by Miescher that salmon, after leaving the sea to ascend the rivers in order to spawn, have a well-developed muscu- lar system, but that in the upper reaches of the stream the muscular system becomes considerably atrophied and the testes enormously developed. As the fish takes no food during the migration, there must be conversion of the protein of the muscles into the cellular tissue of the sexual glands, and nucleic acid must be produced. (2) A hen's egg before its incuba- tion contains practically no nucleic acid, whereas after development has well started nucleic acid increases by leaps and bounds. Similarly the eggs of insects increase in purine content very markedly as development proceeds. (3) Milk contains practically no purine derivative, and yet when it is fed to young growing animals, the organs lay on purine sub- stances abundantly. In general, indeed, it may be said that the combined purine increase is in proportion to the increase in body weight on the milk diet. (4) In Osborne and Mendel's experiments already alluded to, it has been shown that adequate growth depends primarily on the nature of the protein building stones, and not upon the purine content of the food. (5) An objection might be raised to these results on the score that they do not apply to the adult mammal. Investigation of the problem has hitherto been seriously impeded by the fact that no or- dinary laboratory animals were known in which uric acid is excreted in the urine. The discovery that this occurs in the Dalmatian dog has, however, made it possible for S. E. Benedict 41 to show, not only that after increasing the amount of nonpurine food there was a very distinct increase in the uric-acid excretion, but also that when the animal was kept for a year on such foods there was excreted a total amount of uric acid which was at least ten times greater than could have come from the traces unavoidablv included in the food. Eegarding the chemical nature of the substance from which the purine is synthe- tized, we know at present practically nothing. No doubt some of the protein build- ing stones functionate in this capacity, pyrimidine being probably the product that is first formed. Thus, pyrimidine may be produced as a result of the combination of amino-malonic acid with urea, the amino-malonic acid being produced by condensa- tion of hydrocyanic-acid molecules: 080 METABOLISM 3 HCN -^ H 2 N - CH ( CN) 2 + CO ( NH ) 2 - NH - CO CO CNH 2 ! II NH - CNH 2 (hydrocyanic (amino-malonic (urea) (oxy-diamino-pyrimidine) acid) nitrile) Another possible source of pyrimidine is the oxidation of arginine to guanidine-pro- pionic acid, which then condenses to form amino pyrimidine. Purine synthesis undoubtedly occurs in the mammalian body, but it is not easy to recognize for its occurrence is difficult to detect in metab- olism investigations; it is a slow, continuous process. The probabil- ity of its occurrence, however, is indicated by such results as those described on page 648, in which increase in purine excretion is observed after varying the intake of food, even when this is itself entirely free from purine substances. Whether or not changes in the activity of purine synthesis occur in conditions of disease is a question which awaits investigation. The Influence of Various Physiological Conditions, of Drugs, and of Disease on the Endogenous Uric-acid Excretion. Muscular exercise was thought by Burian to cause an increased excretion of uric acid, from which he drew the conclusion that the hypoxanthine present in compara- tively large amount in muscular extract, or its precursor, inosinic acid, must be an important source of endogenous uric acid. Other observers (Leathes, 62 etc.) have found that strenuous exercise causes a distinct in- crease in uric-acid excretion, which, however, is much less marked on repetition of the same kind of exercise on the next day. If some new kind of muscular work is performed, another increase in uric acid will result. There are still other investigators who deny that muscular work has any influence on uric-acid excretion. It has been observed by several investigators that the endogenous purine excretion is distinctly higher during the waking hours than during sleep. This can not be shown to depend on variations in the urinary function, and since it is decidedly doubtful whether ordinary muscular activity has any influence, the diurnal variation is most difficult to account for. The endogenous excretion in man is not the same for different individuals, even when calculated for the same body weight; it varies between 0.12 and 0.20 per cent purine nitrogen in an adult man. It remains remarkably constant for a given individual from time to time, being unaffected by moderate degrees of variation in the amount of food taken, provided this be purine-free; when, however, the amounts are extremely variable, changes are produced (see page 648). In disease, fever causes an increased excretion. This has been most clearly shown by Leathes, 62 who took a large enough dose of antityphoid URIC ACID AND THE PURINE BODIES 681 serum to produce a distinct degree of fever (103 F.), and found that an increase in uric-acid excretion occurred. That increased combustion processes occurring in the tissues were responsible for the uric acid, was shown by the same author, who caused a similar increase by sub- jecting himself to cold baths for a considerable period of time. The in- creased loss of heat thus induced stimulated the combustion processes in the body so as to maintain the body temperature, and as a result there was an increase in uric-acid excretion. It has long been known that an excessive amount of uric acid is excreted in leucemia. The nuclein of disintegrated leucocytes is commonly held responsible for the increase. Naturally, much work has been done on the endogenous and exogenous purine excretion in gout. No very striking anomalies of excretion have, however, been brought to light, except perhaps that after the ingestion of purine-rich foodstuffs it takes longer for the resulting exogenous ex- cretion to develop and pass away. Certain drugs affect the excretion of uric acid. Salicylic acid is said to cause an increased excretion, and citrates certainly have this effect. 63 In both cases the increase is followed by a compensatory fall, which indicates that these drugs act by facilitating the excretion rather than by influencing the metabolic processes that are the source of the uric acid. The effect of caffeine has been very carefully investigated. Given to the Dalmatian dog, referred to above, S. R. Benedict found that a small dose caused a slight decrease, but that a larger dose had practically no effect, although there was a notable retention of nitrogen. On man, however, different results were secured, for it was found that when 1 gram of caffeine was given daily for several days, a slight but definite progressive increase in the endogenous uric-acid excretion occurred, and it lasted for 10 days after the caffeine administration was discontinued. Liberal allowance of this alkaloid may, therefore, not be quite so innocu- ous as it is assumed to be. Uric Acid of Blood. In all of the investigations considered above, the behavior of uric acid is judged from the amount of it excreted in the urine. Valuable though such results must be, their interpretation is always difficult, since two factors that are quite independent of each other have to be kept in mind namely, the production of the uric acid in the organs and tissues and its excretion by the kidneys. In connection with the latter factor, we must also consider the method of transporta- tion of uric acid by the blood from its place of production (or absorp- tion) to the kidneys. These problems have recently been very consider- ably simplified by the elaboration of an accurate method for the estima- tion of the uric-acid content of Hood. By observing changes in the amount of uric acid in the blood rather 682 METABOLISM than in the urine, the excretory factor is partly controlled, and it can be completely so if urine and blood are both investigated. Thanks to the work of Folin, it is now possible to determine with an extreme de- gree of accuracy the uric acid in as little as 10 c.c. of blood. The impor- tance of this achievement will be appreciated when we state that prior to Folin 's work no method existed by which uric acid could be approx- imately measured even when large quantities of blood were available. Much of the work that has been done by the use of this new method has so far applied to the amount of uric acid in the blood of man in various diseases. We shall refer to these results immediately, but meanwhile it is important to call attention to some very suggestive observations concerning the condition of uric acid in the blood. For many years there have been investigators who have thought that uric acid can not be simply dissolved in the blood plasma, like sugar or some inorganic salt. It is believed by many that at least a portion of the uric acid circulates in combination with nucleic (thymic) acid (see page 669), which would account for the fact that some purines are catabolized in the body when they are given in a combined state, as thymic acid, but are excreted unchanged when ingested in a free state. When given freely, certain purines adenine, for example may moreover cause inflamma- tion and calculus formation in the kidneys of dogs, a result not obtained when thymic acid is fed. Other observers have concluded that uric acid exists as two isomeric varieties, lac- tam and lactim, the monosodium salts of which are of unequal stability. The less stable a-salt is much more soluble in the blood serum than the stable (3-salt. It is the a-salt that becomes increased in the blood in gout, the deposition of urates in the tissues, which is the most characteristic symptom of this disease, being caused by conversion of the o-salts into /3-salts. The structural formulas of the two isomers are as follows: H.N-C : O I ! O : C C - NH I !! CO H.N - C - NH [lactam modification forming unstable a-urates] (relatively soluble) N-C.OH HO.C C-NH I I \ C.OH I I / N = C-N [lactim modification forming stable /3-urates] (relatively insoluble) The most recent work of S. R. Benedict has shown that uric acid ex- ists, chiefly in combination in the blood of most mammals but not in that of the bird. It was found, for example, that fresh ox-blood exam- ined by the Folin method contains only 0.0005 gm. free uric acid per 100 gm. of blood; after boiling the protein-free blood filtrate with hydro- chloric acid, however, the uric acid increased by about ten times. This URIC ACID AND THE PURINE BODIES 683 larger amount was also found present in whole blood that had been allowed to stand for some time, indicating that the uric-acid compound can be split by means of an enzyme. The compound exists in the cor- puscles and not in the plasma. It is of some significance that after thus setting free the uric acid, there should be about 50 per cent more of it present in the blood of the ox than in that of the bird, where most exists in a free state in the serum, although the urine of the ox contains only the smallest trace of uric acid, and that of the bird is loaded with it. Investigation of the condition of uric acid in human blood is at present in progress. Uricemia in Gout and Nephritis The practical application of these observations is particularly impor- tant in connection with the etiology of gout. In typical cases of this dis- ease, the uric acid of the blood increases from its normal value of 1 to 3 mg. per cent to nearly 10 mg., indicating a considerable degree of URIC ACID, UREA N AND CREATININE OF BLOOD IN GOUT AND EARLY AND LATE NEPHRITIS UREA N CREATININE SYSTOLIC ACID DIAGNOSIS MG. TO 100 C.C. BLOOD PRESSURE Typical Cases of Gout 9.5 13 1.1 230 8.4 12 2.2 164 7.2 17 2.4 200 6.8 14 1 1.7 Typical Early Interstitial Nephritis 9.5 8.0 25 37 2.5 2.7 185 150 5.0 37 3.9 130 7.1 16 2.0 6.6 24 3.3 185 6.3 18 2.1 8.7 20 3.6 100 7.0 33 2.6 117 6.3 31 2.1 6.3 23 2.4 150 Chronic Diffuse and Chronic Inter- 8.0 80 4.8 240 stitial Nephritis 4.9 8.3 17 72 2.9 3.2 170 238 5.3 21 1.9 145 9.5 44 3.5 210 2.5 19 1.9 120 7.7 67 3.1 6.7 17 1.6 165 8.3 39 2.9 6.5 24 3.0 200 Typical Fatal Chronic Interstitial 22.4 236 16.7 210 Nephritis 15.0 240 20.5 225 14.3 263 22.2 220 13.0 90 11.1 265 8.7 144 11.0 225 (Myers and Fine: Arch. Int. Med., 1916.) 684 METABOLISM renal insufficiency. This uricemia can not in itself, however, be the cause of the deposition of urates in the joints, because it also occurs in other diseases with renal retention, such as nephritis. Moreover, the blood serum is capable of dissolving much larger quantities of uric acid than are ever found present in it in gout. The real cause for the gouty deposits must depend on some change affecting the blood so as to alter the form in which uric acid exists therein, with the result that it passes into the joints and is deposited there. Other diseases showing uricemia are lead poisoning and nephritis. In the latter disease the damaged excretory function of the kidney is manifested first of all by an increase in the uric-acid content of the blood, accompanied later by a retention of urea and later still by one of creatinine. The severity of the renal involvement may therefore be gauged by determining the percentage of these three metabolites. On account of the importance of these facts from a clinical standpoint, we append a table containing results secured by Myers and Fine, in which the behavior of the metabolites in the blood is shown in relationship to the severity of the case as gauged by the blood pressure. Lastly, regarding the influence of drugs on. the blood uric acid in dis- ease, it has been found by Fine that both atophan and salicylates cause a pronounced decrease in the amount, but that it gradually rises to the old level even while administration of the drugs is being continued. Important contributions to the behavior of uric acid in blood are constantly appearing at present, mainly from the laboratories of Foli] in Boston, of S. R. Benedict, and of Myers and Fine in New York. CHAPTER LXXVI THE METABOLISM OF THE CARBOHYDRATES The healthy animal organism is capable of rapidly oxidizing large quantities of carbohydrate, as is evident from the following facts: If carbohydrate is given to a starving animal, (1) the energy output very shortly afterward increases; (2) the respiratory quotient also increases, indicating that, relatively to oxygen intake, more carbon dioxide is being excreted (see page 582) ; and (3) none of the ingested carbohydrate makes its appearance in the excreta. Indeed, of the three proximate principles of food, carbohydrate is the most available for combustion in the animal body. It may therefore be considered as the quickly available fuel for the body furnaces. CAPACITY OF THE BODY TO ASSIMILATE CARBOHYDRATES Assimilation Limits. When the limit to the amount of carbohydrate that the organism can metabolize is overstepped, some of it appears in the urine. The amount that can be tolerated without causing glycosuria is commonly called the assimilation or saturation limit. The use of the term " limit" is, however, very unfortunate, for it implies that beyond this point the organism is capable of dealing with no more carbohy- drate, which is far from being the case, for if a larger amount is taken, only a small trace of the excess will appear in the urine. When the urine is allowed to collect for twenty-four hours, the mixed specimen shows no trace of glucose in the majority of healthy individuals after the ingestion of 200 gm. ; after 300 gm. a somewhat higher percentage of cases develop a mild glycosuria, but frequently none is evident even after 500 gm. Beyond the last mentioned amounts the limit of ingestion is reached, on account of nausea, etc., and it is improbable even if larger amounts could be tolerated, that any more of the glucose would be absorbed than with 300 or 400 gm. The testing of the so-called as- similation limit has been considered an important aid in the diagnosis of early cases of diabetes. It has been found that to make the results of any value, certain conditions must be fulfilled in applying the assimilation test. The most important of these concerns the activities of the gastrointestinal appa- ratus at the time the sugar is given, for it has been found that if other 685 686 METABOLISM foodstuffs are being absorbed at the same time as the sugar, more of the latter can be tolerated than when the sugar alone is being absorbed. It has therefore been customary to give the sugar dissolved in water, or in weak coffee, the first thing in the morning after the patient awakes ; i. e., at least twelve to sixteen hours after the last meal was taken. In making these tests the urine voided before the sugar is administered should of course itself be thoroughly examined for reducing substances, and specimens should be collected about every ninety minutes and examined by a reliable test (Benedict's or Nylander's).* Although a limit is set to the ability of the organism for retaining sugar (mono- or di-saccharides), this is usually considered not to apply, in healthy individuals at least, when starches (polysaccharides) are ingested. Thus, it is a well-known fact that people can eat enormous quantities of po- tatoes or of bread without the appearance of any trace of reducing sub- stances in the twenty-four-hour urine. It should be pointed out, however, that urine collected and examined at short intervals (every half hour) after taking large quantities of polysaccharide-rich food has frequently been found to contain traces of reducing substances in apparently healthy persons. For practical purposes it has been considered that an individual who developes glycos- uria after taking 100 gm. of glucose must be considered as at least a potential diabetic. In the light of the above results and for many other reasons, there is, however, con- siderable doubt as to the value of the assimilation test. Thus, when a solution of glucose is given orally, its rate of absorption will depend very largely on the motility of the stomach. If this is normal, the solution will very quickly find its way past the pyloric sphincter into the intestine, where it will be rapidly absorbed. If, on the other hand, the pyloric sphincter does not open freely, the passage of the glucose into the intestine may be so delayed that no more is present in this place at one time than would be the case after an ordinary diet of polysaccharide. And even after the sugar solution enters the small intestine, differences in the amount of the intestinal contents with which it becomes mixed, in the extent of bacterial growth, and in the absorption process, may very materially affect the rate at which the glucose gains entry to the blood. Although often of doubtful diagnostic value, determination of the assimilation limit is of considerable aid in controlling the treatment of diabetes. For this purpose the patient should first of all be instructed to follow his usual diet, so that, by examination of the amount of sugar excreted in the urine, an opinion may be formed of the severity of the case. The diet should then be changed so as to consist of a part that contains no carbohydrates and another composed entirely of starchy Examination of normal individuals has shown that the assimilation limit for different sugars varies somewhat; for glucose it appears to be from about 150 to 250 gm. ; for leyulose, which, it will be remembered, is the monosaccharide associated with glucose in the construction of the cane- sugar molecule, the assimilation limit is from 100 to 150 gm. ; for cane sugar or saccharose itself the figures seem to vary considerably, but are given as between 50 and 200 gm. ; for lactose, another disaccharide, and the sugar present in milk, the assimilation limit is distinctly lower namely, 100 gm. THE METABOLISM OF THE CARBOHYDRATES 687 food. The former is made up of eggs, fish, green vegetables, fat, etc., and the latter, to start with, should consist of 100 grams of bread, dis- tributed between the two main meals of the day, one of which is break- fast. This diet should be continued until the glycosuria either disappears or attains a constant level. If it disappears, the case is classified as a mild one of diabetes, and the daily allowance of bread may be increased, by 50 grams a day, until the sugar again makes its appearance in the urine, indicating that the assimilation limit has been reached. For therapeutic purposes, the patient should now be instructed to take about three fourths of this amount of carbohydrate in his daily ration, and he should be supplied with explicit instructions in the shape of diet tables as to what variety and quantities of the various carbohydrate materials his food may contain. His urine should be examined at fre- quent intervals once a week and he should be instructed as to the nature of his disease and the importance of his remaining aglycosuric. By further treatment such so-called latent cases of diabetes may be kept in perfect health for many years. When, on the other hand, the glycosuria persists with 100 grams of bread in the daily ration, this must be reduced to 50 grams, and if after some days the first reduction does not suffice to render the urine free from sugar, carbohydrates must be withheld entirely from the diet. If the glycosuria does not now disappear, the case is to be considered severe, and it may be necessary to undertake the starvation treatment, which has recently been developed in this country by Allen 18 and Joslin 19 with apparent success. By the reduction of carbohydrate, or by the starvation treatment, it is usually possible to make even the severest cases of diabetes aglycosuric. When this has been attained, the amount of protein or carbohydrate food may be cautiously increased until just short of the assimilation limit. To avoid error caused by irregular absorption from the intestines, some investigators have recommended the determination of the assimilation limit after intravenous or subcutaneous injections of sugar. But' even this refinement in technic has not, as a rule, had the effect of rendering the results of any very evident value as a criterion of the utilization of glucose in the animal body. The reason for the unreliability of this method is mainly that the period of injection of the glucose solution usually oc- cupies only a few minutes, so that it causes a sudden instead of a very gradual in- crease in the sugar concentration of the blood, the conditions being therefore quite unlike that which exists during the normal absorption of glucose from the intestine. The mechanism by which the body ordinarily disposes of excessive amounts of glucose absorbed into the portal blood, is not adjusted to operate when the systemic blood is suddenly overcharged with this substance. In the one case the glucose is a food- stuff; in the other, because of its excessive concentration in the blood, it is more or less of a poison. Such results, in other words, merely show us how much glucose can be added at one time to the organism without any overflow into the urine, but they 688 METABOLISM furnish us with no information regarding the power of the organism to utilize a con- stant though moderate excess of this substance. In the one case it is the "satura- tion limit," in the other the "utilization limit" of the organism for glucose, that we are really measuring. The Tolerance of the Body for Glucose. Consideration of these prin- ciples has led Woodyatt, Sansum and Wilder 20 to undertake a thorough reinvestigation of the whole problem of the utilization or, as they prefer to call it, the tolerance of the body for glucose. They emphasize the obvi- ous fact that the ability of the organism to utilize glucose ' ' must de- pend on the rate at which the tissues are able to abstract it from the blood by their combined powers, to burn it, to reduce it into fat or to polymerize it into glycogen. " To form any estimate of the combined effect of these processes, we must take into account not only the amount of glucose per unit of body weight (grams per kilogram), but also the rate of injection, for "tolerance must be regarded as a velocity, not as a weight." Briefly summarized, the conclusions which Woodyatt, etc., have so far drawn from their investigations are as follows: In a normal rabbit, dog, or man, 0.8-0.9 gm. of glucose per kilogram body weight and per hour can be utilized by the organism for an indefinite time without causing gly- cosuria. When between 0.8 and 2 gm. are injected, a part of the excess appears in the urine, steadily increasing until a maximum is reached, after which the excreted fraction remains constant (at about one-tenth). If more than about 2 grams per kilogram an hour are injected, "a large percentage of all glucose in excess of the 2 gm. per kilogram an hour appears in the urine when constant conditions are once established." The fact that so much glucose injected intravenously can be used without the appearance of any of it in the urine, indicates a method by which foodstuffs may be supplied to the tissues in cases where, on account of gastrointestinal disturbances, it is impossible to have food absorbed by the usual pathways. The possible value of such a method of treat- ment in cases of extreme weakness has been tested on laboratory animals by Allen, who states that such injection seems to have a valuable nutri- tive and strengthening effect. He found; for example, that in cats starved to extreme weakness the injection of a fraction of a gram per kilogram of glucose had an unmistakable strengthening effect, and sometimes appeared to save life. Such results would seem to indicate that in certain cases where blood transfusion is impracticable, glucose in- fusions should be tried. Subcutaneous injection of sugar, either for the purpose of determining the assimilation limit or with the object of sup- plying foodstuffs parenterally, is impracticable because of the pain and sometimes sloughing produced at the point of injection. THE METABOLISM OF THE CARBOHYDRATES 689 We may now turn our attention to a closer analysis of the changes that take place in carbohydrates during their passage through the animal body and to the more recently introduced methods of diagnosis in which the blood sugar is measured. DIGESTION AND ABSORPTION Digestion. All digestible carbohydrate taken with the food is con- verted by the digestive agencies into the monosaccharides, glucose and levulose, as which it is absorbed into the blood of the portal system. To bring about this resolution of carbohydrate into monosaccharides, several enzymes are employed. The first of these is the ptyalin of saliva. It is not a very powerful enzyme, being capable of acting only on starches that are in a free state, i. e., not surrounded by a cellulose envelope ; but even on free starch, ptyalin displays little of its activity during the time the food is in the mouth. After the food is swallowed and becomes deposited in the fundus of the stomach, there is an interval of time lasting until hydrochloric acid has been secreted to such an extent as to permit some of the acid to exist in a free state during which the ptyalin acts on the starch of the swallowed food. During this time the activity of the ptyalin is actually assisted on account of the fact that a slight increase in hydrogen-ion concentration of the digestive mixture accel- erates the action of ptyalin. The product of ptyalin digestion is maltose, a disaccharide composed of two molecules of glucose. On entering the intestine, the carbohydrates therefore exist partly as undigested starch, partly as glucose, and partly as maltose. In the favorable environment of the duodenum a much stronger diastatic enzyme called amylopsin very quickly hydrolyzes the starch through dextrine into maltose. The maltose derived from the starch and the unchanged sugars, such as cane sugar, maltose and lac- tose, which have been taken with the food, unless they are present in very high concentration in the intestinal contents, are not immediately ab- sorbed into the blood, but become subject to the action of other enzymes contributed by the intestinal juice namely, the inverting enzymes, one of which exists for each of the disaccharides. By their action maltose is converted into two molecules of glucose by the enzyme maltase; lac- tose, into galactose and glucose by lactase; and cane sugar, into levu- lose and glucose by invertase. It is interesting to note that in animals whose food does not contain one or other of those disaccharides, the cor- responding inverting enzyme is absent from the intestinal juice. The her- bivorous animals, for example, do not take any lactose in their food, and the 690 METABOLISM intestinal juice contains therefore no lactase, although it is present in that of the young animals while still suckling. A certain amount of carbohydrate becomes attacked by the intestinal bacteria. These split the monosaccharides into lower fatty acids and gases, such as methane and carbon dioxide. Besides this obviously de- structive process, bacteria also perform a useful function in the digestion of carbohydrates, in that certain strains of them are able to digest cellu- lose, for which no special enzyme is provided. Bacterial digestion is con- sequently essential in herbivorous animals; it takes place in the cecum, which is enormously developed for this purpose (page 497). Absorption. The glucose and levulose produced by digestion are absorbed into the blood of the portal system. When a very large quan- tity of a disaccharide, such as cane sugar, is present in the food, a certain amount of the sugar is absorbed unchanged that is to say, as cane sugar and appears in the blood, from which, since it is an abnormal con- stituent, it is excreted unchanged in the urine. This alimentary glyco- suria is particularly evident when the sugar is taken without any other food; thus, after taking cane sugar in an amount corresponding to 5 grams per kilogram body weight, it was found in one and a half hours afterward that the urine of ten out of seventeen healthy individuals con- tained cane sugar. The urine of three of these men, however, also con- tained invert sugar that is, dextrose and levulose. Cane sugar con- tinued to be excreted for from six to seven hours. The Sugur Level in the Blood. "While no absorption of sugar is going on, the percentage of this substance in the blood of the portal vein is the same as that in the systemic circulation. During absorption the former becomes perceptibly raised to what extent we can not say and in the latter a less marked increase of sugar concentration is usually detectable. Evidently, then, between the point at which the sugar is absorbed and the blood of the systemic circulation, some barrier exists which holds back some of the excess of absorbed sugar. We have very inaccurate information as to how efficiently these barriers hold back the excess of absorbed glucose because of the technical difficulty in collecting blood from the portal vein without serious disturbance to the animal. Indeed, the only way by which the problem can be studied is by comparing the blood of the portal circulation with that of the systemic circulation dur- ing the injection of a solution of glucose into one of the smaller branches of the portal vein. 21 In such experiments it has been found that the percentage of sugar is a little less in the blood of the abdominal vena cava than in that of the portal vein, and is still less in the blood of the systemic veins, such as the femoral results which justify the conclusion that the barriers responsible for taking out some of the absorbed sugar from the blood exist in the liver and in the muscles. The curve in Fig. 190 will illus- trate to what extent the mechanism operates. THE METABOLISM OF THE CARBOHYDRATES 691 It will be observed that, so far as can be judged from changes in the concentration of sugar in the blood, the sugar-retaining power of the liver is about equal to that of the muscles. One objection which may properly be raised to these observations is that the animals on which they were made were under anesthesia, and that the anesthetic may have* had a paralyzing effect on the sugar-retaining power of the liver. In view of this criticism it is important to examine the results obtained on animals that are not under the influence of anesthesia. By collecting blood from the ear veins of rabbits, it has been found, after giving from two to ten grams of glucose by stomach, that the glucose concentration of the systemic blood begins to vise in fifteen minutes, attaining a maximum in about an hour and then returning to the normal level in about three hours. Similar results have been obtained by examination of the venous blood in man. After giving 100 grams of glucose by mouth, for example, there is commonly an increase in blood sugar amounting to from 30 to 34 per cent of the normal and lasting for from one to four hours. The existence of this postprandial hyperglycemia, as we may call it, indicates that the / / / Fig. 190. Curves showing the percentage of glucose in blood after a constant injection of an 18 per cent solution into a mesenteric vein. V.C., vena cava, continuous line; P.O., pan- creaticoduodenal vein, broken line; I, iliac, dotted line. sugar-retaining powers of the liver and muscles are not sufficiently de- veloped to prevent the accumulation of some of the absorbed sugar in the systemic blood. Whenever this increase exceeds a certain limit, some of the sugar begins to escape through the kidney into the urine, producing glycosuria postprandial glycosuria. The concentration to which blood sugar must rise before glycosuria occurs in the case of man is about 0.17 gm. per cent. After damage to the kidney, as in nephritis, or in long-standing cases of mild diabetes, the percentage may probably rise considerably higher in the blood without evidence of glycosuria. Value of Blood Examination.!!! Diagnosis of Diabetes. The determina- tion of the amount of ingested carbohydrate required to bring about post- prandial glycosuria constitutes, as we have already seen, the so-called 692 METABOLISM assimilation limit for sugar, which is often taken as an index of the sugar- metabolizing power of the organism. It is evident, however, that the time of onset, and the extent and duration of postprandial hyperglycemia must serve as a more certain index of the efficiency of sugar metabolism. It is now the usual practice to determine the sugar in samples of venous blood removed immediately before and at half hour intervals following the administration by mouth of 100 gm. glucose dissolved in water. This is done on an empty stomach. In a healthy person the curve of blood sugar rises within an hour to not above 0.15 per cent and the normal is regained in two hours. In early diabetes the curve rises higher, stays up for a longer time and does not return to normal for several hours. Slight deviations from the normal must not be given too much weight in diagnosis since they may occur in other diseases or even in perfectly normal persons. In no disease, probably not even in tuberculosis, is it more important than in diabetes that an early diagnosis should be made. Thus, if we find that the postprandial hyperglycemia after a certain amount of carbo- hydrate develops to an unusually high degree and persists for an unusual length of time, we are justified in curtailing the carbohydrate supply so as to hold these values down to the level they attain in normal individuals. It is almost certain that the earliest sign of diabetes is an unusual degree and duration of postprandial hyperglycemia. At first the excess of sugar leads to no damage and it is insufficient to cause any evident glycosuria, al- though it is quite likely that if the urine in such individuals were collected at very frequent intervals after eating carbohydrate-rich food, glucose would be found present in at least some of the specimens. In incipient diabetes, however, the condition progresses, until the postprandial hyperglycemia after one meal has not become entirely replaced before the next is taken, so that the increase in sugar produced by the second meal becomes super- added on that following the first meal. The curve of blood sugar rises ever higher and higher, until at last permanent hyperglycemia is estab- lished, or rather the normal level from which the postprandial rise occurs has become permanently raised, so that in blood collected at any time a higher percentage of sugar is found. The Relationship Between the Sugar Concentration of the Blood and the Occurrence of Glycosuria. Claude Bernard first pointed out that the percentage of sugar in the blood may rise considerably above its normal level without the appearance of any of the sugar in the urine, or at least without a sufficient amount appearing to give the usual tests for sugar. Thus in man the blood sugar may rise to 0.17 per cent before sugar can be detected in the urine. This has been called the renal threshold. Even when the threshold has been overstepped, however, the sugar which appears is not all of the excess but only a small part of it. THE METABOLISM OF THE CARBOHYDRATES 693 Strong support has been lent to the idea of the renal threshold by the recent work of Woodyatt and his collaborators, who have shown by continuous intravenous glucose injections that as much as 0.8 gm. of glucose per kilo body weight can be injected during an hour into an animal without any glycosuria, although under such conditions a very distinct increase occurs in the percentage of sugar in the blood. To explain the failure of glucose to pass into the urine under normal conditions, it has been supposed by several investigators that the glucose exists in some form of chemical combination in the blood. This compound is believed to behave like a colloid. One of the recent supporters of this view is Allen, who has observed that, when glucose is injected intravenously, it causes diuresis as well as glycosuria; whereas glucose injected subcutaneously or taken by mouth causes neither of these conditions to become developed; indeed it causes for some time after its administration a dimin- ished urinary flow. To explain these differences in behavior between glucose ad- ministered intravenously and that taken in other ways, it is supposed that the glucose molecule in passing through the intervening wall of the capillaries combines with some substance to form a compound which becomes available for incorporation into and utilization by the tissues, glucose in a free state being incapable of utilization. This compound is supposed to be of a colloidal nature, and the substance which combines with glucose to form it is believed to be related to the internal secretion of the pan- creas (see page 710). The difficulty in explaining why the glucose of the blood does not constantly leak into the kidney is, however, the only evidence upon which the hypothesis of a blood sugar compound rests. No chemical evidence can be offered in support of such a view. On the contrary, all experimental work indicates that the sugar exists in a free state; but unfortunately even this evidence is not convincing. Thus, it has been found that, when specimens of perfectly fresh blood are placed in a series of dialyzer sacs sus- pended in isotonic saline solutions, each solution containing a slightly different per- centage of glucose, diffusion of glucose, in one or other direction, occurs in all of them save one namely, that in which the percentage of glucose in the fluid outside the dialyzer is exactly equal to the total sugar content of the blood. Such a result can be explained only by assuming that all of the sugar in the blood exists in a freely diffusible state. In its general nature this experiment is analogous to that by which the tension or partial pressure of CO 2 is determined in blood (see page 355). It has been shown that glycosuria may sometimes become developed because the kidney fails to hold back the blood sugar even when the percentage is not above the normal so-called renal diabetes. For the diagnosis of this condition a comparison must be made between the sugar concentration of the blood and that of the urine. In order to do this at least two samples of blood must be taken, one of them at the beginning and the other at the end of a period during which urine is being collected. Merely to find that one sample of blood collected before or after or during the period of urine collection contains a normal percentage of sugar, does not necessarily indicate that at some other period while the urine was being produced a temporary hyperglycemia may not have ex- isted. Recent contributions have shown that this condition is of more frequent occurrence than it was previously thought to be. 26 CHAPTER LXXVII THE METABOLISM OF THE CARBOHYDRATES (Cont'd) FATE OF ABSORBED GLUCOSE. GLUCONEOGENESIS We may now consider what becomes of the sugar that is retained by the liver and muscles. Two things may happen to it: It may become stored, or it may become oxidized or split up. Of these processes, storage occurs in both the liver and muscles, whereas oxidation occurs mainly if not entirely in the muscles, although a certain amount of splitting of the glucose molecule may also occur in the liver. Storage of Sugar. For the present we shall consider the process of storage of sugar and defer a consideration of its utilization until after we have studied, not only the nature of the process by which the storage occurs, but also the immediate destiny of the stored sugar. The storage of sugar by the liver is brought about by its conversion into a polysac- charide called glycogen. After an animal has been absorbing large quan- tities of glucose, an acidified watery extract of a portion of liver made immediately after death will be found to contain no more sugar than that of a normal liver. On the other hand, it will be observed that the extract is highly opalescent and yields on the addition of alcohol a copious precip- itate, which on further purification can readily be shown to consist of a polysaccharide that is to say, of a starch-like substance which on hydrol- ysis with mineral acid becomes entirely converted into sugar. If instead of examining the liver immediately after death, it is allowed to stand for some time, the yield of glycogen will greatly diminish, and in its place will appear large quantities of glucose, indicating that some enzyme must exist which attacks the glycogen after death and converts it into sugar, This enzyme is called glycogenase. The existence of postmortem glyco- genolysis, as it is called, would seem to indicate that during life a con- stant tendency for the glycogen in the liver to be attacked by glycogenase is held in check by conditions which depend on the vital integrity of the liver cell. It is evident that if anything should happen during life to interfere with this inhibiting influence, the glycogen will become con- verted into glucose, which on escaping into the blood will produce hyper- glycemia and glycosuria. Sources of Glycogen. In studying the sources of sugar in the animal bod}* it is of great importance that we should first of all know exactly the 694 THE METABOLISM OF THE CARBOHYDRATES 695 conditions under which glycogen may be formed in the liver; that is, whether it is formed exclusively from absorbed sugar, or whether other substances, such as protein and fat may also form it. The importance of such knowledge rests in the fact that in severe diabetes, sugar continues to be added to the blood, although no sugar is being taken with the food. To check the hyperglycemia in such cases it becomes necessary, therefore, to curtail the diet not only with regard to its carbohydrate content, but also with regard to whatever other foodstuffs may be capable of causing glycogen formation. The practical question therefore is, What are these foodstuffs? There are two methods by which the problem may be investi- gated. The first, which we may call the direct method, consists in rendering the liver free of glycogen and then feeding the animal with the food- stuff in question, afterward killing it and immediately examining the liver for glycogen. The other, which we may call the indirect method, con- sists in first of all rendering the animal incapable of oxidizing glucose that is, making it diabetic and then proceeding to see whether the in- gestion of a given foodstuff causes an increase in the sugar excretion in the urine. The methods for rendering an animal experimentally diabetic will be considered later; for the present it is important to note that, if a diabetic animal excretes more glucose while fed on a given foodstuff, we may infer that the normal animal would convert it into glycogen. The results of the direct method are much less reliable than those of the indirect for the reason that it is extremely difficult to remove all traces of glycogen from the liver. The methods employed for this pur- pose have-xjonsisted in: (1) starvation of the animal; (2) muscular ex- ercise; (3) exercise and starvation combined; and (4) the production of certain forms of experimental diabetes for example, that produced by phlorhizin. Starvation alone is unsatisfactory, for it has been found that, although at certain stages of this condition the liver may become al- most entirely free from any trace of glycogen, at a later stage glycogen may again make its appearance. It is therefore most difficult to decide at what stage in starvation the animal should be considered as glycogen- free. If the starving animal is made to perform muscular exercise, complete removal of glycogen from the liver can be depended upon. The exercise may be produced by the administration of strychnine in such dosage as just to produce convulsions of the voluntary muscles without permanent contraction of those of respiration. The most useful method, however, consists in starving the animal for a few days and then placing it in a cold, damp room, after giving it a cold bath. The evaporation of mois- ture from the surface so cools the body down that the stores of glycogen a.ll become used up in the attempt to supply fuel for the production of 696 METABOLISM sufficient heat to maintain the body temperature. This method can be rendered still more certain in effecting a removal of all carbohydrate from the body by giving the animal phlorhizin every eight hours. Phlor- hizin, as we shall see, renders the animal diabetic. After removing the glycogen, further deposition in the liver can be readily shown to occur when any of the ordinary sugars or starches are given as food. It does not occur, however, when chemical substances closely related to ordinary sugar, such as the wood sugars (pentoses) or the alcohols and acids corresponding to dextrose, are contained in the diet. Nor does it occur with cellulose or with inulin, a polysaccharide built up from pentose sugar. When proteins are fed the results are not so definite, although many observers have claimed that glycogen is formed. With fat, on the other hand, no glycogen formation can be shown to occur, although we know that a trace of carbohydrate must be formed out of the glycerine of the fat molecule. The results of. the direct method, even when the conditions are per- fectly controlled, are very unreliable, especially when they are of a nega- tive character, because any new sugar that may be produced by the in- gested substance instead of being stored as glycogen is likely to be used by the tissues as it is formed. Where only a slight degree of gluconeo- genesis, as the process of sugar formation is called, is occurring, it is not probable that any of the glucose will be retained in the body as glycogen. The methods employed for producing experimental diabetes in investi- gation of these problems by the indirect method are (1) the entire removal of the pancreas, and (2) the continuous administration of the drug phlorhizin. The animal rendered diabetic by either of these methods is first of all observed for several days to determine the normal daily ex- cretion of sugar. At the same time the nitrogen excretion for the day is determined, the ratio between the total nitrogen and the glucose known as G to N ratio being about 3'.65 to 1 when complete diabetes has become established. The foodstuff in question is then fed to the animal, and the amount of extra glucose excreted thereby is taken to represent that which has been derived from the ingested food. By this method it has been possible to show that, not only the above mentioned carbohydrates, but protein as well produce a very considerable quan- tity of glucose in the animal body. Fats, however, yield only negative results. The indirect method has another great advantage over the direct in that the results are much more quantitative in character; for example, Lusk and his pupils have been able to determine the amount of glucose which can be produced by feeding certain of the building stones of the protein molecule. The great practical importance of such results in THE METABOLISM OF THE CARBOHYDRATES 697 the therapy of diabetes makes it advisable for us to go into the subject a little more in detail here. After a cold bath and exposure in a cold room, dogs are rendered diabetic by phlorhizin. When all of the original glycogen in the body has been got rid of, as evidenced by the constancy of the G to N ratio in the daily quantities of urine excreted, the substance under in- vestigation is fed. If this substance contains no nitrogen and causes no change in the nitrogen excretion, any increase in that of glucose must obviously represent the extent to which the substance has become con- verted into this sugar. On the other hand, if the substance itself con- tains nitrogen, or if it causes a change in the excretion of nitrogen, it becomes necessary to calculate how much of the excreted glucose may have been derived from the body protein, assuming that this can form glucose, and how much from the administered substance.* From the results of this method it has been an easy matter to show that the following substances are converted in the animal body into glucose: (1) Glycol aldehyde (CH 2 OH-CHO). By placing three mol- ecules of this substance together, a hexose molecule results, a synthesis which can be accomplished in the chemical laboratory. The hexose formed in the animal body is glucose. Glycol aldehyde may be formed in normal metabolism out of glycocoll (CH 2 NH 2 COOH). (2) Glycerol (CH 2 OH- CHOH- CH 2 OH) may also readily be con- verted into hexose in the laboratory, the possible intermediary products being dioxyacetone (CH 2 OH - CO - CH 2 OH) and glyceric aldehyde (CH 2 OH-CHOH-CHO). Two molecules of either of these may be polymerized to form a hexose molecule, and when this process occurs in the animal body, the hexose formed is glucose. (3) Lactic acid (CH 3 CHOH - COOH) is completely converted to glu- cose in the diabetic animal, and the process must involve both a re- arrangement of the molecule and subsequent polymerization. The related substance, propyl alcohol (CH 3 - CH 2 - CH 2 OH) is also converted into glucose in the phlorhizinized dog. As to the exact nature of the chemical changes which occur as intermediary stages in the conversion of these substances into glucose, we are not as yet certain, but a clue has been afforded by the discovery that a substance called methylglyoxal (CHgCOCHO) can be obtained from lactic acid and also from glucose, and that this substance is converted into glucose when it is administered to phlor- hizinized dogs. We shall find later an important role for this substance in fat metabolism. It can also readily be produced during the interme- *This calculation^ made as follows: The amount of nitrogen in the administered substance is deducted from the nitrogen excretion, and the difference, which must represent the nitrogen of the body protein, is multiplied by the G to N ratio which prevailed on the day previous to that on which the substance was fed. We obtain in this way the glucose derived from the body. The glucose coming from the administered substance can then be ascertained by deducting that derived from the body protein from the total glucose excretion. 698 METABOLISM diary breakdown of certain of the protein building-stones, such for example as alanine (CH 3 CHNH 2 COOH). These chemical possibilities regarding the nature of the substances that serve as stepping stones between the above sugar-forming sub- stances and sugar itself may be considered as probabilities on account of the discovery that enzymes exist in various tissues which are capable of converting methylglyoxal into lactic acid : CH 3 CH 3 | I CO + H 2 -HCOH I Q<- J CHO COOH (methylglyoxal) (lactic acid) These enzymes are called glyoxalases, and since the reactions which they mediate are undoubtedly reversible in character, it is probable that the conversion into sugar of lactic acid and alanine to take those two as among the commonest of the sugar precursors of the animal body- occurs according to the following equation: CH 3 CHNH 2 COOH v. (alanine) CHXOCHO > C 6 H 12 O 6 CH,CHOHCOOH /* (lactic acid) (methylglyoxal) (hexose) The unique position of methylglyoxal, besides explaining the known resolutions of protein and fat and carbohydrate in intermediary metab- olism, is also of importance in explaining the synthetic production of glucose from fructose (or levulose). Fructose will first of all become converted into methylglyoxal radicles, and these will then become syn- thetized into glucose. The hypothesis of the conversion of glucose into lactic acid as a stepping stone in the metabolism of carbohydrate is difficult to test by direct ex- periment because the lactic acid does not accumulate in the organism, except in cases where there is oxygen deficiency or excess of alkali in the tissue fluids. Coming now to the amino acids, which, it will be remembered repre- sent the building stones of the protein molecule, it has been found that glycocoll, alanine, and aspartic and glutamic acids increase the glucose excretion when given to phlorhizinized dogs, whereas leucine and tyro- sine have no such action. By the method described above, it is possible to determine the exact proportion of the carbon of each of those amino acids which becomes converted to glucose. This is shown in the accom- panying table. It is of further interest to point out that these four amino acids constitute about 26 per cent of all the amino acids in flesh protein, and THE METABOLISM OF THE CARBOHYDRATES 699 TWENTY GRAMS OF THE VARIOUS AMINO BODIES WERE GIVEN TO PHLORHIZIN-DIABETIC DOGS ACID AND FORMULA AVERAGE AMOUNT OF GLUCOSE PRO- DUCED IN BODY PROBABLE GLUCOSE THAT CHANGE WOULD BE PRO- DUCED BY CHANGE Glycocoll CH 2 NH 2 COOH i. alanine CH 3 CHNH 2 COOH Aspartic acid 13.43 (five dogs, one gave 15.77) 18.77 (two dogs) 12.42 (four dogs) All C converted 16.00 to glucose " 20.22 Three of the four 13.52 COOH CH 2 CHNH, COOH Glutamic acid COOH CH 2 . CH a CHNH a COOH 13.31 C atoms converted to glucose Three of the five C atoms converted to glucose 12.24 that the total yield of glucose from them could be 26.3 grams; thus accounting for nearly one half of the 66 grams which a diabetic animal produces from 100 grams of flesh. Gluconeogenesis in Normal Animals. Although it has been clearly shown by the indirect method that not only protein but its decomposi- tion products as well, can be readily converted into glucose, yet this does not necessarily indicate that a similar conversion occurs in the nondia- betic animal. That such is the case, however, can be shown in various ways. Thus, at the end of a period of long starvation considerable quantities of glycogen are quite commonly found in the body, and the blood sugar, although lower than normal, never entirely disappears. Now, since no carbohydrate is being ingested, and the body stores of this foodstuff become exhausted early during starvation (cf. page 695), it is evident that the carbohydrate must be produced from the protein of the animal's body. A still more convincing experiment can be con- ducted by producing strychnine convulsions in a starving animal. If the animal is killed after the convulsions have lasted for a certain time, the tissues will be found almost, if not entirely, free of glycogen, but if the convulsions are made to disappear by giving chloral and the animal allowed to sleep for some time before killing it, glycogen again accumulates in the body. This glycogen must have been manufactured out of noncarbohydrate material. Corroborative evidence of a somewhat different nature is furnished by an examination of the respiratory quotient, which, it will be remem- bered (page 582), varies according to the nature of the foodstuff or body 700 METABOLISM constituent that is undergoing metabolism at the time, being about 1 with carbohydrate and about 0.8 with protein. If the quotient is observed during starvation, it will often be found to fall below 0.7, a figure which can be explained only by assuming that oxygen has been retained in the body beyond the quantity which is necessary for imme- diate purposes of oxidation (cf. equations on page 583). Since it is known that this retained oxygen can not exist in the body in a free state it must be concluded that it has become incorporated into substances having a high oxygen content. Such would be the case if protein or fat, which contains only from 12 to 20 per cent of oxygen, were converted to carbohydrate, which contains about 53 per cent. Utilization of inhaled oxygen for this purpose, as we have seen, becomes very striking in the case of hibernating animals during the winter sleep. CHAPTER LXXVIII THE METABOLISM OF THE CARBOHYDRATES (Cont'd) FATE OF GLYCOGEN Having become familiar with the sources from which glycogen may be derived, we may now proceed to study the fate of the glycogen found in the liver cells and in the muscles. For the present we shall confine our attention to the glycogen of the liver. If a portion of liver removed from a well-fed animal is examined microscopically after staining either with iodine or with carmine by Best's method, it will be found that the cells of the lobules are filled with glycogen except for the nuclei, which are free from this substance. If, on the other hand, the liver is from an animal that has not been recently fed, the lobules will contain no glyco- gen except in an area bordering on the central vein and perhaps a narrow strip at the periphery of the lobule. When it is present the rela- tive amount of glycogen in different lobules, as determined chemically, is the same over the entire, liver that is to say, no one lobe is richer in this substance than another. Nothing definite is known as to how the glycogen is held in the protoplasm of the cells, although some histolo- gists suggest that it is combined with a sustentacular material especially provided for this purpose. The glycogen stored in the liver is gradually given up to the blood of the hepatic vein at such a rate as to maintain in the blood of the sys- temic circulation a more or less constant percentage of glucose. Under ordinary conditions this process of glycogenolysis is relatively slow, but when the requirements of the organism for fuel become increased, as during muscular exercise, it becomes very rapid. The glycogenic func- tion of the liver appears therefore to exist, in part at least, for the purpose of preventing the flooding of the blood of the systemic circu- lation with excess of sugar during absorption from the intestine and of maintaining the normal percentage at other times. This function is analogous to that occurring in plants, in which the sugar produced in the leaves, if not immediately required, is transported to various parts of the plant and there converted into starch, which, when the plant requires it, as during new growth, may again become transformed into glucose. The agency converting the glycogen into glucose is the diastatic 701 702 METABOLISM enzyme glycogenase, which is present, not only in the liver cell, but also in the blood and lymph. It is a difficult matter to explain" why glycogen should be able to exist at all in the liver cells in the presence of this powerful enzyme. The following possibilities may be considered: (1) That glycogenase does not really exist in the living liver cells, but is a postmortem product; (2) that, although present, glycogenase is pre- vented from acting on the glycogen in the living liver cell on account of the latter being protected from its influence by combination with a sustentacular substance; or (3) that some chemical substance in the liver cell prevents the glycogenase from acting on the glycogen an anti- glycogenase. Since the removal of any one of these inhibiting influ- ences would cause glycogenolysis to become excessive, and so bring about hyperglycemia, it is important, in searching for the possible causes of this condition, to examine the evidence that has been brought forward in support of each of these views. Against the first of the above-mentioned possibilities, namely, that glycogenase is a post-mortem product, may be cited the very rapid conversion into glucose that occurs when glycogen is added to living blood, as by injecting some into a vein. On account of the active glycogenolytic action of blood, it has been suggested that during life glycogen does not become transformed into glucose until after it has been discharged into the blood from the liver cell. When increased sugar must be mobilized, glycogen passes unchanged, or perhaps as some dextrine, into the blood and lymph of the liver capillaries and lymphatics, the glycogenase of which converts it into glucose, the conversion being so rapid that, by the time the blood has traveled from the liver through the heart and pulmonary vessels to the arteries, all the glycogen has already become transformed into glucose. Postmortem glycogenolysis, according to this view, is due to the opposite occurrence the transference of glycogenase from the blood into the liver cell. Some facts supporting this view are as follows: (1) It has been found that the amount of free glucose in the blood of the vena cava is sometimes less than in that collected simul- taneously from the carotid artery. (2) After giving certain substances, such as phos- phorus or peptone, there is distinct diminution in the amount of glycogen in the liver, accompanied, it is said, by no increase in the amount of glucose in the blood. And (3) if the liver of an animal that has been rendered diabetic by stimulation of the splanchnic nerve or by puncture of the floor of the fourth ventricle is examined micro- scopically, after staining by the carmine method, masses of stained glycogen can be found present in the capillaries (sinusoids) that lie between the liver cells. According to the second view, the glycogen is removed from the influence of the in- trahepatic glycogenase on account of its combination with a sustentacular material. By disrupting this combination and thus exposing the glycogen to the action of glyco- genase, glycogenolysis will occur. We may call this the mechanical hypothesis and it deserves serious consideration, for it has been shown that very little postmortem glycogenolysis occurs in the intact liver of frogs in winter, even though at this time the organ contains an excess of glycogen, but becomes marked when the liver is broken down by mechanical means. The third view depends on the well-known fact that enzyme activities becomes most markedly altered by slight changes in the chemical nature of the environment in which they act.' Diastatic enzymes are particularly susceptible to the reaction (C H ) of their environment, a very slight degree of acidity favoring and a trace of alkalinity mark- THE METABOLISM OF THE CARBOHYDRATES 703 edly depressing their activities. That a tendency to increasing acidity in the liver cells may accelerate the breakdown of glycogen is suggested by the depressing effect produced on the assimilation limit of sugars by administering acids, and by the ob- servation that postmortem glycogenolysis becomes marked in proportion as the dying liver becomes acid in reaction. It might be thought then that glycogenolysis in the liver cell could be set up by the local production of a certain amount of acid. Such a liberation of free acid could be brought about by a curtailment in the arterial blood supply of the hepatic cell, producing a local accumulation either of carbonic or of other less completely oxidized acids (e. g., lactic). It may be that asphyxia causes hyper- glycemia by such a mechanism. Vasoeonstriction and consequent curtailment of ar- terial blood supply occurs in the liver when the hepatic nerves are stimulated, and it is possible that the glycogenolysis which is also set up by such stimulation is due to the appearance of acids. The accelerating effect of epinephrine on glycogenolysis might also be explained as due to limitation of blood supply on account of vasoconstriction and local asphyxia. THE REGULATION OF THE BLOOD SUGAR LEVEL The level at which the concentration of sugar in the systemic blood is maintained represents the balance between two opposing factors: (1) the consumption of glucose bv the tissues, and (2) the production of glucose by the liver. Since this is the most readily oxidizable of all the proximate principles of food (page 685), muscular activity causes large quantities of it to be consumed, so that its concentration in the blood tends to fall below the physiological level, a tendency which is immediately met by an increased discharge of glucose from the liver. The question therefore arises as to how the muscles or other tissues transmit their requirements for glucose to the liver. There are two possible ways by which this could be done: (1) by means of a nervous reflex, or (2) by changes in the composition of the blood, either with regard to the percentage of sugar itself or because of the appearance in it of decomposition products of glucose or of some special exciting agent or hormone. In order to ascertain the relative importance of these methods of correlation between the places of supply and demand of glucose in the normal animal, it is necessary to investigate the conditions under which an excessive discharge of glucose occurs either because of overstimulation of the nervous control, or because of the presence of exciting substances (hormones) in the blood. The glycogenolytic function can be excited through the nervous system in a variety of ways so as to cause hyperglycemia and glycosuria. This constitutes one form of experimental diabetes. In laboratory animals mechanical irritation of the medulla oblongata and stimulation of the great splanchnic nerves act in this way. Similar stimula- tion may also occur under certain conditions in man. Excitation as a result of changes in the composition of the blood can be produced experimen- tally by certain drugs (phlorhizin), or by the removal of certain of the 704 METABOLISM ductless glands or the injection of extracts prepared from them, such as epinephrine. Nerve Control and the Nervous Forms of Experimental Diabetes. The simplest experimental condition which illustrates the relationship between the nervous system and the blood sugar is electrical stimulation of the great splanchnic nerve in animals in which, by previous feeding with carbohydrates, a large amount of glycogen has been deposited in the liver. By examination of the blood as it is discharged into the vena cava from the hepatic veins, the increase in blood sugar is very evident in from five to ten minutes after the first application of the stimulus; but it is not until later that a general hyperglycemia becomes estab- lished. The conclusion which we may draw from these results is that the splanchnic nerve contains efferent fibers controlling the rate at which glycogen becomes converted to glucose in the liver. The center from which these fibers originate is situated somewhere in the medulla oblongata, for the irritation that is set up by puncturing this portion of the nervous system with a needle yields results similar to those which follow splanchnic stimulation. This " glyco genie" or diabetic center, as it has been called, must be provided with afferent impulses. Such im- pulses have indeed been described in the vagus nerves, but their dem- onstration is by no means an easy matter on account of the disturbance in the respiratory movements coincidently produced by the stimulation. The changes that such disturbances bring about in the aeration of the blood may in themselves be responsible for the hyperglycemia (see page 348). It can at least be said that when the respiratory disturbances are guarded against, as by intratracheal insufflation of oxygen, vagal hyper- glycemia is much less marked, if not entirely absent. But this question awaits more thorough investigation. The increased glycogenolysis which results from stimulation of the efferent fibers in the splanchnic nerves may depend either on a direct control exercised over the glycogenic functions of the hepatic cells, or on the discharge into the blood of some hormone which excites the glycogenolytic process. It must furthermore not be lost sight of that the glycogenolysis may be secondary to local asphyxial conditions in the liver cells resulting from vasoconstriction. From their anatomic position, the adrenals are to be thought of as the source of the hormone, and evidence that splanchnic hyperglycemia is entirely due to hyper- secretion from these glands has seemed to be furnished by the fact that soon after they are extirpated splanchnic stimulation no longer pro- duces hyperglycemia. There is also no doubt that the nervous system, acting by way of the splanchnic nerves, does exercise a control over the discharge of the internal secretion of the adrenal glands and that extracts of the gland, which we must suppose act in the same way as the internal THE METABOLISM OF THE CARBOHYDRATES 705 secretion, cause hyperglycemia when injected intravenously (epinephrine hyperglycemia and glycosuria) (page 776). There are, however, several experimental facts which do not conform with such a view. Thus, after complete severance of the hepatic plexus of nerves, stimulation of the splanchnic nerve does not cause the usual degree of hyperglycemia, whereas electric stimulation of the peripheral end of the cut plexus does cause it. On the one hand, therefore, there is evidence that stimulation of the efferent nerve path above the level of the adrenals has no effect on the sugar production of the liver in the absence of these glands ; and on the other, we see that when the glands are present, stimulation of the nerve supply of the liver is effective, even though the point of stimulation is beyond them. There is but one con- clusion that we may draw namely, that the functional integrity of the efferent nerve-fibers that control the glycogenolytic process of the liver depends on the presence of the adrenals, very probably because of the hormone which the glands secrete into the blood. This conclusion is corroborated by the fact that stimulation of the hepatic plexus, even with a strong electric current, some time after complete removal of both adrenals, is not followed by the usual degree of excitement of the glycogenolytic process. These experiments demonstrate a possible relationship between the nervous control, and at least one form of hormone control, of the sugar output of the liver. They indicate that when a sudden increase of blood sugar is required, the glycogenic center sends out impulses which not only directly excite the breakdown of glycogen in the he- patic cells, but also simultaneously influence the adrenals in such a man- ner as to produce more epinephrine in the blood and so augment the ac- tion of the nerve impulse. It must be pointed out that the less marked hyperglycemia, following piqure and splanchnic stimulation in adrenal- ectomized animals may be due to the moribund condition of the animal, for Stewart and Rogoff have shown that piqure causes the usual degree of hyperglycemia when it is performed on cats some time after removal of one adrenal and complete denervation of the other. At the time of the piqure the animals were well nourished and apparently normal in every way (cf. page 787). We are as yet quite in the dark as to the mechanism by which the nerve impulses or the hormone brings about increased glycogenolysis. It must consist of a removal of the influence that prevents glycogenolysis from occurring in the normal liver, for it has been shown by direct observation that there is no increase in the amount of glycogenase present in extracts of the liver removed from diabetic animals over that present in extracts of the liver of normal animals. The possible nature of this influence has already been discussed (page 702). The change may consist either in a loosening of the combination between the glycogen and the protoplasm of the liver cell, or in a removal of the chemical influence that ordinarily prevents the glycogenase from at- 706 METABOLISM tacking the glyeogen. In the former case the glycogen liberated from its union with the sustentacular substances would either become attacked by the glycogenase present in the liver cell itself or it would first of all migrate, as glyeogen, into the blood capil- laries and there be attacked by the blood glycogenase. Evidence for the possibility of the occurrence of such a process has already been given (page 702). The chemical change referred to under the second possibility might consist in an alteration in the hydrogen-ion concentration of the liver cell, a change, however, which for obvious reasons it is impossible to investigate. Nervous Diabetes in Man. The main interest attaching to the inves- tigation of these nervous forms of experimental diabetes depends on the insight which they afford us into the nature of the mechanism by which a prompt mobilization of glucose may be brought about in the normal animal. There is also some evidence that a relationship may exist be- tween certain of the clinical varieties of the disease in man and repeated excitation of glycogenolysis brought about by nerve stimulation. In- creased glucose output from the liver as a result of nerve excitation may be a normal process, but there is reason to believe that frequent repetition of this process tends to induce a permanent rise in the glucose level of the blood and therefore a tendency to diabetes. There have recently been collected several facts which lend some support to this view. The frequent occurrence of diabetes in those predisposed by inheritance to neurotic conditions, or in those whose daily habits entail much nerve strain, and the aggravation of the symptoms which is likely to follow when a diabetic patient experiences some nervous shock, all point in this direction. Diabetes is common in locomotive engineers and in the captains of ocean liners that is, in men who in the performance of their daily duties are frequently put under a severe nerve strain. It is apparently in- creasing in men engaged in occupations that demand mental concentra- tion and strain, such as in professional and business work. Cannon 23 found glycosuria in four out of nine students after a severe examination, but only in one of them after an easier examination.* In the urines of twenty-four members of a famous football squad, sugar was found pres- ent in twelve immediately after a keenly contested game. Anxiety and excitement must have been responsible for its appearance, since five of the twelve players were substitutes who did not get into the game. Although these nervous conditions, by excitement of hepatic glyco- genolysis, produce at first nothing more than an excessive discharge of sugar into the blood a condition which is exactly duplicated in our laboratory experiments by stimulation of the nerve .supply of the liver their repetition may gradually lead to the development of a permanent form of hyperglycemia. To prevent the repetition of these transient *We have been unable to confirm this observation even though the examinations were made unusually "nerve-racking." THE METABOLISM OF THE CARBOHYDRATES 707 hyperglycemias must be one of our aims in the treatment of early stages of the disease. It is possible that the relationship of nerve strain to the incidence of diabetes has been exaggerated, for it has been stated that there was a marked decrease in the number of cases of this disease in Berlin during the later years of the war. During this period the nerve strain was very great, but the diet was greatly restricted and it may be in light of the latter fact that the disease is related more to dietetic habits than to con- ditions of nerve strain (Magnus Levy). In view of the observations, it is significant that diabetes is said to be increasing in frequency in the United States since prohibition came into effect. The excessive consump- tion of sugar is possibly responsible for this condition. Although there can be no doubt that the glycogenic function of the liver is subject to nerve control, it is probable that its control by hor- mones is of equal if not greater importance. This dual control of a glandular mechanism is by no means unique for the glycogenic function, for we have already seen it to exist in the case of the gastric glands and the pancreas, and it is probable that it also exists in the case of the thyroid. It may well be that the nerve control of the glycogenic function has to do only with those transitory changes in sugar produc- tion that would be demanded by sudden activities of muscle, and that the hormone control has to do with the more permanent process of build- ing up and breaking down of glycogen to meet the general metabolic requirements of the tissues. HORMONE CONTROL AND PERMANENT DIABETES Nervous excitation can explain only transitory increases in blood sugar, the more permanent hyperglycemias being dependent upon some dis- turbance in the hormone control of carbohydrate utilization. This dis- turbance is a much more serious affair than that produced by nervous excitation. In the latter case the hyperglycemia ceases whenever all of the glycogen stores of the liver have been exhausted; whereas a dis- turbance in the hormone control, besides causing as its first step a breakdown of all the available glycogen, goes on to cause a production of sugar out of protein. A process of gluconeogenesis (new formation of glucose) becomes superadded on one of glycogenolysis. To ascertain the nature of this hormone and the mechanism of its action has been the object of most of the researches on those forms of diabetes that are produced by changes in certain of the ductless glands. The following possibilities may be considered: (1) that the controlling agency is the concentration of glucose in the blood; (2) that it is the 708 METABOLISM presence in the blood of decomposition products of glucose ; (3) that it is due to a special hormone produced from some ductless gland. Con- cerning the first of these possibilities, it is supposed that the mechanism involved is dependent on the law of mass action ; namely, that glycogen be- comes converted into glucose whenever the blood flowing to the liver con- tains less than its normal concentration of glucose, and conversely, when this blood contains an excess of glucose, as during absorption, that a glycogen- building process occurs. Although there can be little doubt that the process of glycogen formation or destruction will depend to a certain extent upon the amount of glucose present in the blood flowing to the liver cells, yet it is impossible that this can be an important means in the control that exists between sugar production by the liver and sugar consumption by the tissues, because the sugar that is added to the portal blood during absorption would mask any depletion caused by sugar consumption. The second possibility that the hormone is some decomposition prod- uct of glucose would appear to have some support, if we consider this hormone to be an acid product (carbon dioxide or lactic acid) produced by sugar metabolism, for it is known that an increase in the hydrogen-ion concentration of the blood flowing to the liver cells excites a glycogen- olysis. As we have already seen, however, it is difficult to secure ex- perimental evidence, in anesthetized animals at least, that glycogen- olytic activity is readily excited in this way. The third possibility that some specific hormone may exist in the blood exciting the glycogenolytic process is investigated by producing disturbances involving various of the ductless glands, particularly the pancreas, the adrenals, the parathyroids and the pituitary. The influ- ence of certain of these glands may be closely bound up with that exercised through the nervous control, as we have seen to be the case with the adrenal gland. Whether it is by the production of hormones directly necessary for proper carbohydrate metabolism, or by the re- moval from the -blood of such substances as interfere with this process, that the ductless glands functionate, is one of the main problems we have to consider. , Utilization of Glucose in Tissues. Although the experimental diabetes induced by disturbances in the function of the ductless glands is dependent in the first instance on an upset of the glycogenic function and later on glu- coneogenesis, the utilization of glucose in the tissues ultimately becomes interfered with. It is therefore important that we should digress for a moment to consider briefly what is known regarding the process by which sugar becomes utilized in the organism. That glucose becomes used up by active muscle there can be no doubt. Thus, if the muscles THE METABOLISM OF THE CARBOHYDRATES 709 of one leg in the frog are tetanized, the glycogen content, compared with that of the other leg, will be found to be diminished. At first sight it might appear that the easiest way to study the utiliza- tion of glucose in the muscles would be to compare its concentrations in the blood flowing to and coming from them. The muscle that has been most successfully employed in studies of this kind has been the heart. Some years ago Starling and Knowlton 24 sought to measure the consump- tion of glucose by the excised mammalian heart, by comparing the per- centage in the perfusion fluid at various periods during the observation. Patterson and Starling 25 showed later, however, that the results obtained by such a method can furnish no criterion of the actual consumption of glucose by the tissue on account of the fact that the heart itself may store away large quantities of carbohydrate in an unused state, i.e., as glycogen. After allowing for the glycogen as well as for the glucose consumption by the lungs, Cruikshank and Patterson 64 computed that the heart (of the cat) uses 1.5 mg. glucose per gram per hour. Other investigators have thought to study the utilization of glucose by observing the rate at which it disappears from drawn blood kept in a sterile condition at body temperature for some hours after death. This process is called glycolysis, and it has been assumed that the process is similar to that which occurs in the tissues themselves an assumption, however, for which there is no warranty. Indeed, it may readily be shown that the glycolysis occurring in blood has very little if anything to do with the utilization of glucose in the tissues, for it has been found that glucose disappears from drawn blood very slowly indeed when compared with the rate at which it disappears from the blood of animals in which the addition of glucose from the liver has been prevented by removal of this viscus (Macleod). 26 A third method for studying the utilization of glucose consists in observing the respiratory exchange of animals. In normal animals the injection of glucose causes an increase in the carbon-dioxide excretion and a rise in the respiratory quotient, which it will be remembered is a ratio expressing the relationship between the amount of carbon dioxide excreted and of the oxygen retained in the organism. When carbohy- drate is undergoing combustion, the quotient is nearly 1, whereas with that of protein it is about 0.7 (see page 582). By observing the quotient under given conditions one can compute the proportions of carbohydrate and of fat and protein that are undergoing metabolism. In the hands of Murlin and others, 27 this method has proved of some value in settling certain questions concerning the utilization of glucose in normal and diabetic animals ; but the results must be interpreted with great care on account of the fact that temporary changes in the blood may cause a 710 METABOLISM greater or a less expulsion of carbon dioxide from it. Thus, if acids appear in the blood, they will dislodge carbon dioxide, and apparently cause the respiratory quotient to rise. Alkalies, on the other hand, ap- parently cause the quotient temporarily to fall, and unless the observa- tions are done over a long period of time and with great care, faulty conclusions are very apt to be drawn from the results. Starling and Evans 65 have measured the respiratory exchange in the heart lung prep- aration (see page 163) and have found that the heart uses an average of 3.2 c.c. of oxygen per gram an hour when doing moderate work, the E.Q. being 0.85. This corresponds to 1.6 mg. glucose consumption per gram per hour, thus corroborating the results obtained by direct estima- tion of glucose. Diabetes and the Ductless Glands We are now in a position to consider the forms of experimental dia betes produced by disturbances in the ductless glands. Relationship of the Pancreas to Sugar Metabolism. In no other of the many causes of diabetes has greater interest been shown than in that due to disturbance in the pancreatic function. Many of the earlier clinicians who followed cases of diabetes mellitus into the postmortem room, noted that definite morbid changes in the pancreas were a fre- quent accompaniment of the disease. Prompted by these observations, several investigators attempted experimental extirpation of the gland, but did not succeed in producing glycosuria in the few animals that survived the operation. Their failure, no doubt, was due to incom- plete extirpation. To reduce the severity of the operation, Claude Ber- nard injected oil into the pancreatic duct, and tied it; but he succeeded in keeping only two dogs alive for any length of time, and these did not exhibit glycosuria. Neither were other investigators that adopted similar methods any more successful. It looked as if the pancreas had no relationship to carbohydrate metabolism. In the year 1889 Minkowski and von Mering in Germany, and de Dominicis in Italy, by thorough extirpation of the gland, succeeded in producing in dogs a marked and persistent glycosuria, accompanied by many of the other symptoms of diabetes. The first two authors attributed the condition to removal of an internal secretion. The course of the diabetes produced by complete pancreatectonry is, however, somewhat different from that usually observed in man. It is extremely acute from the start, the G:N ratio being 3.6:1 (see page 696), and it is unaccompanied by any of the classical symptoms seen in the clinical condition. Experimental pancreatic diabetes can, however, be made to simulate very closely the disease in man. This was first of THE METABOLISM OF THE CARBOHYDRATES 711 all demonstrated by Sandemeyer, who found that if the greater part of the pancreas was removed, the animals for some months, if at all, were only occasionally glycosuric, but later became more and more frequently so, until at last the condition typical of complete pancreatectomy super- vened. Similar results have more recently been obtained by Thiroloix and Jacob, in France, and by Allen in this country. These investigators point out that different results are to be expected according to whether the portion of pancreas which is left does, or does not, remain in con- nection with the duodenal duct. When this duct is ligated, atrophy of any remnant of pancreas that is left is bound to occur, and this is asso- ciated with rapid emaciation of the animal, diabetes and death. When the remnant surrounds a still patent duct, a condition much more closely simulating diabetes in man is likely to become developed one, namely, in which there is, for some months following the operation, a more or less mild diabetes, which, however, usually passes later into the fatal type. It is, of course, difficult to state accurately what proportion of the pancreas must be left in order that the above described condition may supervene. Leaving a remnant amounting to from one-fifth to one-eighth of the entire gland is commonly followed by a mild diabetes, whereas if only one-ninth or less is left, a rapidly fatal type de- velops. As in clinical experience, the distinguishing feature between the mild and the severe types of experimental pancreatic diabetes is the tolerance toward carbohydrates. In the mild form, no glycosuria develops unless carbohydrate food is taken; in the severe form, it is present when the diet is composed entirely of flesh. It is thus shown that "by removal of a suitable proportion of the pancreas, it is possible to bring an animal to the verge of diabetes, yet to know that the animal will never of itself become diabetic. . . . Such animals, therefore, constitute valuable test objects for judging the effects of various agencies with respect to diabetes' 7 (Allenis). By these means it becomes theoretically possible to investigate, on the one hand, other conditions which will have an influence similar to removal of more of the gland, or, on the other, condi- tions which might prevent the incidence of diabetes, even though this extra portion of pancreas is removed. Allen has shown that the continued feeding of a partially depancreatized dog with excess of carbohydrate food will surely convert a mild into a ?vere case of diabetes, and in one experiment he succeeded in bringing ibout the same transition by performing puncture of the medulla that is, by creating an irritative nervous lesion. By none of the other means usually employed to produce experimental glycosuria could the mild case be made severely diabetic, although this was accomplished in one inimal after ligation of the portal vein. To the clinical worker the value )f these results lies in the fact that they furnish experimental proof that so-called latent case of diabetes that is, one that has a low tolerance *alue for carbohydrates may be prevented from developing into a severe case by proper control of the diet. 712 METABOLISM The Pathogenesis of Pancreatic Diabetes The certainty with which diabetes results from pancreatectomy in dogs, as well as the frequent occurrence of demonstrable lesions of the pan- creas in diabetes in man, leaves no doubt that this gland must be in some way essential in the physiological breakdown of carbohydrates in the normal animal, but how, we can not at present tell. All we know is that the first change to occur after the gland is removed is a sweeping out of all 'but a trace of the glycogen of the liver, although the glycogen of the muscles may remain ; indeed, in the cardiac muscle there may be more than the usual amount. 28 Nor can any glycogen be stored in the liver when excess of carbohydrates is fed. After the glycogen has disappeared, gluconeogenesis sets in, so that the tissues come to melt away into sugar, and all the symptoms of acute starvation, associated with certain others that are possibly due to a toxic action of the excess of sugar and of other abnormal products in the blood such as ketone bodies make their appear- ance. Accompanying the gluconeogenesis another very definite abnormality in metabolism becomes evident namely, an inability of the tissues to ~burn sugar. This fact is ascertained by observing the respiratory quotient. When glucose is added to the blood in the case of a completely diabetic animal, no change occurs in the quotient, whereas in normal animals under such conditions it rises almost to 1.0 (cf. page 582). There are, therefore, two essential disturbances of carbohydrate metabolism in pancreatic diabetes overproduction of sugar and aboli- tion of the ability of the tissues to use it. It becomes important for UP to see whether the tissues exhibit this inability to use sugar when they are isolated from the animal ; for if they should, a much more searching investigation of the essential cause of their inability would be possible than is the case when they are functioning along with the other organs and tissues. The earlier experiments, of Lepine and his pupils, which seemed to show that diabetic blood did not possess the glycolytic power of normal blood; and those of Cohnheim, from which it was concluded that mixtures of the expressed juices of muscle and pancreas, although ordinarily destroying glucose, failed to do so when they were taken from a diabetic animal, are now known to be erroneous. The failure to show a depres- sion of glycolytic power by these methods prompted Knowlton and Starling 2 * to investigate the question whether any difference is evident in the rate with which glucose disappears from a mixture of blood and saline solution used to perfuse a heart outside the body, according to whether the heart was from a normal or a diabetic dog. In the first series of observations which these workers made, it was thought that the normal heart used glucose at the rate of about 4 mg. per gram of heart substance per hour; whereas that of a diabetic (depancreatized) animal used less than 1 mg. If such striking differences in the rate of sugar consumption could make themselves manifest for so relatively small a mass of muscular tissue as that of the heart, it is THE METABOLISM OF THE CARBOHYDRATES 713 permissible to assume that a much more striking difference could be demonstrated when the perfusion fluid is made to traverse all or practically all of the skeletal muscles, as well as the heart. For this purpose an eviscerated animal may be employed that is, one in which the abdominal viscera are removed after ligation of the celiac axis and mesenteric arteries, and the liver is eliminated by mass ligation of its lobes. Using such preparations, E. G. Pearce and Macleod29 found that the rate at which glucose disappears from the blood, although very irregular, is in no way different in completely diabetic as compared with normal dogs. They were thus unable to confirm any of Knowlton and Starling's earlier conclusions. As has already been stated Patterson and Starling subsequently pointed out that a serious error was involved in the earlier perfusion experiments, partly on account of a remarkable but irregular disappearance of glucose from the lungs, and partly because the diabetic heart may contain a considerable excess of glycogen, from which its demands for sugar may be met without calling on that of the perfusion fluid. More recent work by Starling and Evans, in which the respiratory ex- change of the heart in heart lung preparations from diabetic (pancreatic) dogs was determined, has revealed a low R.Q. (0.71) although the oxygen consumption was normal. These workers consider that their results indi- cate "a depression or abolition of the power of the diabetic tissue to utilize carbohydrate." This conclusion, however, awaits confirmation by more direct methods. Whatever may be the fundamental cause for the development of dia- betes following pancreatectomy it is important to know whether the condition supervenes because of the absence of an internal secretion, or hormone, furnished by the pancreas or merely because some local action of the gland has been removed. Attempts have been made (by Hedon, Carlson and Drennan and others) to demonstrate the presence of such a hormone in the blood of normal animals but with doubtful success. I That one may be present has however been shown by A. H. Clark, 65 who perfused the isolated pancreas of a dog for some time with Locke's solu- tion and then used this fluid to perfuse the isolated heart of the same animal. He found under these conditions (i.e., of combined perfusion) that sugar disappeared much more rapidly from the perfusion fluid than when fresh Locke's solution was perfused through the heart from the beginning. This indicates that the pancreas must contribute some sub- stance which is carried by the blood to the heart, and presumably other muscles, where it enables the muscle to consume sugar. Until recently, however, no constant success has attended the injection of pancreatic extracts in reducing the hyperglycemia or other symptoms of diabetes. Thinking that this might be due to the destruction of the hor- mone by the trypsin present in such extracts, Banting and Best, working in the author's laboratory, made extracts either from the residue which remains some weeks after ligation of the pancreatic ducts or from early fetal pancreas. In both these cases trypsin production in the gland is absent, or nearly so, but it is believed that the Isles of Langerhans are 714 METABOLISM functioning. Injected subcutaneously into completely depancreated dogs, these extracts caused a marked lowering of blood sugar with great re- duction in the sugar excretion by the urine and when the injections were repeated daily the animals survived the operation for several weeks longer than is usually the case and were in excellent condition. Later, in collaboration with Collip, extracts were prepared by alcohol from fcl. 15 16 17 Fig. 190-A. Curves showing effect of "insulin" in a case of diabetes mellitus. I, Daily excretion of glucose in urine for periods before, during and after administration of Insulin; //, Daily excretion of total ketone bodies before and during administration (from Jan. 23rd on); ///, The effect of one injection (4 c.c. subcutaneously) of insulin on the blood sugar; IV, The two hourly excretion of glucose for several days during administration. adult beef pancreas and their effects on human diabetes observed by Campbell and Fletcher. The results as shown in Fig. 190-A justify the ifiope that when this extract, for which the name "insulin" is suggested, \is given subcutaneously for some time to diabetic patients, the pancreas may reacquire its normal function, or at least, with control of the diet, THE METABOLISM OF THE CARBOHYDRATES 715 may be restored sufficiently to effect a practical cure. In any case "insulin" has a profound effect on carbohydrate metabolism for we have found (in collaboration with Hepburn, Latchford and Noble) that it causes the respiratory quotient to rise almost to unity when given along with sugar to diabetic animals (and also to diabetic patients), in which also it causes large amounts of glycogen to appear in the liver. Insulin also greatly reduces the blood sugar in normal rabbits and in rabbits showing hyperglycemia as a result of piqure, injection of adrenalin or asphyxia. DIABETIC ACIDOSIS OR KETOSIS Nature and Cause. Much confusion has existed in medical literature over the correct definition of acidosis, mainly because the term was first used for the particular variety of the condition observed in the later stages of diabetes mellitus. The acids which accumulate in the tissue fluids in this disease are acetoacetic and /3-oxybutyric, which are re- lated to acetone and are derived from fatty acids by a faulty metabolism (see page 737). The essential cause of the acidosis is therefore en- tirely different from that in nephritis; in diabetes foreign acids are added to the blood, whereas in nephritis the acids of a normal metab- olism accumulate because of faulty excretion through the kidneys. The usual signs of acidosis exist in both cases, because the surplus of acid depletes the store of bicarbonate and causes changes in the alveolar C0 2 , in the C0 2 -absorbing power of the blood, in the reserve alkalinity, and in the acid excretion by the kidney. It is important to recognize the special nature of diabetic acidosis by a separate name ketosis. The chemical processes by which the kQtone bodies are produced are discussed elsewhere (page 737). It remains for us to consider the general nature of the metabolic disturbance responsible for the appear- ance of ketone bodies in diabetes. For the thorough combustion of fat in the animal body a certain amount of carbohydrate must be simultaneously burned. Fat evidently is a less readily oxidized foodstuff than sugar; it needs the fire of the burning sugar to consume it. If the carbohydrate fires do not burn briskly enough, the fat is incompletely consumed; it smokes, as it were, and the smoke is represented in metabolism by the ketones and derived acids. Such a closing down of the carbohydrate furnaces may be brought about either by curtailment 4 of the intake of carbohydrates, as in starvation (page 600), or by some fault in the mechanism of the furnace itself, as in diabetes. Besides fat, protein may also contribute to the production of ketones when carbohydrate combustion is de- 716 METABOLISM pressed. Fundamentally, therefore ketosis in diabetes is due to the same cause as in starvation namely, an improper adjustment between the metabolisms of fat and carbohydrate. Bearing these principles in mind, it is easy to see how the intensity of acidosis which develops during starvation will depend upon the re- lative metabolism of carbohydrate, on the one hand, and of fat and protein, on the other; it will therefore depend on the amounts of these foodstuffs which have been stored in the organism, and this again will depend on the nature of the diet previous to the starvation period. For the first few days following entire abstinence from food in a healthy, well-nourished individual, very few if any ketones will be excreted in the urine, because the carbohydrate stored in the body as glycogen has sufficed during this time to maintain the proper proportion between fat and carbohydrate. Afterwards, however, their appearance is to be ex- pected, because the glycogen stores become exhausted long before those of fat. If starvation is still further prolonged, a stage will come when the fat, as well as the carbohydrate, is used up so that the organism has now to subsist on protein alone. When this stage arrives, the ketones will diminish, for, although they might be derived from certain of the amino acids, yet this does not actually occur, because a large part of the protein molecule (nearly half) also becomes changed into glucose, which by burning, as above explained, prevents the formation of ketones from the other part of the molecule. For the same reasons, marked acidosis will not be expected to occur during any stage of starvation in lean persons, who from the start must utilize mainly their stored protein to supply the fuel upon which to live. In diabetes exactly the same principles apply, but to an organism in which the ability to metabolize carbohydrate has been depressed, so that "the maximum rate at which dextrose can be oxidized is fixed at some level which is absolutely lower than in health. ' ' 30 Therefore, since a cer- tain proportionality must exist between the rates of combustion of fat and carbohydrate, the diabetic can thoroughly oxidize less fat; in other words, an amount of fat which could readily be burned in a healthy body is improperly burned by the diabetic, and ketones and their acids ac- cumulate. Starvation Treatment. "In order to check a diabetic acidosis, it is necessary to restore the proper ratio of fatty acid to glucose oxidation/' which can best be done by starvation, rest in bed and warmth. But this treatment may not at first suffice, because we have to deal not only with the acidosis bodies derived from fat, but with those which can be derived from protein on account of the diabetic organism having lost the power THE METABOLISM OF THE CARBOHYDRATES 717 even of burning the glucose which is derived from this foodstuff. By persistence in the starvation, however, the ability of the organism to utilize carbohydrate usually becomes so far restored that enough burns to prevent acidosis. Every case of diabetes can not, therefore, be expected to react in the same way to starvation, the determining condition being the relation between the quantities of glycogen and fat stored in the body at the outset of the fasting period. This relationship depends on the nature of the previous diet. To sum up, "fasting will lower acidosis either in health or in diabetes, if it has the effect of stopping a one-sided metabolism and throwing the tissues on a more nearly balanced ratio of fatty acids and glucose" (Woodyatt). A practical point may be noted here namely, that there is likely to be more danger of serious acidosis developing during starva- tion in fat than in lean diabetics. The importance of our appreciation of these facts in the starvation treatment of diabetes will be self-evident. Insulin Treatment. As already pointed out, subcutaneous injections of properly prepared extracts of pancreas remove all the cardinal symp- toms of diabetes both in man and laboratory animals and also cause the ketone bodies to disappear from the urine. Life has also been consider- ably prolonged in depancreated dogs by such injections and the subjec- tive symptoms and muscular vigor of diabetic patients markedly improved. CHAPTER LXXIX FAT METABOLISM Before considering the physiology of fats, a few of the most essential points regarding their chemistry may be of assistance. THE CHEMISTRY OF FATTY SUBSTANCES It is usual to classify all substances that are soluble in ether as lipoids. They in- elude fatty acids, neutral fats, cholesterols, cholesterol esters, and phospholipins. The fatty acids belong to two main homologous series, which differ from each other with regard to whether they are saturated or unsaturated. A saturated fatty acid is typified by palmitic, whose formula is CH 3 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH o - CH^-CH^CI^ CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -COOH, or CH 3 -(CH 2 ) 14 -COOH; that is to 2 say 2 it i s 2 a MgheJ member of the series to which acetic acid (CH -COOH) belongs, differing from the latter in having fourteen extra methyl radicles, each joined to its neighbor by one bond or saturated linking on either side. Another member of this series is stearic, in which there are sixteen extra CH 2 groups (CH 3 (CH 2 ) i6 -COOH). An unsaturated fatty acid is oleic (CH 3 (CH 2 ) 7 CHr=CH-(CH 2 ) 7 -COOH). Its unsaturation is rep- resented in the formula by the double bond or unsaturated linking, which it will be seen occupies a position in the middle of the molecule, the other methyl radicles being linked together by single bonds. The fatty acids readily combine with alkali to form soaps; thus, CH 3 (CH 2 ) i4 -COOH + KOH = CH 3 (CH 2 ) i4 -COOK + H 2 0, (palmitic acid) (soap) the reaction being analogous to that by which acetic acid forms an acetate with alkalies. In place of being combined with alkali, the COOH (carboxyl) group of fatty acids may combine with alcohols to form substances called esters. Thus, acetic acid and ethyl alcohol from ethyl acetate, CHCOO 2o = (acetic (ethyl' (ethyl acetate) acid) alcohol) When the alcohol thus united with fatty acid is glycerol (glycerine), in which there are three OH (hydroxyl) groups, the resulting ester called triglyceride is neutral fat. Tripalmitin has the formula: CH,-OOC-C 15 H 31 CH -OOC-C ]5 H 31 CH 2 -OOC-C 15 H 31 . By boiling neutral fats with alkali the fatty acid radicles are split off as soaps, leaving the glycerol. This process is called saponification, and it may be effected in 718 FAT METABOLISM 719 many othor ways, as for example by heating with steam or by the action of special enzymes called Upases, which are widely distributed in plants and animals. The natural fats are usually a mixture of triglycerides, and their differences in properties are dependent upon the relative amounts of fatty acids present. The three most important in animal fats are tripalmitin, tristearin and triolein. It is essential in the study of fat metabolism that we should know the most important methods "by which the proportion of fatty acids present in a mixed fat is determined. These methods are as follows: 1. The melting point. Olein is liquid at C.; palmitic acid melts at 62.6 C. ; and stearic at 69.3 C. The solidity of animal fats depends on the proportion of olein, palmitin and stearin present. Mutton fat, for example, is much stiffer than pig fat because it contains less olein and more stearin. The melting points of fats from different parts of the body may also vary. 2. The acid number indicates the amount of free fatty acid mixed with the fat, and is determined by titrating a solution of a weighed quantity of the fat in alcohol with a N/10 alcoholic solution of KOH, phenolphthalein being used as indicator. 3. The saponification value indicates the total amount of fatty acid present, both that which is free and that combined with glycerol. It is determined by heating a weighed amount of fat with an exactly known amount of alcoholic KOH (determined by titration with standard acid). After saponification is complete, titration of the mixture shows how much alkali has been used to combine with the fatty acid. This is the saponification value. 4. The ester value indicates the amount of fatty acid combined with glycerol, and is obtained by subtracting the acid value from the saponifieation value. Besides these there are two values, known as the iodine and the Beichert-Meissl val- ues, that are of importance because they depend on certain cliar act eristics of the fatty- acid radicles. 5. The iodine value indicates the amount of unsaturated fatty acids present, or the number of double bonds. It depends on the fact that iodine, like many other substances, is capable of directly attaching itself to the fatty-acid chain wherever double bonds exist. 6. The Eeichert-Meissl value indicates the amount of volatile soluble acid present in the fat. It is determined by first of all saponifying the fat, then decomposing the soap by mixing it with mineral acid and distilling the liberated fatty acid, the distil- late being collected in a known amount of standard alkali and titrated. It is a value that is not of very great use in physiological investigations, but it is so in connection with food chemistry. Since volatile acids are present in butter, the Beichert-Meissl value helps us to distinguish between butter and margarine. Fat is insoluble in water but soap is soluble, forming a colloidal solution which )resents phenomenon 'of surface aggregation of molecules. This consists in the con- centration of the soap both at the free surface of the liquid, where a skin may form, id at the interfaces between the soap solution and any undissolved particles present in it. This pellicle-formation around the particles prevents them from running together that they remain suspended, thus forming an emulsion. An emulsion may there- fore be formed either of neutral fat of any other physically similar substance. When fat itself is used, there is usually enough free fatty acid admixed with it to make it unnecessary in forming the emulsion to do more than shake the fat with weak sodium- irbonate solution. With other substances not containing any free fatty acid, some soap should be added. To preserve the emulsion it is often useful to add some mucilage, the emulsified state, neutral fats are much more readily attacked by lipases than rtien they are present in an unemulsified state. Thus, emulsified fats are "digested" 720 METABOLISM by the relatively small amounts of lipase present in the stomach, whereas neutral fats themselves are not so. Fatty acids also exist in nature in combination not only with the triatomic alcohol, glycerol, but also with monatomic alcohols such as cholesterol. These cholesterol fats differ from the glycerol fats in being very resistant towards enzymes and microorgan- isms. They are therefore used for protective purposes in the animal economy; for example, they occur in the sebum, the secretion of the sebaceous glands, where they serve to moisten the hairs and skin. They are also present in cells, in which it is prob- able they take an important part in forming the skeleton of the cell. Cholesterol is absorbed from the intestine; it is always present in the blood both in plasma and in corpuscles; and it is an important constituent of bile, from which it may separate out in the bile passages and form calculi (gallstones). In the cells themselves the lipoids are represented mainly by compounds of a some- what more complex structure namely, the phospliolipins. As their name indicates, these consist chemically of phosphoric acid combined with neutral fat and with a nitro- genous base, cholin. The best known of the phospholipins is lecithin, which is widely distributed in the animal body (present in blood and bile as well as in all cells). Other phospholipins present in nervous tissue are cephalin, cuorin and sphyngomyelin. There are various lecithins distinguished from one another by the fatty-acid radicles which they contain. Distearyl-lecithin, for example, has the formula: CH 2 -0-OC(CH 2 ) 14 -CH 3 CH -0-OC(CH 2 ) 14 -CH 3 (stearic acid) CH 2 -0 O (glycerol) P OH OCH 2 -CH 2 -N(CH 3 ) 2 (phosphoric I acid) OH (choline) This complex molecule can readily be split up by hydrolysis (warming with baryta water) into: glycero-phosphoric acid, CH 2 -OH CH -OH CH 2 - O O \ ; choline, N OH C 2 H 4 OH (CH 3 ) 3 (oxy-ethyl-ammonium OH hydroxide) ; and fatty acids. With hydrochloric acid, choline forms a salt which readily forms a double salt with plantinic chloride. Since this double salt forms characteristic crystals, it is used to identify and separate lecithins. For quantitative purposes, however, it is more suitable to determine lecithin indirectly by the amount of phosphoric acid present in an ethereal extract of the organ or tissue. Evidence is constantly accumulating to show that lecithin is an extremely impor- tant constituent of cells; indeed, it seems to be the intermediate stage in the utiliza- tion of neutral fats by protoplasm. Its phosphorus also probably serves as the source of this element for the construction of nucleie acid (see page 669). In nervous tissues FAT METABOLISM 721 it is often associated with carbohydrate molecules (galactose), forming the substance known as cerebrin. It may therefore have some role to play in carbohydrate metabo- lism. Some workers also attribute to lecithin an important function in the transference of substances through cell membranes. When mixed with water it swells up by imbibi- tion, and if crystalloids or other substances are dissolved in the water, a means is offered for bringing water-soluble and fat-soluble substances into intimate contact. DIGESTION OF FATS A certain amount of fat, especially when it is in an emulsified condi- tion, can be digested in the stomach by the lipase contained in the gas- tric juice. Most of it, however, is digested in the small intestine, into which as we have seen, it is gradually discharged suspended in the chyme. For this intestinal digestion of fat both pancreatic juice and bile are nec- essary. This is easily shown in the rabbit, in which the pancreatic duct enters the intestine at a considerable distance below the bile duct. If the mesentery is inspected during the absorption of fatty food, no fat in- jection of the lymphatics will be noted between the bile and the pan- creatic ducts but only below the latter. In the dog, in which both the bile and the main pancreatic ducts enter the intestine at about the same level, fat injection of the lymphatics starts at this point, but if the bile duct (or rather the gall bladder) is transplanted at some distance down the intestine, it will be found that the injection of the lymphatics with fat occurs only below the new point of insertion of the bile duct. Removal of the pancreas interferes very materially with the absorption of fat. In man, for example, absence of the pancreatic juice alone di- minishes the absorption of fat by 50 or 60 per cent. If the bile is also absent, the diminution amounts to 80 or 90 per cent, and in such cases, as is well known, the administration of bile or pancreas powder greatly tproves fat absorption. This interference is no doubt mainly dependent >n the secretion of pancreatic juice. Pratt, McClure and Vincent, 48 for example, found that completely depancreated dogs could still absorb con- siderable quantities of fat. On the other hand, the internal secretion of the pancreas has a considerable influence on fat metabolism as is evi- denced by the fact, observed by us, that there is usually 10-12 per cent of total fatty acid in the livers of depancreated animals but only a small amount after the animals have been treated with pancreatic extract (insulin, page 717). As to the relative roles of pancreatic juice and bile in the digestion of fat, we know of course that in the pancreatic juice there exists a lipolytic enzyme, lipase, which, under suitable conditions has the power of split- ting neutral fat into fatty acids and glycerol. If bile is examined, no lipolytic enzyme will be found in it. It is entirely inactive on fat, but 722 METABOLISM if we mix bile with fresh pancreatic juice, which by itself only slowly digests fat, we shall find that the bile very materially increases the lipo- lytic activity of the pancreatic juice. It has been found that the salts of cholalic acid, the so-called bile salts, are the constituents of bile that are responsible for this activation of lipase, this fact having been demonstrated with bile salts prepared in such a way that there was no possible chance of any other biliary constituent being present as an impurity. It is important to remember, however, that lipase itself be- comes slowly activated on standing, which explains why it should be that bile added to pancreatic juice that has stood for some time, has a less evident activating influence than bile added to fresh juice. It is probable that the activating influence of bile salts is due to some physico- chemical change induced in the digestion mixture. One may ask how it happens that, when bile and pancreatic juice are both absent from the intestine, the fat which appears in the feces is not neutral fat but fatty acid. The reason is that the neutral fat that has escaped digestion in the small intestine becomes acted on by the intestinal bacteria, particularly in the large intestine. Under these conditions, however, the fatty acid that is split off is not absorbed, because the epithelium of the lower parts on the intestinal tract can not perform this function. Besides assisting the action of lipase, bile facilitates fat digestion in other ways. Thus, by its containing alkali and mucin-like substances it assists in the emulsification of fat. Although emulsification is no es- sential part of fat absorption, yet it greatly facilitates the process by breaking up the fat into small globules on which the lipase can act much more efficiently. The alkali also combines with the fatty acids, as they are liberated by the digestive process, to form water-soluble soaps, which are readily absorbed by the epithelial cells. The bile salts further assist in the solution of the fatty acids, and they lower the sur- face tension of fluids in which they are contained and so bring the fat and lipase into closer contact. ABSORPTION OF FATS After its digestion fat lies in contact with the intestinal border of the epithelial cells as fatty acid and glycerol. The fatty acid is combined either with alkali to form a water-soluble soap, or with bile salts to form a compound, which is also soluble. The glycerol and the dissolved fatty acids are separately absorbed into the epithelial cells of the in- testine, in the protoplasm of which after the fatty acid has been set free from the alkali or bile salt they become united or resynthetized FAT METABOLISM 723 to form neutral fat, which gradually finds its way by the central lac- teals into the lymphatics and then to the thoracic duct. No evidence can be obtained that any of the fat is absorbed into the portal blood although only about 60 per cent of ingested fat can be recovered from the thoracic duct. The chemical explanation of the absorption of fat is very different from that formerly held by histologists who maintained that the fine particles of emulsified fat in the intestine penetrate by a mechanical process through the striated border of the epithelial cell into its protoplasm. The histolog- ical evidence for this view seemed very convincing, for fine fat globules can readily be seen in the epithelial cells of the intestine after fatty food has been taken, while they are absent during starvation. These par- ticles seemed to have passed directly from the intestinal canal into the epithelial cells because, when the fat was stained with characteristic fat dyes before feeding it to the animal, the globules in the epithelial cells were found to be similarly stained. The supporters of this mechanistic view of fat absorption maintained that the appearance of the stained fat globules in the epithelial cells could not be explained in any other way than by supposing that the fat globules had wandered unbroken into the epithelial cells. Such a conclusion is, however, unwarranted, for the stains that are soluble in fat are also soluble in soap, so that when the fat splits up, the stain will remain attached to the soap and be carried along with it into the intestinal epithelium. Proof that the chemical theory is the correct one has been supplied by a large number of experiments. The following may be cited: (1) When the lymph flowing from the thoracic duct is examined after feeding with fatty acids instead of neutral fat, it is found to contain only neutral fat, indicating that a synthesis must have occurred between glycerol and fatty acid during the absorption. The glycerol for this synthesis is furnished from sources which will be described later. (2) When an emulsion made partly of neutral fats, and partly of some hydrocarbon, such as albo- lene, is fed and the feces are examined for these substances, it has been found that all the fat but none of the hydrocarbon is absorbed; the feces contain all of the albo- lene but none of the fat. This experiment supplies very strong evidence against the mechanistic theory, for microscopic examination of the above described emulsion shows the particles of neutral fat and hydrocarbon to be of exactly the same size. (3) By examining the properties of the fatty substances in the thoracic lymph collected 'during the absorption of such an emulsion as that described above, nothing but neutral fat has been found present. (4) Similar results are obtained when wool fat, which is an ester of cholesterol and fatty acid, is fed. We may conclude that fatty substances which are insoluble in water or can not be changed by digestion into substances (soap) that are soluble in water, are not absorbed, however like fat they may be in other particulars. The chemical theory of fat absorption further explains why there should be such large quantities of soapy substances in the intestinal contents, and also why the globules of fat present in the epithelial cells of the 724 METABOLISM intestine are so very much smaller than those which lie on the surface of the epithelium. It might be objected to the conclusions just stated that, although undetectable, there is really some essential physical difference between emulsified fat and emulsified hydrocarbon. In order entirely to prove the case for the chemical theory, it is neces- sary to feed a neutral fat possessing some characteristic that depends on the manner of union existing between fatty acid and glycerol, and then to see whether it appears in an unchanged condition in the thoracic duct. If it does so, the fat must have been absorbed through the intestinal epithelium in an unbroken, unsaponified condition, for it is unlikely that, in 'the resynthesis which occurs in the intestinal epithelium, the fatty- acid molecules would recombine with the glycerol molecules in exactly the same manner as before. There are, however, but very few qualities of neutral fats, apart from those of the fatty acids which compose them, by which they can be characterized. The most likely one is that of optical activity. None of the ordinary fats is optically active, although from chemical considerations it is quite conceivable that some ought to be so. In order to obtain such a fat Bloor^ conducted numerous experiments with the esters of stearic acid.* In a series of experiments Bloor fed isomannid-dilaurate a synthetic fat of dextrorotatory power and as readily absorbed as natural fats and by examination of the neutral fat present in the chyle flowing from the thoracic duct, found no evi- dence of 'the dextrorotatory fat. This result confirms previous work by Frank, who found that the ethyl esters of fatty acids are not absorbed unchanged. The results of both workers emphasize the probability that readily saponifiable fatty-acid esters do not escape saponification under the favorable conditions of the normal intestine. In other words, had the fats been absorbed unchanged, as would be required by the mechanistic theory of fat absorption, they would have appeared in the chyle in optic- ally active conditions. These most important conclusions lead us to inquire as to the reason for the change in fat during its absorption. It can not be for the purpose of preventing the absorption of undesirable fatty substances, such as the petroleum hydrocarbons or the wool fats, because such substances are so rarely present in our food. It is most probable that the breakdown and resynthesis of neutral fat occurs for the same reason that similar processes occur during the absorption and assimilation of protein. It will be remembered that protein is entirely disintegrated in the intestine into its so-called building stones. These are absorbed separately into the blood, which carries them to the tissues, in which they become re- synthesized to form the body protein. And so it appears to be in the case of fats. The process, in other words, permits of the rearrangement of fatty-acid molecules, as a result of which the newly formed fat is more adaptable for use in the organism. It comes to be more like the char- acteristic fat of the animal. There may be another reason for the proc- *Bloor prepared an optically active mannitan distearate, but found it to have a very high melt- ing point and to be only half as digestible as the ordinary fats. Its absorption was too slow and unsatisfactory to make it suitable for the above purposes. He, therefore, proceeded to prepare the di-ester of isomannitan with lauric acid, and he found the resulting compounds to be as well-ab- sorbed as ordinary fat, and yet to possess very marked dextrorotatory power, which, of course, they lose on saponification. This fat seemed suitable, therefore, for testing the above question. FAT METABOLISM 725 ess. It will be remembered that lecithins, which constitute the most important of the fatty substances of the cell itself, are mixed glycerides that is to say, are compounds containing a variety of fatty acids. The rearrangement of the molecules of neutral fat which occurs during ab- sorption may be the first step in the transformation of fat into lecithin. In order to throw further light on the question, Bloor has performed a number of interesting experiments in which the chemical properties of fats before and after absorption were compared. The criteria which he took were melting point, iodine value, and mean molecular weight; the melting point representing the solidity of the fat, and the iodine value, its degree of unsaturation that is, the number of double links in the fatty-acid chain. It was found that during absorption very considerable changes occur in these two characteristics; for example, when fat with high melting point and low iodine value was fed, the fat in the thoracic lymph was of distinctly lower melting point and higher iodine value. When fat with a low melting point and a high iodine value was fed, the reverse change occurred, for the melting point of the thoracic lymph fat was higher and the iodine value lower. These results' could be explained as due in the first case to the addition of oleic acid to the fat during its synthesis in the intestinal epithelium, and in the second case to the addition of some saturated fatty acid. When a fat consisting mainly of glyceride and saturated fatty acid, but with a low melting point, was fed, the addition of oleic acid was still found to occair, as judged from the iodine value. This indicates that the change is, not merely in order that the melting point of the absorbed fat may be lowered, but also for some chemical reason. In a fourth series of experiments, a lowering of iodine value occurred after feeding with cod-liver oil, which contains a high percentage of glycerides of highly unsaturated fatty acids. Evidently, then, the intestine possesses the power of modifying the com- position of fat during its absorption, and this modification is apparently of such a nature that it causes a change toward the production of a uniform chyle fat, presumably characteristic of the animal body. The changes are probably greater than could be produced by admixture of the absorbed fat present in the normal fasting chyle, but the source of the oleic acid or of the saturated acid required for this synthesis is at present unknown. Regarding the transference of the synthesized fat globules from the epithelial cells to the central lacteal it is believed, with Schafer, that leucocytes play an important role. They engulf the fat globules and carry them to the lacteal where they break down and liberate the glob- ules. In support of this view Clark and Clark have observed, after injec- tion of fats into the tails of tadpoles, that leucocytes pass out from the blood vessels and accumulate around the oil globules, ultimately absorb- ing the latter and then wandering to near-by lymph vessels into which apparently they disgorge their loads of fat. CHAPTER LXXX FAT METABOLISM (Cont'd) THE FAT OF BLOOD Methods of Determination. Normally the blood contains only a small percentage of fat, but after a fatty meal it may contain so large an amount that the fat actually rises to the surface of the blood like a cream. By means of the ultramicroscope, ex- amination of the blood in the dark field after a fat-rich meal reveals the presence of glancing particles, the so-called "fat dust." These particles are most abundant about six hours after the meal has been taken, and they gradually disappear by the twelfth hour. They do not appear after a meal when the thoracic duct is ligated. They dis- appear when oxygen is bubbled through the blood. Fat dust has also been found abundantly present in the blood of embryo guinea pigs at full time, but not in the mother 's blood. This would indicate that the placenta must have the power of taking the constituents of fat from the mother's blood and building them into fat, which then passes into the blood of the fetus. The placenta under these conditions acts like the mammary gland. In this connection it is of interest that there is also much fat present in the blood of pregnant women. The fat content of the placenta is, however, greater in the early stages of pregnancy than later. Although these facts have been known for some time, it has been impossible, either on account of the large quantities of blood required for a chemical examination or because of the difficulty in estimating the amount of fat from the density of the "fat dust," to follow with any great degree of accuracy the exact chemical changes that take place in the fat of the blood. Recently, however, Bloor has succeeded in elaborating methods by which the fat content of the blood can be determined with satisfactory accuracy in small quantities of blood, so that a continuous series of observations can be made over a considerable period. The fat is extracted from the blood by an alcohol-ether mixture with moderate heat. An aliquot portion of the filtrate is evaporated in the presence of sodium ethylate , which saponifies the fat. The residue, consisting of soap, is well washed and then treated with hydrochloric acid so as to precipitate the fatty acid. The density of the precipitate thus produced is compared in an optical apparatus, called a nephelometer, with a standard solution of two milligrams of oleic acid treated in the same way. The fatty acids in human blood are mainly oleic and palmitic. The lecithin and cholesterol may also be estimated in the same blood extract. For lecithin the above extract of blood, after the removal of the alcohol and ether, is digested by heating with concentrated HNO 3 and H 2 SO 4 . This decomposes the lecithin, liberating the phosphorus, a solution of the resulting ash being rendered faintly alkaline to phenol- phthalein and then slowly added to a silver nitrate solution. The density of the pre- cipitate thus produced is compared in the nephelometer with that of a precipitate pro- duced in the same amount of silver nitrate by adding to it a standard phosphoric acid solution. For cholesterol an aliquot portion of the above extract is saponified with sodium ethylate and then saturated with chloroform ; the chloroform extract is mixed with acetic 726 FAT METABOLISM 727 anhydrid and H 2 SO 4 (con.) until the bluish color is fully developed (Liebermann reac- tion), the intensity of which is then compared in a colorimeter with that obtained by similar treatment from a standard cholesterol solution. Variations in Blood Fat. In the dog the percentage of fat in the blood is remarkably constant under normal conditions. After a fatty meal the increase in fat begins in about an hour, and reaches its maxi- mum in about six. The increase is not found in animals in which the thoracic duct has been ligated. Although this result would seem to support the view that fat absorption occurs exclusively into the tho- racic duct (page 723) it must be remembered that the evidence is not convincing since the shock caused by so severe an operation may account for the faulty fat absorption. Very interesting results have been obtained following the intravenous injection of emulsions of oil, either the so-called casein emulsion or col- loidal suspensions. Up to a dose of 0.4 gram per kilogram of body weight which by calculation would suffice to raise the fat content of the blood by 100 per cent there was no increase in fat content. In or- der to explain this disappearance of fat, it might be imagined that the injected fat particles formed emboli in the smaller capillaries. Against such a view, however, is the fact that the particles of fat in these emul- sions are one-half to one-seventh the size of a red corpuscle. Although this argument is no doubt of some weight, it should be remembered that the physical condition of these fine fat globules is not the same as that of the red blood corpuscle. Their surface condition may be such that they readily agglutinate so as to form small masses, which may stick at the branching of the smaller arterioles and capillaries. Bloor himself suggests that the injected fat may be stored, possibly in the liver, since the fat in this organ, as we shall see later, increases under similar conditions. When twice the above quantity was fed in the form of egg- yolk fat, some of it persisted in the blood for several hours. This in- crease may have been owing to the flooding of the temporary storehouses with fat, or, more probably, to a retarding influence that lecithin may have on fat assimilation, for lecithin itself persists in the blood for a long time after intravenous injection. During fasting, no increase in blood fat was found unless the animal, by special feeding, had been stuffed with excess of fat prior to the fast- ing period. The lipemia in this case indicates that fat is being trans- ited from one place to another to serve as fuel for the starving tissues. Narcotics were found to produce an increase in blood fat. Ether pro- duced this increase during the narcosis, whereas morphine and chloro- form did not do so until after recovery. The explanation given for the 728 METABOLISM ether effect is that a mixture of blood and ether has a higher solvent power for fat than blood alone. The explanation for the chloroform and mor- phine effects is that a certain amount of breakdown of the tissue cells, in which lipins are set free, supervenes upon the action of these narcotics. The blood fat also becomes enormously increased in about forty hours after the administration of phlorhizin, and on the second or third day after the administration of phosphorus. The special significance of these facts we shall consider later in connection with the relationship of the liver to fat metabolism. By comparison of the fatty acid, lecithin, and cholesterol contents of blood during fat absorption, it has been found that there is a steady but very variable increase in fatty acid, accompanied by an increase in lecithin, which varies from 10 to 35 per cent, but does not run strictly parallel with the fatty-acid increase. At a later stage there may also be an increase in cholesterol; indeed in persistent lipemia cholesterol may become more abundant than lecithin. It looks as if fat were absorbed into the corpuscles where it is transformed into lecithin which is then returned to the plasma, cholesterol also appearing when the lecithin reaches a certain concentration. Separate analyses of blood plasma and whole blood show the increase of lecithin to be much more marked in the corpuscles than in the plasma, whereas the fatty-acid increase is most pronounced in the plasma. To illustrate some of these points the following table will be of value. In it is shown the average distribution of fatty acid, lecithin and choles- terol in normal individuals and in cases of diabetes, in which disease, BLOOD LIPOIDS IN NORMAL AND IN DIABETIC PERSONS NORMAL PER CENT MILD DIABETES PER CENT MODERATE DIABETES PER CENT SEVERE DIABETES PER CENT Fat by Bloor's J Method 1 Whole Blood Plasma 0.59 0.62 0.83 0.90 0.91 1.06 1.41 1.80 Total Fatty AcidsJ Whole Blood Plasma Corpuscles 0.37 0.39 . 0.34 0.59 0.64 0.45 0.65 0.75 0.48 1.01 1.28 0.62 Lecithin Whole Blood Plasma Corpuscles 0.30 0.21 0.42 0.32 0.24 0.42 0.33 0.28 0.40 0.40 0.40 0.40 Cholesterol J Whole Blood Plasma Corpuscles 0.22 0.23 0.20 0.24 0.26 0.21 0.26 0.30 0.20 0.41 0.51 0.24 Glycerides j Plasma Corpuscles 0.10 0.38 0.18 0.46 0.23 0.84 0.38 Total Lipoids Plasma 0.68 0.98 1.16 1.98 FAT METABOLISM 729 as has been known for long, there is marked disturbance of fat metab- olism. The table shows that this increase affects all types of lipoids in proportion to the severity of the disease. Bloor considers that lecithin which is soluble in water, is the main form in which fat is transported in the blood. The lecithin is taken up by the tissue cells in which there are enzymes (esterases) capable of hydrolysing it so as to produce fat, or by reversible action, of converting the fat stored in the cells to lecithin which is then sent into the blood. The fat secreted in the milk can, for example, all be accounted for by the difference between the lecithin content of the blood plasma passing through the gland (Meigs). Some of the fat is also transported in the blood by the leucocytes acting as phagocytes towards it. The Destination of the Fat of the Blood. In general, it may be said that the blood fat is transported to three places: (1) the depots for fat; (2) the liver; and (3) the tissues. The fat present in each of these places differs from that in the others, as is revealed by chemical examination by the methods described on page 719. The depot fat usually yields about 95 per cent of its total weight as fatty acid. The tissue fat, on the other hand, yields only about 60 per cent of its total weight as fatty acid. This difference indicates that the fatty acid must be combined in the tissues with a much larger molecule than is the case in the fat of the depots. This large molecule is probably that of lecithin or other phos- pholipin, and the smaller molecule in the depots, that of neutral fat. The liver fat takes an intermediate position between depot fat and tissue fat in its yield of fatty acid. When no active metabolism of fat is go- ing on, the liver fat is like that of the tissues; but when fat metabolism is active, the liver fat occupies an intermediate position between tissue fat and depot fat. Another difference among the fats in these three places is with regard to the degree of saturation of the fatty-acid radicles. This, it will be remembered, is indicated by the iodine value; the higher the iodine value, the greater the desaturation of fatty acid. In depot fat this value is relatively low for example, about 30 in the goat and about 65 in man ; depending somewhat on the fat taken in the food, compared with which it is usually a little higher. The fat in the tissues, on the other hand, has a high iodine value, possibly 110 to 130. The iodine value of the fat of the liver is remarkably inconstant, being about the same as that of the tissues when fatty-acid metabolism is not particularly active, but approximating that of the depots when fat mobilization is proceeding. The significance of this fact we shall consider later. 730 METABOLISM The Depot Fat The places in the animal body where depot fat is deposited in great- est amount are the subcutaneous and retroperitoneal tissues. These fat depots may sometimes become of enormous size, as in the case of the famous dog of Pfliiger, of whose total body weight 40 per cent was due to fat. Bloor suggests that there may really be two different types of fat storage : one of a purely temporary character, which readily takes up and liberates the fat, but which is of limited capacity and possibly under the control of some quickly acting regulating mech- anism, like that of the glycogenic function of the liver; and another of a more permanent nature, into which the fat is slowly taken up, but the capacity of which is very much greater. Two questions present themselves concerning the depot fat: (1) where does it come from, and (2) what becomes of it? Regarding the source of the depot fat, there is no doubt that it comes partly from the fat and partly from the carbohydrate of the food; in other words, it is either taken ready-made with the food or manufactured in the organism. That some of it comes from the fat of food is now a well-established fact, the evidence for which need not detain us long. The best-known experiment consists in first of all starving an animal until its stores of fat are nearly exhausted and then feeding it with some "ear-marked" fat- that is, with some fat having a characteristic property which it will not lose during absorption. It will be found that the depot fat thereby deposited presents many of the qualities of the fed fat. The "ear- marking" of the fat may be secured by using fats of different melting points, such as mutton fat, which has a high M.P., or olive oil, which has a low M.P. On feeding a previously starved dog with mutton fat, the M.P. of the depot fat approaches that of mutton fat he becomes a dog in sheep's clothing; whereas when olive oil is fed, the subcutaneous fat becomes oily. Or again we may "ear-mark" the fat by combining it with bromine, when the deposited fat will likewise be brominized fat. It must not be imagined, however, that no change takes place in the fat during its absorption and before it becomes deposited in the tissues. On the contrary, the stamp of individuality is put upon the fat, for, as we have already seen, its iodine value may become altered and its melt- ing point changed during the process of absorption. In other words, although the absorbed fat does not become entirely adapted to conform with the ordinary qualities of the depot fat, yet it tends to change in this direction. That some of the depot fat comes from carbohydrate is well known to stock raisers. When, for example, an animal is fed on large quantities of carbohydrate and kept without doing muscular exercise, its tissues FAT METABOLISM 731 become loaded with fat. Strict scientific proof of its production from car- bohydrate is given in the old experiments of Lawes and Gilbert, who, it will be remembered, showed that the fat deposited in the tissues of a growing pig is greatly in excess of the fat that could have been derived from the fat or protein which was meanwhile metabolized. The experiment was performed on two young pigs from the same litter and of approximately equal weight ; one was killed and the exact amounts of fat and nitrogen in the body determined; the other was fed for several months on a diet the fat and protein contents of which were accurately ascertained. When after four months this pig was killed and the fat determined, it was found that much more had become deposited than could be accounted for by the fat and protein of the food, even suppos- ing that all the available carbon of the protein had become converted into fat. The only conclusion is that the carbohydrate must have been an important source of the extra fat. The Destination of the Depot Fat. The depot fat becomes mobilized and transported by the blood to the active tissues whenever the energy requirements of the body demand it. During starvation, as we have seen, the depot fat is used to supply 90 per cent of the energy on which the animal maintains its existence. Before the fat is transported, it is possibly reconverted into lecithin, as which it passes through the cell walls. Enzymes capable of accelerating the hydrolysis of simple esters and of lecithin (esterases) are abundantly present in practically all tis- sues while there are only traces of true lipases except in the pancreas md intestines. These esterases would seem to be the responsible agents for the transfer of lipoids, as lecithin, in and out of the tissue cells al- though some may pass in particulate form as neutral fat. It is probably mly in the intestinal epithelium that the latter is synthetized through :he influence of lipase. The Fat in the Liver The physiology of the liver fat has been very diligently studied, particularly by Leathes and his pupils. 50 The outcome of this work has been to show that the liver occupies an extremely important posi- tion in the metabolism of fat, being, as it were, the half way house in the preparation of the fatty-acid molecule for consumption in the tissues. Fat is a material containing large quantities of potential en- ergy. While in the depots this potential energy is so locked away as to be unavailable for tissue use. To make it available the depot fat is carried to the liver, where the energy becomes unlocked but not actu- ally liberated. The fat is then transported to the tissues, and the libera- tion of the energy occurs. Neutral fat is like wet gunpowder: it con- 732 METABOLISM tains much potential energy, but not in a suitable condition for explo- sion. The liver, as it were, dries this gunpowder, whence it is sent to the tissues to be exploded. The great importance of the liver in fat metabolism is indicated by comparison of the percentages of fat or better of fatty acid contained in it under different conditions of nutrition. In the ordinary run of slaughter-house animals the liver contains from 2 to 4 per cent of higher fatty acid, but in about one in every eight animals a much higher per- centage will be found to occur. The same is true in laboratory animals. In the case of the human liver as obtained from autopsies in certain classes of patients, from 60 to 70 per cent of the dry weight of the organ, or 23 per cent of the moist weight, may be fatty acid. There is no other organ in the animal body that is ever loaded with fat to this extent. As in the depots, this liver fat might be derived either from fat carried to the organ from elsewhere in the body, or it might represent a surplus of manufactured fat. Transportation of Fat to the Liver. About forty hours after giving phlorhizin to a dog, it has been found that enormous quantities of fat appear in the liver; a few hours later, however, this excess of fat may have entirely disappeared. Fatty infiltration of the liver is also observed in phosphorus poisoning, although in this case the fat usu- ally persists till the death of the animal. In man, in delayed chlo- roform poisoning and in cyclical vomiting, considerable quantities of fat may be present in the liver within a very short period of time after the onset of the condition. There can therefore be no doubt that fat is transported to the liver under abnormal conditions, but this can not be taken as evidence that the liver has anything to do with fat metab- olism in the normal animal. Such evidence has, however, been supplied by Coope and Mottram, 51 who have shown, at least in rabbits, that a sim- ilar invasion of the liver with fat occurs in late pregnancy and early lactation. They also found that the fatty acid deposited in the liver in late pregnancy gives an iodine value which lies nearer to that of the mesenteric fatty acid than is the case in normal animals. Mottram con- cludes that "wherever . . . there is abundant fat metabolism, the liver is found to be infiltrated with fats, presumably to be handed on elsewhere when worked up." It is interesting that the fetus is greedy of unsaturated fatty acids. The most likely source of the fat transported to the liver is the fat pres- ent in the depots, unless when digestion is in progress, when it may be the fat from the intestine. That much of it comes from the depots is easily demonstrated. Thus, the more extensive the infiltration of the liver with fat, the more closely will this fat be found to agree with the depot fat in its chemical characteristics. This has been very clearly FAT METABOLISM 733 shown by, first of all, starving an animal so as to clear the depots of fat as much as possible; then feeding it on some "ear-marked" fat (unusual melting-point or a brominized fat) ; and after another day or so of starvation, so as to clear the liver itself of fat, poisoning the animal with phosphorus or phlorhizin. The liver will be found shortly after- wards to be invaded with fat which has all the ear-marks of that on which the animal had been fed. Evidence of the same character has been furnished in a series of clin- ical cases by observations on the amount of fat and the iodine value of the fatty acid of the liver. This is shown in the accompanying table. FATTY ACIDS OF LIVER CAUSE OF DEATH HIGHER FATTY ACIDS PER CENT OF DRY WEIGHT IODINE VALUE OF FATTY ACIDS 1. Pernicious anemia 12.1 116.8 XTrV"rTYlQl < 2. Lobar pneumonia 13.7 116.8 JiN \Jl Jlldl figures 3. 4. Pernicious anemia Diabetes 14.25 14.4 116.0 119.6 5. Toxemic jaundice 15.6 109.5 Commencing 6. Accident 17.2 103.5 fatty 7. Empyema 21.5 96.0 change 8. Phthisis 25.4 96.4 : 9. Broncho-pneumonia 38.4 84.9 10. Appendicitis 44.0 91.1 Marked 11. Carcinoma of bladder 47.2 77.8 fatty 12. Broncho-pneumonia 54.6 71.8 change 13. Ulcerative colitis 60.9 80.3 14. Accident 66.3 63.0 15. Dysentery 73.5 69.1 This table clearly shows that the more fat there is in the liver, the nearer this fat approaches in character that stored in the depots (viz. 65). That some of the fat in the liver may come directly from the fat re- cently absorbed from the intestine is also very readily demonstrable. Thus, when cocoanut oil was placed in the intestine of anesthetized an- imals, along with bile salts and glycerine, it was found by Raper 52 that per cent of the absorbed oil appeared in the liver. The characteristic feature of cocoanut oil is that its fatty acids are also volatile in jam and are saturated. Some of the fatty acids of the liver are volatile in steam, )ut they are unsaturated. By distillation in steam of the fatty acids obtained by iponification of the liver, it is possible to determine how much of the cocoanut oil las passed to the liver. Similar results have been obtained when unsaturated fatty acids, such those contained in cod-liver oil, are fed. In all these cases the rela- tionship of the fat of the liver to that of the food is even more evident than that between food fat and depot fat, because in the liver the newly absorbed 734 METABOLISM fat is not diluted by that deposited, it may be months, previously, as is the case in the connective tissues. Changes in the Fat Deposited in the Liver. An indication of the nature of the change is furnished by observing the iodine value of the fat. This, it will be remembered, indicates the degree to which the fatty acid is unsatu- rated. It does not necessarily indicate the number of unsaturated bonds present in the fatty-acid molecule, for the difference in iodine-absorbing power may depend not on the number of such bonds but on the position in the chain at which a given double bond is inserted. Even with this reserva- tion, however, it is evident that the increase observed in the iodine values shows that the liver has the power of desaturating fat. The advantage of this change depends on the fact that the desaturated fatty acid will be more liable to break up than the saturated fatty acid. In other words, the double linkage will weaken the chain with the consequence that it is liable to fall apart at this place; such at least is the natural interpreta- tion which the chemist would put on the result. It may not, however, be the correct interpretation, for it has been shown that, although un- saturated fatty acids are more susceptible to chemical change than sat- urated in the laboratory, yet when fed to animals they appear to be more stable than many saturated acids. It may then be wrong to con- clude that the introduction of a double linkage in fat necessarily means the liability of the fatty-acid chain to break at that point. However this may be, it seems likely that one function of the liver consists in introducing double linkages at places in the fatty-acid chain, as a result of which the chain breaks at these places, and the fragments then undergo further oxidation. Double linkages may be introduced not only in one place in a fatty-acid chain,, but in several. For example, it has been found in the liver of the pig, after oxidizing the fatty acids with permanganate, that oxidation products are obtained indicating the ex- istence of unsaturated acid with four double links. Permanganate (in alkaline solution) is used for detecting the position of these double bonds, because, when it is allowed to act on unsaturated fatty acids in the cold, it causes hydroxyl groups to be introduced in the position of the double bonds. When the oxidation is performed at a moderate temperature, the fatty acid falls apart at the hydroxyl groups. A fatty acid with eight hydroxyl groups has been obtained in this way from the liver of the pig. The presence of the hydroxyl groups has been confirmed by finding that an octobromide is obtained by treatment with bromine. An acid of the same formula is said to be present in cod- liver oil. To sum up, we may conclude that there are certain positions, in the chains of carbon atoms which constitute the fatty-acid radicle, where the liver introduces double bonds, and that the weakened radicles then circulate to the tissues, where they break up at those positions. Desaturation is probably not the only process by which the liver assists in the metabolism of fat. It may also take part in the building of fatty- acid radicles into the complex molecule of lecithin. The process of de- FAT METABOLISM 735 saturation is probably a preliminary step to this incorporation of the fatty- acid molecule into lecithin, for it is well known that lecithin contains highly unsaturated fatty-acid radicles. In support of such a view it is interesting to note that in alcohol-ether extracts from normal and pathological livers, the lecithins, which are precipitated by acetone, have higher iodine values (i.e., are more unsaturated) than the neutral fats extracted from the same liver, which also have higher iodine values than the depot fat of the same animal. Conversion of fat into lecithin also occurs extensively in the blood itself (page 728). Desaturation of fatty acids occurs in other places besides the liver. The relative activity of the different tissues in this regard has been studied by feeding cats with fatty fish and then determining the iodine value of fat from various places in the body. The absorbed fat was more obvious in the liver than in the subcutaneous tissues, because it had not become diluted with fat deposited, it may have been months, previously, which would be the case in the fat of the fat depots; and it was found that, although the iodine value of the subcutaneous fat was slightly raised, that of the liver was much more so, indicating that the desaturation process had been more active in this organ, but had also occurred to a certain extent in the depots. Before leaving this subject of fat in the liver, it is important to re- call the old observation of Rosenthal, that a more or less reciprocal relationship exists between glycogen and fat in the liver. When much glycogen is present there is little or no fat, and vice versa. It is impor- tant to note that the exact locations of fat and carbohydrate in the he- patic lobule are somewhat different. A practical clinical application of the above work is found in the fact that fats will be more readily utilized by the body when they contain a high percentage of unsaturated fatty acids. It is possibly for this reason that Norwegian cod-liver oil is of such undoubted nutritive value. It is much more so than Newfoundland cod-liver oil, because in the prep- aration of this variety oxidation occurs, which makes it no longer unsat- urated. Fish oils in general are more unsaturated than other animal oils, and are for this reason more nutritious. The presence of vitamines rather than their assimilative properties may, however, be the factor which determines the nutritive value of different brands of cod-liver oil. The fat in the tissues differs very materially from that of the liver or the depots. Only 60 per cent of this fat consists of fatty acid, which is present very largely as part of the lecithin molecule, thus accounting for the high iodine value. Some is probably also present as simple glyceride, in a highly unsaturated and therefore very fragile condition. CHAPTER LXXXI FAT METABOLISM (Cont'd) Two very important questions of fatty-acid metabolism may now be considered: namely, (1) how is fatty acid formed from carbohydrate? and (2) what becomes of the fragments into which the fatty-acid molecule is split as the result of the desaturation process? Although these prob- lems involve chemical details of a somewhat complex nature, we must not on this account fail to consider them; for, as we shall see, much of what is known has an important practical application depending on the fact that certain of the intermediary substances may accumulate in the organism and develop a toxic action. The Production of Fatty Acid out of Carbohydrate. If we place the formulas for glucose and palmitic acid side by side, thus: CH 2 OH-(CHOH) 4 -CHO (glucose), and CH 3 -(CH 2 ) 14 -COOH (palmitic acid); we shall see that this transformation must involve: (1) a considerable alteration in the structure of the molecule, (2) the removal of oxygen, and (3) the fusion of several glucose molecules into one molecule of fatty acid. The conversion of carbohydrate to fat therefore involves a process of reduction, and the resulting molecule must be capable of yielding more energy when it is oxidized than the original one of carbohydrate, for obviously the system 2 -CH 2 (which corresponds to fat) will develop more energy than that of 2 - CHO (which corresponds to carbohydrate) ; just as a piece of wood when it is burned will develop more heat than a piece of charcoal. This explains why one gram of fat yields 9.3 calories of heat, and one gram of carbohydrate, only 4.1 (page 571). Fatty acid therefore contains more potential energy than sugar, and in explain- ing its synthesis from sugar in the animal body we must indicate the source of the extra energy. This is dependent on oxidation of some sugar molecules which do not themselves become changed to fatty acid proceeding side by side with the reduction which affects the others and is represented in the outcome of the reaction by the combustion products C0 2 and H 2 0, thus: 6C 6 H 12 6 + 13 2 = 20 C0 2 + C 16 H 32 2 + 20 H 2 0. (glucose) (fatty acid) 736 FAT METABOLISM 737 What evidence have we that such a process actually occurs in the body? If we compare the intake of oxygen with the output of carbon dioxide in the respired air, we shall find that usually there is less of the latter; that is to say, the respiratory quotient, as this ratio is called, is usually less than unity. During the extensive conversion of carbohydrate into fat, however, which occurs during the fall months in hibernating animals, the R.Q. has been found to rise as high as 1.4. The great excess of C0 2 - output over 2 - intake which such a quotient indicates conforms with the above equation. The entire dissimilarity in chemical structure between the molecules of fat and carbohydrate suggests that the primary step in the conversion must be a thorough breakdown of the carbohydrate chain into compara- tively simple molecules, from which the fat molecules are then recon- structed and the unnecessary oxygen set free. The problem is to ascer- tain the chemical structure of these simpler molecules and the manner of their union into fatty acid. The Method by Which the Fatty Acid is Broken Down. In the chemi- cal laboratory, ordinary oxidizing agents attack the fatty-acid chain at the C-atom next the carboxyl (COOH) group (the alpha C-atom). But this can not occur in the animal body, because it would leave behind a smaller chain containing an uneven number of C-atoms, and such chains are never found present in the animal fats. On the contrary, the com- moner fats all contain an even number of C-atoms, thus : Butyric, C 4 H 8 2 ; palmitic, C lfi H 32 2 ; stearic, C 18 H 3G 2 ; oleic, C 18 H 34 2 . The intermediary substances which are produced during the gradual breakdown of the fatty-acid molecule in the normal animal are of a very transitory character so much so indeed that it is impossible for any one of them to accumulate in sufficient amount to permit of isolation, or even detection, by chemical means. How then are we to identify the inter- mediary products? This has been rendered possible by the discovery that, when anything occurs to disturb the normal course of fat metabolism, as, for example, when the tissues are deprived of carbohydrates (as in star- vation or in severe diabetes), the oxidation of the fatty-acid chain stops short when a chain of four C-atoms still remains unbroken. These last four C-atoms seem to form a residue that is more resistant to oxidation than the remainder of the fatty-acid molecule. It is a residue, therefore, which is quite readily further oxidized to C0 2 and H 2 under normal con- ditions, but which, undergoes only a partial oxidation when the metab- bolism is upset, resulting in the production of various intermediary prod- ucts. These accumulate in the body in sufficient amount to overflow into the urine, from which they can be isolated and identified. The fatty acid with 4 C-atoms is "butyric, CH 3 CH 2 CH 2 COOH, and the 738 METABOLISM first oxidation product formed from it in the body seems to be (3-oxybuty- ric acid, CH 3 CHOHCH 2 COOH. This then becomes oxidized to form a body having the formula CH 3 COCH 2 COOH, acetoacetic acid, which, on further oxidation, readily yields CH 3 COCH 3 , or acetone. These sub- stances (/?-oxybutyric acid, acetoacetic acid and acetone) appear in the urine during carbohydrate starvation, as in diabetes. It might be objected, however, that a chemical process occurring under abnormal conditions need not also occur in the normal animal. That it probably does, however, is indicated by the results of the experiments of Knoop and of Embden and his coworkers. Knoop conceived the idea of introducing into the fatty-acid molecule some group which is resistant to oxidation in the body. The phenyl group (C 6 H 5 ) was found to have this effect. By feeding an animal with the phenyl derivatives of acetic, propionic, butyric, and valeric acids, it was found that the urine con- tained either hippuric (see page 663) or phenaceturic acid. Both of these are compounds of aromatic acids with glycocoll or aminoacetic acid (CH 2 NH 2 COOH), one of the protein building-stones and always available in the organism to form such compounds, thus : ( 1 ) C C H 5 COOH + CH 2 NH 2 COOH = C 6 H 5 CONHCH 2 COOH. (benzole (glycocoll) (hippuric acid) acid) ( 2 ) C 6 H 5 CH 2 COOH + CH 2 NH 2 COOH C C H 5 CH 2 CONHCH 2 COOH. (phenylacetic (glycocoll) (phenaceturic acid) acid) When either benzoic acid (C 6 H 5 COOH) or phenylacetic acid (C 6 H 5 CH 2 - COOH) is formed in the body as a result of the oxidation of phenyl derivatives of the higher fatty acids, the acid combines with glycocoll according to the above equations. From this it follows that if oxidation occurs so that two C-atoms are thrown off at a time (/3-oxidation), fatty acids with an even C-atom chain should yield hippuric acid, and those with an uneven chain, phenaceturic. This was found to be the case, as the accompanying table shows. ACID FED OXIDATION PRODUCT EXCRETED AS Benzoic acid, C 6 H 6 .COOH Phenylacetic acid, C 6 H 6 . CH 2 . COOH Not oxidized Not oxidized Hippuric acid Phenaceturic acid Phenylpropionic acid, C 6 H 5 . CH 2 . CH 2 . COOH C 6 H 5 .COOH Hippuric acid Phenylbuty ric acid, C 6 H B . CH 2 . CH 2 . CH 2 . COOH C H 5 . CH 2 . COOH Phenaceturic acid Phenylvaleric acid, C 6 H 5 . CH 2 . CH 2 . CH . CH 2 . COOH C H 5 . COOH Hippuric acid (From Dakin.) FAT METABOLISM 739 Embden's experiments are equally convincing. He studied the forma- tion of acetone in defibrinated blood perfused through the freshly excised liver. Normally only a trace of this substance is formed, but when fatty acids with an even number of carbon atoms were added to the blood, they gave rise to a marked increase in acetone, whereas those with an uneven chain failed to cause any change. The acetone was found to be derived immediately from acetoacetic acid. The following table shows the results. NORMAL FATTY ACID FORMATION OF ACETOACETIC ACID Acetic acid CH 3 .COOH Propionic acid CH 3 .CH 2 .COOH Butyric acid CH 3 . CH 2 . CH 2 . COOH + Valeric acid CH 3 .CH 2 .CH 2 .CH 2 .COOH Caproic acid CH 3 . CH 2 . CH 2 . CH 2 . CH, . COOH + Heptylic acid CH 3 . CH, . CH 2 . CH 2 . CH~ 2 . CH., . COOH Octoic acid CH 3 .CH 2 .CH 2 .CH 2 .CH 2 .CH 2 .CH 2 .COOH + Nonoic acid CH 3 . CH 2 . CH 2 . CH 2 . CH 2 . CH 2 . CH 2 . CH 2 . COOH Decoie acid CH 3 . CH 2 . CH 2 . CH 2 . CH 2 . CH 2 . CH 2 . CH 2 . CH 2 . CH 2 . COOH + (From Dakin.) For a long time it was difficult for chemists to understand how such a process of oxidation at the /?-C-atom could occur, since they were unable to bring it about in the laboratory by the use of the ordinary oxidizing agents, but recently Dakin has removed the difficulty by show- ing that hydrogen peroxide (H 2 2 ) oxidizes fatty acids just exactly in this way. We may sum up the results of these experiments and observations by stating that normal saturated fatty acids and their phenyl derivatives can undergo oxidation, not only in the animal body, but also in vitro, in such a manner that the two (or some multiple thereof) terminal C -atoms are removed at each successive step in their decomposition. But we must not be too hasty in concluding from these experiments that the steps in the process are necessarily in the order of first, the production of a /3-hydroxy acid, and second the oxidation of this to a Ketone group. The mere presence, side by side, of /3-hydroxybutyric acid and of acetone in the above experiments does not indicate which is the antecedent of the other, and indeed there are several experimental facts that seem to show that the hydroxy acid may be derived from the ketone. For example when acetoacetie acid is added to minced liver and the mixture incubated, ^3-hydroxy- butyric acid is formed (a reduction process), although less usually the reverse action (oxidation) may occur when /3-hydroxy acid is added. A reversible reaction must there- fore be capable of occurring between these two substances, thus : reduction CH 3 .CHOH.CH 2 .COOH < CH 3 . CO . CH 2 . COOH. oxidation (^-oxy butyric acid) > (acetoacetie acid) 740 METABOLISM We know practically nothing as to the conditions determining whether oxidation or reduction shall predominate, but there are two significant facts that one should bear in mind: (1) that a plentiful supply of oxygen is necessary for the oxidative process, and (2) that the presence of readily oxidizable material in the liver (e.g., carbohydrates) may determine the direction which the reaction shall take. It is commonly said that fats burn in the fire of carbohydrates, and it may be that the undoubted diminution in acidosis which occurs in diabetes when carbohydrate food is given is dependent upon the directive influence which its combustion in the liver has on the above processes. But we must be cautious not to transfer results obtained by experiments with minced liver in judging of the reactions which occur during life. Provisionally, then, we must assume either that /3-hydroxybutyric acid is a necessary stage in the oxidation of butyric acid or that it is formed by reduction of acetoacetic acid, which is really the first step in that process. Of course there is no evidence in the above experiments that the higher fatty acids are also broken down by the removal of two C-atoms at a time, nor has it been possible to detect any ketonic or /3-hydroxy derivatives of them in the animal body. We can only reason from analogy that a similar process may occur, although some support is fur- nished for such a view by the fact that ketonic fatty acids have been found in vegetable organisms. What, then, it may be asked, is the relation of the desaturation of fatty acids which we have seen occurs in the liver (and probably elsewhere) to the /3 oxidation? There can be no doubt that both processes can occur in the animal body, indeed in the same organ, e.g., the liver; and it is important to ascertain their relationship to each other. The conclusion which would seem to conform best with the known facts is that the de- saturation process occurs (in the liver) so as to break up the long fatty-acid chain into smaller chains, which are then capable of /3 oxidation (in the tissues) ; desaturation may be the process by which the molecule is rough hewn, and /3 oxidation that by which the resulting pieces are finally split to their smallest pieces that is, to molecules of the size of acetic acid, which are finally completely burnt to carbonic acid and water. The increase of iodine value observed by Leathes and his coworkers need not, as has already been pointed out, necessarily indicate that new double linkages have been intro- duced in the fatty-acid chain; it may merely indicate that structurally isomeric deriva- tives which absorb iodine more readily have been formed. Direct evidence of desatura- tion has, however, been offered by Hartley, who isolated the unsaturated fatty acids (by dissolving the lead soaps in ether) from pig's liver and then proceeded to oxidize them with alkaline permanganate. When the olein of the depot fat is thus treated at a low temperature, two hydroxyl groups become attached where the double linkage existed (forming dioxystearic acid), and when the mixture is now warmed, the molecule splits into two at this place, forming two lower acids (pelargonic and azelaic) : (1) CH 3 -(CH 2 ) 7 CH:CH(CH 2 ) 7 COOH; (oleic acid) OH OH / / (2) CH 3 - (CH 2 ) 7 -CH- -CH (CH 2 ) 7 COOH; (dioxystearic acid) (3) CH 3 (CH 2 ) 7 COOH + COOH-(CH 2 ) 7 COOH. (pelargonic acid) (azelaic acid) We may conclude from this that the double linkage in the oleic acid of the depot fat exists between the ninth and tenth C-atoms. But it is otherwise in the case of the un- saturated acid from the liver (pig's), for under the above process of oxidation this yielded caproic acid, which, since this acid has six C-atoms, would indicate that the FAT METABOLISM 741 double linkage existed between the sixth and seventh C-atoms. Another interesting fact brought to light by the experiments was that a tetraoxystearic acid was formed, which fell apart in such a way as to indicate that the hydroxyl groups occurred between the sixth and seventh and between the ninth and tenth C-atoms. The occurrence of this substance would be satisfactorily explained by the introduction into the molecule of oleic acid of a second double bond i. e., between the sixth and seventh C-atoms. ' ' The acids found in the pig's liver may be accounted for, in other words, by supposing that desaturation of stearic acid and of the ordinary (depot) oleic acid 'Occurs by the introduction of a double link between the sixth and seventh carbon atoms in each case" (Leathes). Still other double links may, however, be introduced into the fatty-acid chain, for acids of the linolic acid series are present in cod-liver oil. Finally, it is of interest to note that caproic acid is a product of the above oxidation process, for it has an even number of C-atoms and therefore will form /3-oxybutyric acid. To go into these chemical problems any further here would be out of place. One other fact, should, however, be borne in mind namely, that the unsaturated acids may be formed from saturated acids through the intermediate formation of /?-hydroxy and /?-ketonic acids. Their mere presence, in other words, should not be taken as evidence that the oxida- tion of fatty acids is initiated by the introduction of an hydroxyl group at the ft position in the chain. CHAPTER LXXXII CONTEOL OF BODY TEMPERATURE AND FEVER The classification of animals into two groups warm-blooded and cold- blooded according to their ability to maintain the body temperature at a constant level, is more or less arbitrary. Between the two groups an- other exists, represented mainly by hibernating animals, in which at certain times of the year the animal is warm-blooded and at other times cold-blooded. The ability of the higher mammals to maintain a constant body temperature may or may not be present at the time of birth. The heat-regulating mechanism of the human infant for example remains ill developed for some time, so that exposure to cold is liable to lower the body temperature to a dangerous degree. VARIATIONS IN BODY TEMPERATURE In animals in which the heat-regulating mechanism is fully developed, there is not, even during perfect health, entire constancy in temperature in the different parts of the body or in the same part at different periods of the day. The average rectal temperature of man is usually stated as being 37 C. (98.6 F.), but the diurnal variation may amount to 1 C., being highest in the late afternoon and lowest during the night. There are probably several causes for this variation, and they are in part at least dependent upon the greater metabolic activities of the waking hours and upon the taking of food. Apart from these influences, how- ever, others which are less evident appear to operate; for it has been found that, when the daily program is reversed by night work, the usual diurnal variation, although much less pronounced, still remains evident even after this reversal in habit may have been kept up for years. It is of interest to note in this connection that nocturnal birds have their maximum temperature at night and their minimum during the day. Regarding the temperature in different parts of the body, that of the rectum is usually about 1 C. higher than that of the mouth, and this again higher than that of the axilla. Of these three the mouth tempera- ture is the most variable, for many conditions, such as mouth breathing, talking, drinking cool liquids and even exposure to cold air, are sufficient to lower markedly the temperature of this region. When the mouth temperature is carefully taken by leaving the bulb of the thermometer 742 CONTROL OF BODY TEMPERATURE AND FEVER 743 under the tongue for a minute or more, it is practically the same as the temperature of the arterial blood of the hand when this is exposed to the ordinary conditions of .outside temperature. Greater differences than 1 C. in the temperature of different regions of the body are often ob- served in feeble individuals and in those with some circulatory disturb- ance. FACTORS IN MAINTAINING THE BODY TEMPERATURE The body temperature represents the balance between heat production and heat loss. The production is effected mainly in the muscles by the oxidative processes which are constantly ensuing there. When the activity of the muscles is abolished by paralyzing the terminations of the motor nerves with curare, the temperature of warm-blooded animals immediately falls or rises according to the temperature of the environ- ment. A curarized warm-blooded animal is thus made to behave like a cold-blooded one. Increased muscular activity, on the other hand, promptly raises the body temperature by 1 or 2 C., above which, how- ever, a further rise does not occur, provided nothing has been done to interfere with the mechanism by which the excess of heat is got rid of from the body. The temperature in such cases adjusts itself at a higher level, at which it remains fairly constant however strenuous the exer- cise. It is possible that a certain amount of heat may also be generated by the chemical processes occurring in the liver and other viscera, but when compared with the muscles this source of heat is undoubtedly in- significant. Many of these chemical processes, as in the liver, instead of producing, actually absorb heat, so that the balance between heat- producing and heat-evolving mechanisms may or may not come out in favor of the liberation of heat. The production of heat goes on all the time in muscles on account of the condition of tonic contraction in which they are held (see page 905), and which is also necessary for keeping the joints in the proper degree of flexion or extension. When more heat is required by the animal body, the tone of the muscles increases independently of the function which they may be performing in controlling the position of the joints. This increased tone may become so pronounced that it causes visible contrac- tions, which we recognize as shivering. Whenever the insensible hyper- tonicity and the shivering are inadequate to produce a sufficient amount of heat, the animal instinctively moves about in order that the greater contractions may liberate more heat. The heat is produced in the muscles by oxidation of the foodstuffs that have been assimilated from the blood. Even during the process of as- similation itself a certain amount of heat is generated; this is known 744 METABOLISM as the specific dynamic action of the foodstuff, and is most pronounced with protein and least so with carbohydrate (page 574). Advantage may be taken of this heating power of protein to produce more heat when the cooling conditions are excessive ; in winter, for example, there is an inclination to take more protein food than during summer, and the per capita consumption of such food is much greater in peoples living in temperate zones than in those living in the tropics. The ultimate amount of heat produced by oxidation is greatest with fat and least with carbo- hydrate. Heat loss in man is effected partly through the lungs, but mainly through the skin. Through the latter pathway heat is lost by the physical processes of heat conduction and radiation and by the evaporation of the sweat. Through the lungs it is lost mainly in the vaporization of the water contained in the expired air (latent heat of vapor). The amount of heat lost from the skin by conduction and radiation depends on the temperature of the skin, which again depends on the rate at which the blood is circulating through the cutaneous vessels. Under ordinary con- ditions of external temperature two or three times as much heat is lost by these methods as by evaporation. The losses by evaporation, under conditions of rest and average external temperature, are about equally divided between the lungs and the skin. Under average conditions in man the main regulation of heat loss is ef- fected ~by variations in the skin temperature brought about by peripheral vaso-constriction and dilatation. The marked sensitivity of the cutaneous blood supply to changes in the temperature of the environment has been very clearly shown by observations made with the hand calorimeter of Stewart described elsewhere (page 296). When the bloodflow through the hand is examined in a person who has been exposed to the outside air, it may be little more than half that which it attains after he has been in a warm room for some time. In the outside air the vessels con- strict to prevent heat loss by conduction and radiation; in the warm room they dilate to facilitate this loss. The afferent impulses which reflexly control the change in the cutaneous blood circulation may be set up by local applications of heat or cold, as can be shown in the hand-calorim- eter experiments by applying a cold pad to the skin of the correspond- ing forearm, or allowing an electric fan to blow on the arm when an immediate curtailment of bloodflow takes place. Or the reflex may be excited from distant skin areas, as illustrated in the curtailment in blood- flow observed when the opposite hand to that on which the observation is being made is placed in cold water. The magnitude of the change in cutaneous circulation is more or less dependent upon the extent of the area of the body that is exposed to the change in temperature. CONTROL OF BODY TEMPERATURE AND FEVER 745 it has recently been shown that the blood becomes diluted with tissue fluid when more surface cooling has to occur. Although afferent impulses from the skin are therefore of great im- portance in adjusting the cutaneous blood supply according to the amount of surface cooling that has to occur, a further effect is also pro- duced on them by the action on the nerve centers of temperature dif- ferences in the blood itself. Thus, when the temperature of blood going to the brain is raised by placing the carotid arteries on some heating de- vice or when the region of the corpora striata is directly warmed, the skin vessels become dilated as if the animal had been exposed to general warmth, and the rectal temperature tends to fall (Barbour 56 ). When the loss of heat by radiation and conduction is no longer ade- quate to prevent a rise in body temperature, or when the processes can not operate on account of a high temperature in the environment, the loss of heat from the skin is mainly dependent upon the evaporation of sweat. Under ordinary conditions this evaporation takes place at such a rate that there is no visible perspiration on the surface of the body the so-called insensible perspiration. When the heat loss by this channel must become greater, the perspiration is produced in larger amount, so that it collects on the surface of the body ; and, provided the conditions of the environment are such that evaporation can readily take place (low relative humidity), the amount of cooling of the body that can be effected becomes very great. A man may exist without any marked rise in body temperature in a very hot environment even when he is exposed to an out- side temperature that is the same as that of his body, or even greater. To encourage evaporation, however, he should be naked or very lightly clad, and the air should be kept in constant motion so that the layers of air next to the skin, which ordinarily very quickly become saturated with vapor, are transferred and replaced by dryer air. Movement of the air also increases the heat loss by convection, provided the temperature of the air is not too near that of the body. The importance of the movement of air in the regulation of heat loss has been clearly demonstrated by Leonard Hill, 54 F. S. Lee, and others, and will be fully discussed in the chapter on ventilation (page 754). The stimulus to increased sweating has been thought to be dependent mainly on changes in the temperature of the blood since sweating does not immediately set in when the body is subjected to heat, as by a warm bath or a hot pack. It usually takes from ten to twenty minutes after the person has been placed in the bath or surrounded by the warm blan- kets of the pack before sweating becomes pronounced. It has been shown, however, that the body temperature does not require to be raised before sweating sets in. In a hot bath sweating starts on the forehead 746 METABOLISM before there is any change in rectal temperature and if the hand be plunged in cold water the sweating stops at once (cf. Barbour). Loss of heat by evaporation of sweat occurs only in certain animals. It is practically absent, for example, in the dog. The degree to which it may occur also varies in different individuals of the same species. Dilution of the blood accompanies it. The power of withstanding high temperatures is proportional in man to the facility with which he per- spires. Where sweating is interfered with by skin diseases, by ichthy- osis, for example, exposure to heat or increased heat production, as by muscular activity, may raise the body temperature to a dangerous degree. Another factor upon which the efficiency of evaporation of sweat in cooling the body depends is the relative humidity of the air. When this is high, evaporation of water into it can not occur, and it is on this account that an increase in body temperature is much more likely to occur in warm, humid atmospheres than in those that are dry. At the same temperature people can live in perfect comfort in the dry air of the open plains, but suffer immediately from rise of temperature when they go into the humid air of the river valleys. Similarly, work in hot fac- tories or in mines is quite possible at very high temperatures if the air is kept dry and in motion, but becomes impossible when the air is moist. In judging of the adequacy of air from this point of view, it is there- fore important to take not the ordinary dry-bulb thermometer reading but that of the wet-bulb.* In animals, like the dog, that do not perspire over the surface of the body, vaporization of the water in the expired air is the most important method of regulation of heat loss. When such an animal is exposed to warmth or when the region of the corpora striata is artificially warmed, the breathing immediately becomes much quicker and deeper, so that pulmonic ventilation is greatly increased and much more water is carried out as vapor with the expired air. To vaporize the water large quanti- ties of heat are required (seen in the latent heat of steam). In man this method is, ordinarily, not of great importance, but it may become so when sweating is interfered with, as in ichthyosis. The more rapid breathing also facilitates cooling by increasing the conduction of heat from the mucous membranes of the tongue, mouth, throat, etc. The im- portance of this method of cooling has been shown by finding that after the introduction of a tracheal cannula a dog can not withstand an in- crease of external temperature nearly so well as a normal animal. *The wet-bulb thermometer registers a temperature that is lower than that of the dry-bulb in inverse proportion to the relative humidity of the air. When the air is completely saturated with moisture, the temperature recorded by the two instruments will be the same; when it is perfectly dry, the difference will be maximal. CONTROL OF BODY TEMPERATURE AND FEVER 747 THE CONTROL OF TEMPERATURE In the case of man the body temperature is very largely under volun- tary control, as by the choice of clothing and the artificial heating of the room. Desirable as this voluntary control of heat loss may be, there can be little doubt that it is often managed to the detriment of good health. Living in overheated rooms during the cooler months of the year so diminishes the loss of heat from the body that the tone and heat-produc- ing powers of the muscular system are lowered. Not only does this diminish the resistance to cold, but it causes the food' to be incompletely metabolized so that it is stored away as fat. The superficial capillaries also become constricted and the skin bloodless and " pasty." It is not looks alone that suffer, however, but health as well, for by having so little to do the heat-regulating mechanism gets, as it were, out of gear, so that when it is required to act, as when the person goes outside to the cold air, it may not do so as promptly as it should, with the result that the body temperature falls somewhat and catarrh, etc., are the result. There can be little doubt that much of the benefit of open-air sleeping is owing to the constant stimulation of the metabolic processes which it causes. As will be inferred from what has been said above, the control between heat production and heat loss is effected through a nerve center located in or near the corpora striata. In most animals, when the spinal cord is cut in the cervical region, the body temperature quickly falls unless artificially maintained. In the case of man, on the other hand, it has usually been observed, after accidental section of the spinal cord in the cervical region, that a rise in temperature occurs. In twenty-four un- complicated cases of spinal-cord injury in man, collected from the rec- ords of Guy's Hospital by Gardiner and Pembrey, it was found that nineteen showed hyperthermia (sometimes amounting to 43.9 C.), and only five, hypothermia (sometimes 27.6 C.). If the patient lived, the ultimate effect of the section, as in the lower animals, would no doubt be the loss of the power of maintaining a constant temperature. The extent to which the animal comes to behave as if cold-blooded, after section of the spinal cord, varies considerably according to the level of the lesion; if the cord is cut in the upper thoracic region, for example, the regulation against cold, although distinctly less efficient than normal, is far better than when the section is made through the cervical cord. This difference is dependent on the fact that after the lower lesion much larger muscular groups and skin areas are left intact, so as to make regulation possible. Section of the dorsal cord in mice has been found by Pembrey to abolish entirely the increased metabolism which occurs in normal mice when they are exposed to cold. 748 METABOLISM In the light of these experiments it is probable that the difference in the effects produced on body temperature by section of the cervical spinal cord in man and the lower animals depends on the relative im- portance of the heat-producing and heat-dissipating mechanisms. When the control of heat loss is paralyzed in the smaller animals, the cooling of the body becomes excessive in relation to the amount of heat produced in the paralyzed muscles, because the body surface is extensive in com- parison with the body weight (see page 586). In the larger animals such as man, on the other hand, the cooling effect is much less marked, espe- cially when, as is common after spinal cord injuries, the patient is kept unusually warm. FEVER The clinical application of a knowledge of the mechanism of heat regu- lation in the animal body concerns the causes of fever. In the most familiar form fever is produced by infectious processes, but it may also be owing to various other causes, among which may be mentioned the parenteral injection of foreign protein, excessive destruction of protein substances in the body itself, the action of certain drugs, and lastly, injury to the base of the brain or lesions of the upper levels of the spinal cord. Various types of fever are recognized: when the temperature re- mains constantly above the normal, it is known as continuous fever; when oscillations occur but the temperature never falls to the normal level, it is known as remittent; when it attains the normal level at cer- tain periods during the day, it is known as intermittent. The Mechanism of Fever During a sudden rise in body temperature there is, on the one hand, in- creased heat production in the muscles, and on the other, dimin- ished heat loss from the surface of the body. The fever is therefore due to an exaggeration of the processes ~by which the body normally re- acts to conditions which tend to lower the body temperature. The increased muscular tone thus induced often causes visible contractions, familiar as shivering; and the constriction of the cutaneous blood vessels pro- duces the subjective sensation of chills, and causes the skin to become pale and cold to the touch. The skin muscles also contract, producing " goose skin." During this stage, objective demonstration of the cur- tailment of the skin circulation can be secured by observation of the bloodflow through the hands and feet (page 296). When the temperature suddenly falls again, (the crisis, as it is called) the muscles become flaccid and produce less heat, and the cutaneous blood vessels dilate, as has been shown by measurements of the bloodflow of the hands and feet. CONTROL OF BODY TEMPERATURE AND FEVER 749 At the same time also, the sweat glands are stimulated and marked per- spiration occurs. It is significant that fever can be caused by injection of very strong sugar solutions intravenously. These attract water from the tissues and so interfere with the giving off of heat. Concerning the cause of continuous fever, it must be assumed that the balance between heat production and heat loss has been adjusted at a higher plane than normal. We cannot explain the fever on the basis either that heat production alone is increased or that heat loss alone is diminished, for in neither of these cases would the temperature stand at a permanent level but would steadily rise or fall according to which mechanism was disturbed. The thermogenic nerve centers, while set at the higher plane of fever, are still capable of responding in the usual way to the influences which cause the body temperature to change in a normal person. For example, when a fever patient is subjected to a hot bath so that his body temperature rises about 0.2 to 0.5 degrees C., sweating occurs just as in a normal individual; or if exercise is taken the increased amount of heat thereby produced in the muscles is dissi- pated in the usual way. When, on the other hand, the patient is exposed to cold, the vessels of the skin contract and he shivers. Although fever is not caused by an actual disturbance of balance be- tween heat production and heat loss, neither of these processes is pro- ceeding at its normal rate. That there is a distinct increase in the total heat production of the body in acute fevers in well-developed persons has been shown by means of the respiration calorimeter. This increased heat production is not observed in patients who have been brought into a weakened condition and in whom the muscular tissues have become atrophied by long-continued fever. The increased heat production in continuous fever is mainly dependent upon the increase in body tem- perature and is not one of its causes, as is evident from the fact that far larger quantities of heat are frequently produced in normal individuals as a result of muscular exercise or the taking of large quantities of protein-rich food. The heat thus produced is, however, very quickly dissipated, so that only a temporary rise in temperature occurs, (cf. Hewlett. 57 ) A large heat production also occurs in hyperthyroidism without a corresponding rise in body temperature (page 578). Similarly, it can be shown that in continuous fever there is a relative inefficiency in the mechanism of heat dissipation. When the temperature of a normal person is artificially raised through about 1 C., a marked increase in cutaneous bloodflow and profuse perspiration are invariably noted. In a patient with fever of the same degree, on the other hand, there is practically no change in the skin circulation; indeed, it is usually diminished, and there is no unusual perspiration. The heat-regulating 750 METABOLISM mechanism is now fixed on a plane that is higher than the normal, so that although further increase in body temperature, as we have seen, calls forth responses like those in a normal individual, yet at the fever temperature itself there are none of the reactions which a normal individ- ual would exhibit if his temperature were artificially raised to that level. 57 The adjustment of the temperature at the higher level is by no means so perfect as it is at the normal level of health, so that a normal subject is more resistant to the effects of cold than is a patient with fever. The degree of response of the fever patient, however, varies considerably from time to time ; a cold bath in typhoid fever, for example, lowers the body temperature much less effectively at an early stage in the disease, when the fever is more or less continuous, than later when it is becoming of the intermittent type. In the third week of the disease the cold bath more readily brings down the temperature and keeps it down for a longer time than during the first or second week. The mechanism for heat loss is also deranged in fever, which explains the rise in temperature that is likely to follow the performance of even moderate muscular exercise or the taking of too hearty a meal in tuberculous and convalescent typhoid patients. Changes in the Body During Fever In seeking for the cause of fever which is evidently of an obscure nature, it is necessary to collect all the information we can regarding the metabolic changes that are then occurring in the animal body. A few of the most significant facts that have so far been collected may be mentioned here. Some of the most important concern the dis- turbance in nitrogenous equilibrium caused by the considerable loss of nitrogen which takes place in fever patients when they are fed on the usual hospital diet prescribed for such cases. This loss of nitro- gen is no doubt the result of the partial starvation in which the pa- tient is kept; for it has been shown by Shaffer and Coleman 55 that patients with typhoid fever may be maintained in nitrogenous equi- librium by feeding them with relatively large amounts of carbohy- drate, which acts by protecting the protein of the body from disintegra- tion (see page 605). The protein minimum to which fever patients can be reduced is nevertheless considerably higher than the minimum in normal individuals. From the above results as a whole, it is probably safe to conclude that there is a specific destruction of protein going on in the body during fever. Further evidence of such a destruction is furnished by the presence in the urine of excessive amounts of creatinin, of purine bases, and, it is said, of incompletely hydrolyzed proteins, such as the albumoses (pro- CONTROL OF BODY TEMPERATURE AND FEVER 751 teoses.) Moreover, when the fever suddenly terminates in crisis, there is a marked increase in the excretion of urea (the epicritical urea in- crease), which indicates that an extensive deamination of protein build- ing stones (amino acids) is occurring. The so-called "diazo reaction" obtained in the urine during the fever is also believed to depend on the presence of abnormal protein-disintegration products. As to the specific cause of the increased protein disintegration, little is known. Several factors may operate: (1) the partial starvation of the patient, entailing an increased breakdown of protein to meet the calorie requirements; (2) the high temperature, which in itself may stimulate increased protein metabolism, for it has been shown that, when normal animals are artificially warmed, protein metabolism becomes increased; and (3) toxic protein-decomposition products specifically causing an ex- cessive breakdown of protein. Although there is increased protein breakdown during fever, it must not.be forgotten that when food is refused only about 20 per cent of the total expenditure of the body is derived from this foodstuff, 80 per cent coming from non-nitrogenous material, which must be fat, because the available carbohydrates are used up at an early stage. Since the general metabolism is increased, the excessive breakdown of the fatty substances, occurring as it does in the presence of a diminished combustion of carbohydrates, interferes with the proper oxidation of the fatty-acid molecules and leads to the appearance of so-called acidosis products in the urine, and consequently to a relative increase in the urinary ammonia (page 650). A tendency to acidosis therefore exists. The acidosis may reach a considerable degree of severity and cause the tension of carbon dioxide in the alveolar air to become diminished. Since a similar degree of acidosis may be produced in partially starved ani- mals by overheating them with moist air, but not so if the animals are liberally fed with carbohydrates, it is probably safe to conclude that abundance of carbohydrate is advisable in the food that is furnished to fever patients. Another interesting metabolic change in fever concerns the salt bal- ance. This is studied by observing the amount of sodium chloride excreted by the urine. As is well known, this becomes markedly diminished until the crisis of the fever, when it suddenly increases. The salt retention is related to the concentration of the blood, a marked rise in which is usually accompanied by a rise in temperature and is an unfavorable prognostic sign. Salt retention is more marked in certain types of fever than in others, and it is essentially different in nature from the salt retention that has been ob- served to occur in nephritis. This difference has been brought to light by examination of the chloride content of the blood. In nephritis, the concen- 752 METABOLISM tration of chlorides in the blood is considerably increased, whereas in fever it is markedly diminished. The deficiency in salt elimination cannot be at- tributed to a deficiency of salt in the food, for it sets in before the diet has been curtailed and, when salt is given to a febrile patient, it is re- tained in the body to a greater degree than is the case in the normal individual. Belonging to this group of fevers must also be considered the im- portant ones produced by the intravenous injection of certain forms of protein, as those of egg white or those derived from the bodies of bac- teria or from the laked corpuscles of a foreign blood. The fever in these cases is no doubt caused by a mechanism closely related to that responsible for anaphylaxis (see page 90). It is believed that many cases of so-called aseptic fever, occurring after severe contusions or other wounds, may be the result of destruction of proteins within the body. Similarly the rise in temperature during infections may be owing to the breakdown of protein by microorganisms within the cells. The Essential Cause of Fever It is possible that the essential cause for fever is that poisonous sub- stances (such as toxins) cause the tissues to break down and so increase the osmotic pressure of their protoplasm. Water is therefore attracted into them from the blood, the volume of which in circulation is thereby reduced. This reduction affects the superficial capillaries relatively more than the deeper ones and so tends to cool down the skin, as a result of which the nervous reflexes against cold are aroused and the superficial capillaries constricted. The view that removal of water from the blood is a most important element in fever is supported not only by the fact, already stated, that the blood has been found to be more concentrated in fever but also by numerous observations on the influence of drugs. Those which cause concentration of blood are likely also to cause rise in body temperature, such, for example, as certain diuretics (purines, sugars, etc.), intravenous injection of hypertonic salt solutions (3 per cent Nad) and certain cathartics, such as aloin. Cocaine causes fever partly by blood concentration. Antipyretics, on the other hand, (such as acetyl salicylic acid) act mainly by increasing heat elimination, although under certain conditions (as in coli fever) they may also cause dilution of the blood (Barbour). While increased concentration of the blood is undoubtedly an impor- tant factor in fever it must also be remembered that upset of the control of heat production and loss through action on the heat regulating or thermogenic centers may also be involved. CONTROL OF BODY TEMPERATURE AND FEVER 753 The Heat-regulating Center In all discussions on the regulation of body temperature and the causes of fever, it is assumed that a heat-regulating or thermogenic center exists somewhere in the brain. It is believed to be located about the optic thalami or corpora striata, for it has been found in rabbits that destruction of the brain anterior to this region does not cause any change in body temperature, whereas destruction behind it is followed by an entire upset in the heat-regulating mechanism. Fur- thermore, artificial puncture of this part of the brain causes marked elevation in body temperature in rabbits (heat puncture). Most in- teresting experiments have been recorded by Barbour, 56 who succeeded in applying heat or cold locally in the region of the centers. By the application of cold, increased muscular metabolism, on the one hand, and diminished heat loss, on the other, were excited; and conversely, when warmth was applied, an increased heat loss and a diminished heat production were observed. Irritation of this region of the brain in man, as after cerebral hemorrhage, is also accompanied by remarkable dis- turbances in heat regulation. It is believed by many that the essential cause of fever in infective conditions, is an action on these centers by toxic substances which develop in the blood. Significance of Fever to the Organism It is impossible at present to state definitely whether fever is a re- action of the organism against some infection and therefore of benefit in assisting the organism to combat it, or whether it is in itself an un- favorable condition. The question can certainly not be answered by observing the behavior of bacteria growing at different temperatures in various media outside the body. That certain bacteria should be found not to thrive at incubator temperatures equal to those found in the body during fever, does not at all prove that this fever is of sig- nificance as a means of combating the growth of the bacteria in the body. It is undoubted that, where the body temperature becomes ex- cessively high, the correct treatment is to keep it down as much as possible. On the other hand, the reduced mortality that has followed the introduction of the cold-bath treatment in typhoid fever may not be due so much to the reduction in body temperature itself as to the favorable effect produced on the nervous system and circulation. We certainly know that in normal animals moderate degrees of hyper- pyrexia brought about by exposure to moist heat are well borne for consider- able periods of time, thus indicating that it is the infection and not the hyperthermia that causes the serious damage to the body in infectious fevers. CHAPTER LXXXIII THE PHYSIOLOGICAL PRINCIPLES OF VENTILATION The well-being of a conscious animal in relationship to its environment constitutes the main problem of the study of ventilation. In the case of animals leading an outdoor life, it is a problem of relatively little im- portance, but for those like man which spend much of their time in confined spaces, it is a problem of great importance, because it becomes necessary to determine the limits within which the outside influences may be altered without detriment to health or comfort. This problem is con- sidered here because it is closely related to that of the body temperature. The Relationship Between the Chemical Composition of the Air and the Well Being of the Body. When our knowledge of the function of breathing became developed to the extent of showing that an animal requires the oxygen of the air for the living processes of its body, and as a result of these processes that it produces carbonic acid, which is then added to the air, it was natural to suppose that the unfavorable ef- fect of overcrowded confined spaces was due either to the using up of the available oxygen or to a poisonous action of the carbonic acid. It needs only a few words to point out how utterly erroneous were these earlier explanations. That deficiency of oxygen is no factor is in- dicated by the facts, first, that this gas is seldom reduced by more than one per cent, even in the most crowded places; and secondly, that people live a normal existence at altitudes at which the oxygen percentage, meas- ured at sea level, is reduced to less than two-thirds the normal. It is not altogether easy to understand why excess of C0 2 was thought to be responsible for the evil effects of vitiated atmospheres. No doubt the chief reason was that the percentage of this gas is often raised in such atmospheres, but this is nothing more than coincidence, for on the one hand most unsuitable conditions may exist when the percentage of C0 2 is normal, and on the other, air loaded with almost a hundred times the percentage found even in the most polluted atmosphere can be breathed for indefinite periods of time without any unfavorable symptoms. As a matter of fact, even in the open, we are constantly taking into the pulmonary alveoli large percentages of CO 2 , for obviously with each inspiration the first air to be drawn in is that which remains over in the air passage from the preceding expiration. This air contains somewhere about 5 per cent of CO,, and in quiet breathing it amounts in volume to about one-third of all the air that is drawn in from the outside. This in itself indicates that CO, per se can not be poisonous, and when we consider further the 754 THE PHYSIOLOGICAL PRINCIPLES OF VENTILATION 755 now well-known fact that a certain amount of this gas in the alveoli is absolutely es- sential to the well-being of the animal, the whole hypothesis of its toxic action becomes, to say the least of it, absurd. Indeed so important is the presence of this constant amount of CO 2 in the alveolar air that whenever there comes to be a marked increase in the amount of CO 2 in the atmosphere, the breathing becomes greater, so as to ventilate the air sacs more thoroughly, and thus keep the relative amount of CO 2 in them at the normal level. The extent of this increase in respiration is usually so small as to be unnoticed by the individual, and certainly increased breathing is not one of the symp- toms of which persons complain who are living in polluted atmospheres. In the face of such evidence, even the most ardent supporters of the theory that the vitiated air owes its evil influence to C0 2 , were compelled to abandon their position, but they did not do so without a final attempt to retain for determinations of C0 2 a certain significance in the appraisement of the healthfulness of air. Their new interpretation was to the effect that the C0 2 percentage is proportional to the amount of deleterious organic matter, and for many years this view prevailed. It is still believed by some that an increase from the normal of 3 to 10 parts of C0 2 per 10,000 parts of air indicates a degree of organic pollution which is dangerous to health. More recent work definitely shows, however, that this view also must be abandoned, and there remains for C0 2 -analysis only the secondary value that it indicates, in a readily measurable way, to what extent the inside air is being mixed by ventilation with pure air from the outside. However free this dilution may be, the atmosphere may still be deleterious to health and comfort unless certain other properties of it are incidentally altered. This interpretation of the value of C0 2 analysis naturally leads to a con- sideration of the next possibility, namely, that the air in confined spaces is contaminated by the accumulation of organic poisons derived from the exhaled air of the persons living in it. It is many years ago now since experiments apparently proving this hypothesis were published. These have been shown to be entirely fallacious, and we need refer to only one group of them here, namely, those that were devised to show that inhalation by one animal of volatile proteins contained in the exhaled air of others caused anaphylactic reactions (page 90). As proof for this hypothesis, experiments were performed in which a man breathed through a filter of glass wool (to catch any saliva) into a cooled vessel, and the condensed vapor was' then inoculated in appropriate dosage into guinea pigs, so as to sensitize them, and a month or so later the animals were inoculated with a minute trace of human blood serum. The injected animal showed decided symptoms of anaphylactic shock, whereas other animals not previously sensitized were unaffected by the injection of the same amount of serum. Such results taken by themselves did seem to afford substantial support for the new hypothesis, but it is almost certain that they depended on contamination of the condensed vapor by traces of saliva which it is im- possible to keep out by any kind of filter. This saliva contains traces of soluble protein (mucin) which had been responsible for the anaphylactic reaction. The symptoms are, however, entirely dissimilar from those of a vitiated atmosphere. Hay fever, some forms 756 METABOLISM of asthma, and the reaction which some persons show when near to horses may be due to anaphylaxis, but the symptoms are not at all like those of persons breathing polluted air. Once and for all, the toxic theory, as we may call it, both in its new and its old form, is disproved by a very simple series of experiments per- formed a few years ago by Leonard Hill, Flack and others. 72 These ob- servers kept rats and guinea pigs in deep boxes so that they were huddled together in a very poorly ventilated place, the atmosphere of which indeed often contained 1 per cent of C0 2 ten times more than the legal limit. The animals lived and thrived for months, although they must have been breathing air which was highly contaminated by the supposed volatile proteins. Not only did the animals show no symptoms while in the box, but they failed to exhibit any anaphylactic reaction when, after some time, they were inoculated subcutaneously with the serum of animals of the other species living with them in the box. This was really a most excellent test of the anaphylactic theory because there are probably no two animals in which anaphylaxis is more pronounced than in the rat and guinea pig. The only things that were found to be of importance in maintaining the animals in a thriving condition were cleanliness and plenty of food. By an eliminative process we are gradually approaching the correct solution of our problem, but before we proceed to consider this, it may be well to remark that the odor of polluted air has nothing whatever to do with its unhealthy influence, except in so far as it excites disgust and puts one off his appetite. Indeed one very soon becomes so accustomed to these odors that they fail entirely to be sensed after a short period in contact with them. Their influence is entirely psychological. In many trades and occupations people are constantly exposed to odors that are almost un- bearable to one who is unused to them, and these people are perfectly heal- thy, and indeed do not complain at all of the smells. We have so far considered in what is approximately their chronological order the various hypotheses that have been brought forward to account for the harmful influence of vitiated atmospheres. We have done this mainly in order to correct any false conclusions that may still exist in connection with the subject. And if further evidence be demanded to justify this position, there is one crucial experiment which once and for all shows that changes in the chemical composition of the atmosphere has no relationship whatsoever to the unhealthful influence of vitiated air. This experiment is all the more convincing because it was performed on healthy young men. In its simplest form it consists in crowding as many persons as possible into an airtight cabinet, provided with an electric fan, and with the necessary apparatus for measurements of the physical and chemical conditions of the air. The following is a description of the results of such an experiment: THE PHYSIOLOGICAL PRINCIPLES OF VENTILATION 757 "After 44 minutes the dry-bulb thermometer stood at 87 F., the wet bulb at 83 F. The carbon dioxide had risen to 5.26 per cent. The oxygen had fallen to 15.1 per cent. The discomfort felt was great; all were wet with sweat and the skin of all was flushed. The talking and laughing of the occupants had gradually become less and then ceased. On putting on the electric fans and whirling the air in the chamber the relief was im- mediate and very great, and this in spite of the temperature of the chamber continuing to rise. On putting off the fans the discomfort returned. The occupants cried out for the fans. No headache or after effects have followed this type of experiment which has been repeated five times. " (Leonard Hill) Long before the discomfort had become extreme the oxygen percentage became so low that matches would not light. The disin- clination to smoke cigarettes was not noticed until some time after it was impossible to light them. In other experiments of similar type the person in the cabinet was allowed to breathe outside air through a tube, but with no amelioration of the un- comfortable feeling, or a person outside the chamber breathed for hours the air inside it through a tube without suffering any discomfort. Clearly therefore neither the chemical nature of the air, nor the presence of toxic substances in it, has any relationship to its evil influence. But the experi- ment is not merely destructive of previously held hypotheses ; it also points the way to the true solution of the problem, for it indicates that stagna- tion of air loaded with moisture has some very close relationship to the discomfort. It shows that a change in the physical rather than the chemical properties of the air is the real cause of its deleterious action. THE RELATIONSHIP BETWEEN THE PHYSICAL CONDITION OF THE AIR AND THE WELL-BEING OF THE BODY The changes observed in the preceding experiment can affect but one function of the body, namely, that of heat dissipation, and by so doing cause disturbances in the mechanism of heat control. This does not nec- essarily imply that this disturbance is so great as actually to cause an in- crease in the body temperature, although this is very commonly observed in persons who have been for some time in crowded places, but it interferes with a mechanism which is responsible not alone for proper heat regulation but also for the maintenance of a correct relationship of blood supply to different parts of the body, and for tonic stimulation of the nervous system. It is in connection with this phase of the subject, more than any other, that many people find it difficult to understand the true significance of relative humidity to the well-being of, the body. The difficulty depends on the fact that the relative humidity has an opposite influence at low and high temperatures. In the former case it increases the conductivity of the atmosphere for heat and has a cooling influence, and in the latter it interferes with the evaporation of sweat, and has a heating influence. Below about 65 F. the cooling effect of moist air is prominent because there is little sweating, therefore a cold wet atmosphere is chilling it 758 METABOLISM conducts heat away. At about 70 F. the cooling effect of air disappears and sweating occurs. The evaporation of the sweat now causes cooling, the degree of which varies inversely with the relative humidity. Be- tween these two temperatures, i.e., 65 and 70 there is a range in which humidity has little influence a neutral region. The influence of high relative humidity on bodily comfort at temperatures above the neutral temperature becomes very marked indeed at 85 F. and at a relative hu- midity of 90 per cent, for example, unfavorable symptoms appear in a few minutes, when there is no movement of the air. Relative humidity and temperature alone are not, however, the only physical conditions to be considered. Another is the movement of the air, for even under the unfavorable conditions just cited, immediate relief is afforded if an electric fan be started, as it will be recalled was the result in Hill's experiment. The movement of the air enables it, though nearly loaded to its full capacity with moisture, to carry away considerable quantities of heat in small loads. The wearing of clothes greatly affects the rate with which these changes occur. The clothes act as barriers, preventing the movement and exchange of air around the body. The garment next the skin entraps a layer of air which is more or less at the same temperature as the skin, and which soon becomes saturated with moisture at that tem- perature. Between the inner garments and those over them other layers of air are en- trapped, each one being at a somewhat lower temperature and containing less moisture than the one inside. These layers of air, therefore, form stepping stones, as it were, be- tween the extreme conditions next the surface of the skin, and the environment of the clothed body. Obviously if the layers of air next the skin are to be renewed at such a rate that they remain cooler than the skin and unsaturated with moisture the clothing must be adjusted to suit the outside conditions. There is every reason for believing that it is because of interference with the processes of heat loss that improperly ventilated and over- crowded places are uncomfortable. The moisture exhaled and evapo- rated from the bodies soon raises the relative humidity so that heat loss is retarded from the skin, and the heat that is actually given off raises the temperature so that loss from the body by radiation and convection becomes suppressed. As the temperature steadily rises, the air takes up more and more moisture, with the result that less and less heat comes to be lost from the lungs in saturating the expired air with vapor. The physical conditions of the environment become unsuitable for the physi- ological mechanism of heat loss, although meanwhile heat production goes steadily on. The body furnaces are not damped down in propor- tion as the loss of heat diminishes, and the consequence is a rise in the temperature of the blood a mild fever. Now it is well-known that the cellular activities, which, taken together, make up the life process of the body are extraordinarily sensitive to change of temperature; their chemical activities become interfered with, they demand more oxygen. THE PHYSIOLOGICAL PRINCIPLES OF VENTILATION 759 they fail to get rid of effete products properly, substances which have no action on them under the ordinary conditions of temperature become toxic and so forth. A highly abnormal internal environment therefore becomes created around the living tissues of the body. The Relationship between the Conditions of Ventilation and Suscepti- bility to Infections. But short of a measurable rise in the temperature, improperly ventilated places cause reactions in the human body that are responsible not only for the discomfort which is experienced, but also for a lowering of resistance to infections. These reactions are due in the first instance to alteration in the temperature differences between the skin and the underlying tissues. Normally, as has been remarked before, this difference maintains at the skin a constant stimulation of the ther- mic nerves, and this stimulation is important in maintaining the tone of the nerve centers. The nerve cells that control the functions of the body do not originate impulses; they only act when other afferent im- pulses arrive at them. There are many varieties of stimuli which may excite these afferent impulses, but none more important than those which excite the heat nerves of the skin. This stimulation depends on the rate at which heat is passing through the sense organs (or receptors), in which these nerves terminate. It is necessary to emphasize that it is the rate of change that acts as the stimulus, and this depends on the dif- ference between the deep and superficial temperatures. When the skin vessels become dilated, so large a volume of blood reaches the surface that this difference becomes slight, and the thermic receptors are not stimulated. There are many practical applications of these principles; thus it is because of stimulation of the thermic skin nerves that cold baths have a bracing effect, that the open-air treatment, as in tuberculo- sis, tones up the body and enables it the better to hold its own against the tubercle bacillus, and that sleeping out of doors is the best tonic for maintaining good health. In the open-air treatment it is true that the body is closely wrapped up that is essential but this does not elim- inate the cooling influence, for not only does the cool air play on the exposed face and hands, in the skin of both of which the thermic nerves are very sensitive, but it acts also on these nerves in the skin, under the clothes, for the clothing merely serves to regulate the rate of cooling. This still goes on very much more than it would with much less clothing in an atmosphere that is stagnant, hot, and humid. Open windows in bedrooms are never so healthy as open-air porches, because there is no draft. It is the draft that is important. Naturally it must be regulated so that it is not restricted to one part of the body only that obviously would introduce conditions to which the body is unaccustomed it must blow equally all over. There is probably no greater fallacy in pop- ular hygiene than that drafts are dangerous. Like all good and desirable 760 METABOLISM things they become so only when they are improperly used when a per- son, overheated by being in a hot atmosphere, is suddenly subjected to a restricted draft, of course there is danger that the sudden change of conditions, affecting one part of the body only, will cause vascular dis- turbances that may be undesirable, but if the conditions be properly con- trolled, drafts are the healthiest things and the best tonics. It is a common experience not only that ordinary colds, but more serious infections as well, can be directly traced to some unsuitable condition of ventilation; such as sudden exposure to a draft while over- heated, or going out into a cold, damp atmosphere from an overheated room. What is the reason for the infection under these conditions? At the outset we must recognize that all these conditions, colds, catarrhs, bronchitis, just like the more acute infectious diseases, as diphtheria, pneumonia, cerebrospinal fever, etc., are due to microorganisms, and the question therefore is why should unfavorable ventilating conditions so frequently be the immediate cause of the attack. There are two methods by which the infection might occur. First, by a great increase in the number of organisms in the air, and secondly by a lowering of the resistance of the body towards the organisms, which would not then require to become increased in numbers. The former method is usually known as mass infection, and there can be no doubt that it is very common, perhaps, indeed, is the commonest cause for infection. The organisms, of course, come from infected individuals, who add them to the atmosphere in the exhaled air, particularly when this is forcibly discharged as in coughing or sneezing, or even in speaking. Evidence of the importance of this factor is as follows. If the mouth be rinsed with a culture of some readily recognizable organism not commonly present in detectable amounts in the atmosphere, and the person, standing in front of a row of P'etri dishes each containing some culture medium upon which the organism will grow, then speaks at ordinary pitch, the plates after proper incubation develop colonies of the organism, those nearest the speaker having most, but even those at a distance of several feet also show- n.g them. A serious problem in zoological gardens has been to keep animals that are highly susceptible to tuberculosis free from this disease. The higher apes, for example, inevi- tably succumb to this disease, being infected by the bacilli exhaled by persons standing in front of their cages. Many of the latter harbor these bacilli, though they may not show any of the symptoms of tuberculosis. Now it has been found that if glass screens are erected in front of the cages, the animals remain almost free from the disease. But mass infection does not suffice to explain the cause for the onset of attacks of many conditions that are, nevertheless, fundamentally due to bacteria, such as ordinary colds. These can frequently be traced to some chill, or wet feet, or exposure to sudden change in temperature. In such cases it is believed that the bacteria are present on the mucous mem- branes of the upper respiratory passages, but that they remain inactive THE PHYSIOLOGICAL PRINCIPLES OF VENTILATION 761 because of the normal protective influences which exist on these surfaces. So long as the blood supply is normal, these protective influences are adequate to protect the body from invasion, but if this should become cur- tailed, then the bacteria become active and set up pathological processes. Evidence favoring this view has been obtained by several recent investi- gators by finding that the blood supply of the upper respiratory passages becomes decidedly curtailed when the surface of the body is cooled. For example Leonard Hill and Muecke some years ago examined with a spec- ulum the mucous membranes of the nose under various conditions, par- ticularly out of doors, and in rooms which were ventilated and heated to an average degree. Out of doors the mucosa was pale and taut, and when touched by a probe did not show any pitting. This is the normal con- dition. Indoors it was common to find the membrane decidedly swollen, flushed with blood and covered with thick secretion, and when a probe was pressed on it a depression resulted lasting for some time. In one case that was frequently examined during these observations there was a deflected septum which only partly blocked the nasal passage on one side when the person was outside, but which did so completely under un- favorable conditions of ventilation. It is this swelling of the nasal mucosa and probably of that of the cavities which extend upward from it on to the forehead that causes the sense of stuffiness and probably also the headaches which are common in crowded, overheated places. The conditions found to bring about these changes with greatest cer- tainty were when the feet were cold and the air round the head was warm, conditions which are just exactly the opposite of those obtaining out of doors. Here the head is usually more quickly cooled than the feet scause convection currents of cool air play around it freely, whereas lext the ground the air is more stagnant. Besides, if the sun is shining. ie earth becomes heated by absorbing the heat. The temperature as igistered by a thermometer, either wet or dry bulb, may be the same at the feet as at the head. It is not this that counts, however, it is the rate of cooling which is dependent, mainly, on the movement of the air. Now in a poorly ventilated room, such for example as one heated by a stove, or even by radiators, and in which there is no movement of air, the feet become colder than the head, and it is under these conditions that the nasal membranes become swollen. It ought to be emphasized that the cause for these changes is not cold feet alone. It is the com- bination of cold feet and hot head. Out of doors, it is well known, that any one may stand with cold feet for hours without any risk of catching cold, but then the head is really cooling as fast as the feet, because of .convection currents. The ideal system of warming a room is to supply radiant heat near 762 METABOLISM the floor level; open fires, properly flued modern gas fires, and electric heaters at floor level are the best methods to attain this. Suppose the person subjected to conditions which cause the mucous membrane to become swollen and congested should go outside, then the membrane at once becomes pale because the blood vessels constrict, but for some time it remains swollen and boggy and continues to show pitting with a probe. It is while in this state that it offers favorable conditions for the growth of bacteria. The membrane is swollen and covered with secretion, and the blood flow is cut down. The natural defensive agen- cies that are normally carried by the blood do not succeed in combating the multiplication of the bacteria in the swollen membrane. After some time out of doors the blood supply returns because it is required to warm up the cool air, but this reaction does not occur before the mucosa has regained its normal condition.* The protective influence of a rapid blood flow through the nasal mem- brane is possibly the explanation of the relative immunity from infec- tious colds of those who work in air containing irritating gases, such as workers in various kinds of chemical factories. Even the irritation set up by coal dust may, by similar methods, afford some protection against in- fection by the tubercle bacillus for phthisis is relatively infrequent among coal miners. The supposedly antiseptic action of ozone is prob- ably due to a similar irritating effect. Any benefit that may be derived from its presence in the atmosphere can not otherwise be explained. It is possible that a useful prophylactic practice to avoid infection, such as that of influenza, would be to stimulate the nasal mucosa at intervals by snuff, but this may be an unwise suggestion. After becoming acclimatized to outdoor conditions, the nasal mucous membrane is in a much more favorable condition to withstand infection than indoors because of the very rapid blood flow that is necessary in or- der to supply heat with which to warm up the inspired air. This more rapid blood flow, and the freer flow of lymph which accompanies it, is reinforced by increased secretion, which assists to wash away invading bacteria. Mass infection being equal inside and outside, the animal body can withstand it much less satisfactorily in the former case. Many other observations bearing on the relationship between chilling and immunity to infection have been recorded, but it would take us be- yond our subject to discuss them here. Because of their accuracy and the excellent control of possible fallacies, it is important, however, to say something about the recent investigations of Mudd and Grant. 73 These observers measured the temperature of the mucous membranes of the palate, tonsils and pharynx by means of thermo-couples before and dur- *The congestion of the mucous membrane brought about by warm moist air does not probably depend on dilatation of the small arteries entailing- increased flow of blood, but rather on dila- tation of the capillaries and therefore a stagnation of blood. 1 THE PHYSIOLOGICAL PRINCIPLES OF VENTILATION 763 ing application to the skin of cold towels, or while cold air from a fan was allowed to play on it. A rise in temperature would indicate that the part had become more vascular, and a fall, the contrary. That this in- terpretation was the correct one was confirmed by direct inspection of the degree of flushing (redness). It was found that chilling the body surface immediately caused a fall in the temperature of the mucous mem- branes which could not be accounted for by any accompanying change in blood pressure, or, entirely at least, by changes in respiration or by lowering of the temperature of the blood. The conclusions are "that chill- ing of the body surface causes reflex vasoconstriction and ischemia in the mucous membranes of the palate, f aucial tonsils, oropharynx and naso- pharynx. ' ' The Methods for Determining the Healthfulness of Air. Although the present review does not venture to discuss the methods that are employed for the measurement of the various physical properties which have to be considered in gauging its influence on health, or the engineering problem of how ideal conditions may be maintained, it may not be out of place to mention, in connection with the former of these, that the physical property to which most attention should be devoted is the cool- ing power. This can not be done by reading an ordinary thermometer, for this instrument only registers the temperature of the piece of wood and of the wall against which it is hung. It registers the same whether the air is dry or moist, or whether it is stagnant or moving. Somewhat more information regarding cooling power is afforded by readings of a wet-bulb thermometer, an instrument in which the bulb is kept constantly moist, so that evaporation occurs from it. This evaporation tends to cool the thermometer, in proportion to its rate, and since this is dependent mainly on the degree to which the air can take up more moisture, we can tell by the use of a formula or tables the relative degree of humidity of the air. Still this does not tell us the real degree of cooling which the atmosphere can bring about. It does not adequately register the cooling which is dependent upon the movement in the air, the so-called convec- tion currents. To afford this information Leonard Hill has invented what he calls the Kata thermometer, by which the rate of cooling is directly measured. The instrument consists of an alcohol thermometer with a rel- atively large bulb, and with the scale registering between 105 F and 90 F. It is placed in warm water at about the former temperature, and is then removed, and the time required for the temperature to fall from 100 F. to 95 F. is measured by means of a stop watch. This time di- vided by a factor determined for each instrument, and written on the stem, gives the actual amount of heat in millicalories per square centi- meter per second which would be given off from, say the surface of the human body, under similar environmental conditions. Hill and his as- 764 METABOLISM sociates 3 have shown that much important information concerning the cooling power of the atmosphere can be gained in this way, which can not be gained by any other. METABOLISM REFEKENCES (Monographs and Original Papers) iLusk, Graham: The Elements of the Science of Nutrition, W. B. Saunders Co.. ed. 3, 1917. ^Cathcart, E. P.: The Physiology of Protein Metabolism, Monographs on Bio- chemistry, Longmans, Green & Co., 1912. sTaylor, A. E.: Digestion and Metabolism, Lea & Febiger, New York, 1912. ^Underbill, F. P. : The Physiology of the Amino Acids, Yale Press, New Haven, 1915. sMacleod, J. J. E.: Diabetes, Its Pathological Physiology, E. Arnold, 1913. saFiirth, von: The Problems of Physiological and Pathological Chemistry, etc., J. B. Lippincott Co., 1916. sbJones, W.: Nucleic Acids, Monographs in Biochemistry, Longmans, Green & Co., 1914. ecMendel, Lafayette B.: Ergebnisse der Physiologic, 1911. s^Leathes, J. B.: The Fats, Monographs in Biochemistry, Longmans, Green & Co. seMathews, A. P.: Physiological Chemistry, Wm. Wood & Co., 1917. sfDakin, H. K. : Oxidations and Eeductions in the Animal Body, Monographs in Bio- chemistry, Longmans, Green & Co., 1912. sgLeathes, J. B.: Problems in Animal Metabolism, 1906. 6Du Bois, E. F., and collaborators : Clinical Chemistry, Papers 1 to 25, Arch. Int. Med., 1915-17, xvi-xix. 7Benedict, F. G.: Am. Jour. Physiol., 1916, xli, 275 and 292. sMendel, Lafayette B.: Harvey Lecture, J. B. Lippincott Co., 1914-1915, p. 101. sMcCollum, E. V., and collaborators: Numerous papers in Jour. Biol. Chem., be- ginning 1913. "Hopkins, F. Gowland, and Willcock, E. G.: Jour. Physiol., 1906, xxxv, 88. nBayliss, W. M.: The Physiology of Food and Economy in Diet, Longmans, Green & Co., 1917. i2McCollum, E. V.: Harvey Lecture, Jour. Am. Med. Assn., 1917. , J. E., Carson- White, E. P., and Saxon, G. J.: Jour. Biol. Chem., 1913, xv, 181; ibid., 1915, xxi, 309. , W.: Biochem. Ztschr., 1909, xxii, 452. isFunk, Casimir: Ergebnisse der Physiologic, 1915. isMcKillop, M.: Food Values: What They Are and How to Calculate Them, Eutledge. isaMcCay, D. Major: The Protein Element in Nutrition, E. Arnold, London, 1912. irPembrey, M. S.: Chemistry of Eespiration, in Schafer's Text Book of Physiology, 1898, i. isAllen, F. P.: Glycosuria and Diabetes, Boston, 1913. isJoslin: Diabetes. zoWoodyatt, E. T., Sansum, W. D., and Wilder, E. M.: Jour. Am. Med. Assn., 1915, Ixv, 2067. Also Taylor, A. E., and Hulton, F.: Jour. Biol. Chem., 1916, xxv, 173. 2iMacleod, J. J. E., and Fulk, M. E.: Am. Jour. Physiol., 1917, xlii, 193. 22Hamman, L., and Hirschmann: Arch. Int. Med., 1917, xx, 761-788. 23Cannon, W. B.: Bodily Changes in Pain, Hunger, Fear and Eage, D. Appleton & Co., 1915. 24Knowlton, F. P., and Starling, E. H.: Jour. Physiol., 1912, xlv, 146. 25p a tterson, S. W., and Starling, E. H.: Jour. Physiol., 1913, xlvii, 135; also Cruick- shank and Patterson: Ibid., p. 113. 26Macleod, J. J. E.: (a) Jour. Biol. Chem., 1913, xv, 497; (b) Physiol. Rev., 1921, i, 208. 27Murlin, J. E.: Jour. Biol. Chem., 1913, xvi, 79. 28Cruickshank: Jour. Physiol., 1913, xlvii, 1. 29Macleod, J. J. E., and Pearce, E. G.: Zentralbl. f. Physiol., 1913, xxvi, 1311. sowoodyatt, E. T.: Jour. Am. Med. Assn., 1916, Ixvi, 1910. THE PHYSIOLOGICAL PRINCIPLES OF VENTILATION 765 Slyke, D. D.: The Present Significance of the Amino Acids in Physiology and Pathology, Harvey Lectures, J. B. Lippincott & Co., 1915-1916, p. 146. Also papers in Jour. Biol. Chem., 1911, ix, 185; xii, 275; ibid., 1912, xii, 301 and 399; ibid., 1913, xiii, 121, 125 and 187. in, O., and Denis, W.: Jour. Biol. Chem., xi, 87 and 493; ibid., 1912, xii, 14 and 253. 33 Abel, J. J.: The Mellon Lecture, Science, 1915, xlii, 135. 3*Hewlett, A. W., Gilbert, L. O., Wickett, A. D.: Arch. Int. Med. r 1916, xviii, 636. ssLosee, J. E., and Van Slyke, D. D.: Jour. Am. Med. Assn., 1917, cliii, 94. 36Shaffer, P. A.: Am. Jour. Physiol., 1908, xxviii, 1. 37Cathcart, E. P.: Jour. Physiol., 1907, xxxv, 500. ssMyers and Fine: Jour. Biol. Chem., 1913, xiv, 9. ssLevene, P. A.: Cf. W. Jones.* 4oJones, W.: Nucleic Acids, Monographs on Biochemistry, Longmans, Green & Co., 1914. ^Benedict, S. E.: Harvey Lecture, 1915-16. *2Hunter, A., and Givens, M. H.: Jour. Biol. Chem., 1914, xviii, 403. 43Burian, E., and Schur, H.: Cf. Macleod in Eecent Advances in Physiology and Bio- chemistry, ed. by Leonard Hill, E. Arnold, London, 1905. 44Mendel, Lafayette B., and Lyman, J. F.: Jour. Biol. Chem., 1910, viii, 115. 45Taylor, A. E., and Eose, W. C. : Jour. Biol. Chem., 1913, xiv, 419. 46Hopkins, F. G., and Hope, W. B.: Jour. Physiol., 1899, xxiii, 277. 47Ascoli, M., and Izar, G. : Ztschr. f . Physiol. Chem., 1909, Iviii, 529 ; ibid., 1911, Ixiii, 319. *8McClure, C. W., Vincent, B., and Pratt, J. H.: Am. Jour. Physiol., 1916, xlii, 596. *9Bloor, W. E.: Jour. Biol. Chem., 1912, xi, 429; ibid., 1913, xv, 105; ibid., 1914, xvi, 517; ibid., 1912, xi, 141; ibid., 1915, xxi, 421; ibid., 1914, xix, 1; ibid., 1915, xxiii, 317; ibid., 1914, xvii, 317; ibid, 1915, xxii, 133. Also Bloor and Knudson: Jour. Biol. Chem., 1916, xxvii, 107; ibid., 1916, xxiv, 447; Bloor, Joslin and Homer: Ibid., 1916, xxvi, 417; ibid.j, 1916, xxv, 577. (49b) Physiol. Eev., 1922, ii, 92. soLeathes, J. B.: The Fats, Monographs on Biochemistry, Longmans, Green & Co. siCoope, E., and Mottram, V. H.: Jour. Physiol., 1914, xlix, 23; ibid., 1915, xlix, 157. 52Eaper, H. S.: Jour. Biol. Chem., 1913, xiv, 117. ssSmedley, I. D.: Proc. Phys. Soc., Jour. Physiol., 1912, xiv, 25. s*Hill, Leonard: Address to the Phys. Sec. Brit. Assn. for the Adv. of Sci., Section, J, 1912. ssShaffer, P. A., and Coleman, W.: Arch. Int. Med., 1909, iv, 538. seBarbour, H. G. : Arch. f. Exper. Path. u. Pharmac., 1912, Ixx, 1; Jour. Pharmac. and Exper. Therap., 1913, v, 105 ; Physiol. Eev., 1921, i, 295. ^Hewlett, A. W.: Monographic Medicine, D. Appleton Co., 1917, i. ssHunter, A., and Campbell, W. E.: Jour. Biol. Chem., 1918, xxxiii, 169. 59Thompson, Sir W. H. : Biochem. Jour., 1917, xi, 307. eoRingsbury, F. B., and Bell, E. T.: Jour. Biol. Chem., 1915, xxi, 297. siLynch, V.: Am. Jour. Physiol., 1919, xlviii, 258. 62Leathes, J. B. : Problems of Animal Metabolism, (London, 1906). 63Maeleod, J. J. E. : Jour. Biol. Chem., 1906, ii, 231. e^Cruikshank and Patterson: Jour. Physiol., 1913, xlvii, 381; ibid., 1914, xlix, 67. <55Clark, A. H.: Johns Hopkins Hos. Eeports, 1919, xviii. eeHarris, J. A., and Benedict, T. G. : Eeport Carnegie Institution of Washington, 1919. G7Means, J. H., and Aub, J. C. : Arch. Int. Med., 1919, xxiv, 645. esDavis, N. C., and Hall, C. C., and Whipple, G. H. : Arch. Int. Med., 1919, xxiii, 689. 69Banting, C. F., Best, C. E., Collip, J. B., Campbell, W. E., and Fletcher, A. A.: Canadian Med. Jour., 1922 (March). 7oHopkins, F. Gowland, and Chick, Hariette: Medical Eesearch Committee National Health Insurance, Special Eeport No. 38, 1920, H. M. Stationary Office, Imperial House, Kingsway, London, W.C. 2. 7iOsborne, T. B., and Mendel, J. : Jour. Biol. Chem., 1917, xxxi, 144. 72Hill, L.: Special Eeport No. 32. The Science of Ventilation and Open Air Treat- ment, Medical Eesearch Committee, H. M. Stationary Office, London, 1919. 73Mudd, S., and Grant, L. B.: Jour. Med. Eeseareh, 1919, xl, 53. 74Allen, F. M., Wishart, M. B., and Smith, L. M. : Arch. Int. Med., 1919, xxiv, 523. Also Murlin, J. E., and Nites, W. I. : Am. Jour. Med. Se., 1917, cliii, 79. 75McCarrison, E. : Food and Deficiency Disease, 1921. PART VIII THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS (Revised by N. B. Taylor) CHAPTER LXXXIV GENERAL CONSIDERATIONS, THE ADRENAL GLANDS In order that the various activities of the animal organism may act efficiently as a whole, it is necessary that those of one part be correlated with those of another. This correlation of function is mediated either through the nervous system or through the action on one part of the body of substances produced in another part and carried between them by the blood. Control through the nervous system is especially developed for those functions which have to be brought promptly into play, such as muscular movement and the other physiological processes concerned in the adjustment of the organism to quickly changing conditions of its environ- ment. Control through the blood is the mechanism by which the metabolic activities of different organs are mainly correlated. The chemical sub- stances involved are often called internal secretions. Some of these internal secretions are merely by-products of metabolism, and are only incidentally used for the purpose of bringing about control between different parts of the body. To this group belong carbon dioxide, which may act on the respiratory and other nerve centers, and urea, which may stimulate increased activity of the kidneys. Indeed, the list of sub- stances included under such a definition of internal secretions is almost illimitable, and to designate by the special name of hormone every con- stituent that can affect physiological functions, as some have done, can lead only to confusion. The internal secretions with which we are more directly concerned are those that are specially produced for the purpose of controlling the metabolic functions. They are given the general name of autacoids (E. A. Schafer). 3 Autacoids may be either the sole product of some special gland or a secondary product of glands which have other functions. To the former class belong the autacoids produced by the para- thyroid, thyroid, pituitary and adrenal glands, and to the latter, those produced by the pancreas and generative glands. 766 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS 767 Autacoids have further been subdivided by Schafer into two classes according to whether they excite metabolic processes or depress them. Examples of excitatory autacoids, also designated as hormones, are the epinephrine produced by the adrenal glands, which excites the termina- tions of the sympathetic nervous system, and pituitrin produced by the posterior lobe of the pituitary gland, which excites plain muscular fiber. Inhibiting autacoids, also called chalones, are not so commonly known, but are illustrated by the substance contained in extract of the placenta, which tends to prevent the secretion of milk. Autacoids may have either an immediate or a delayed action ; the effect which they produce may be like that with which we are familiar as the result of stimulation of the nerve supply of a gland, being illustrated again by the effect of epinephrine, or they may act so slowly that it is only after a considerable period of time during which they have been in the organism in excess, that any apparent effect is produced. The slowly acting autacoids have been called morpho genetic, and they are well illustrated in the internal secretions of the anterior lobe of the pituitary and of the generative glands secretions which affect growth. Regarding the chemical nature of autacoids, certain facts stand out prominently. Being very largely the products of glands, it might be imagined that they would be enzymic in nature, for enzymes are now known to be the most important active agents in bioplasm as well as the active agents in many of the external secretions, like those of the salivary, gastric and intestinal glands. Autaeoids, however, are not enzymes. They are far simpler in chemical structure, and are not destroyed by heat in the presence of water. They are represented by a comparatively small molecule, and are therefore dialyzable. This latter fact justifies the hope that it may be possible to prepare them or their simpler salts in crystalline form a hope which has already been realized in the case of thyroxin and epinephrine. Progress has likewise been made in isolating the active principles of the anterior and posterior lobes of the pituitary gland. To sum up, then, we may say that an autacoid is a specific organic substance, formed by the cells of one organ and secreted into the circulating fluid, which carries it to other organs, upon which it produces effects similar to those of drugs. Methods of Investigation To investigate the function of an autacoid, careful studies are made of the effects produced (1) by excision of the gland which furnishes the autacoid and (2) by administering intravenously or subcutaneously or >rally extracts prepared from the gland. Frequently, also light is thrown on the function of the autacoid by observing the effect which fol- lows prolonged feeding with the endocrine organ that manufactures it and by observing the pathological changes in the various endocrine organs in diseased conditions. Embryological and histological studies are also of the greatest importance. A difficulty in investigating the function of an endocrine organ lies in the fact that the secretion of no one gland 768 THE ADRENAL GLANDS acts independently of those from other glands. On the contrary, there is undoubtedly a close association of function, so that we can not tell whether a change of function observed after removal of some gland or administration of some extract is a direct consequence of the experi- mental procedure, or is induced by some secondary effect developed on another endocrine organ. It will no doubt take many years before suf- ficient data have been collected to enable us definitely to state what the particular function of each endocrine organ may be. Since most progress has been made in connection with the adrenal gland, it will be advan- tageous to consider the functions of this gland first. ADRENAL GLAND In mammals the adrenal gland is composed of two parts, the cortex and the medulla. The origins of these two are quite different, and though in mammals they are intimately associated in anatomical position, in other groups of animals they are more or less separate, being completely so in fishes. This not infrequent separation of cortex and medulla, together with their distinctive origins, suggests different functions for the two. Experimental investigation supports this view. The Cortex The cortex on microscopic examination is seen to be composed of rows of epithelial cells arranged more or less in columns except at the periphery, where they form glomeru- lar masses, and next the medulla, where they assume a reticular formation. The cells of the greater part of the cortex, unlike those of the medulla, contain no granules with special staining qualities, but they do contain particles which are believed to be com- posed of cholesterol esters and lecithin. In the cells of the reticular portion of the cortex, however, pigment particles are not infrequently observed. The blood supply of the cortex is not nearly so rich as that of the medulla, being represented by fine arterioles which run inwards from the capsule towards the medulla in the connective tissue that Des between the columns of cortical cells. Nerves similarly penetrate into the cortex, some supplying its blood vessels and cell columns, but most of them proceeding to the medulla. They are derived from a network of nerve fibers in the capsule of the organ, and the nerve supply of this network comes partly from the suprarenal plexus, and partly from the splanchnic nerve. Embryologically the cortex is developed from the cells of the genital ridge, that is, from mesodermic cells. Very little is known concerning the function of the adrenal cortex al- though there is little doubt that it is closely related to the development of the sexual organs. The evidence for this is as follows: (1) It arises from the mesoderm in common with the sexual organs. There is also a remarkable similarity between the cortical cells and those of the corpus luteum. (2) In cases of sexual precocity it is found that the adrenal cortex is much hypertrophied. Also, certain tumors of the cortex occur- ring in young children are associated with premature development of the THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS 769 secondary sexual characters. The subjects of these growths (Pig. 191), which are termed hypernephromata, present, in many cases, a most re- markable appearance. A boy, for instance, of four or five years may possess the sexual development of a mature male, the testicles are en- larged, hair grows upon the chest and pubis, and a moustache or a beard may develop. There may be also undue muscular development or ex- treme obesity of an adult type, so that these prodigies have been likened in appearance to "an infant Hercules" (Weber) or "a burly brewer's Fig. 191. Child aged 4J4 years suffering from hypernephroma. (Guthrie.) drayman" (Guthrie). 4 In female children the breasts hypertrophy, hair appears upon the mons veneris and labia majora, the uterus tends toward the mature type, and menstruation may occur. In appearance these patients resemble stout little women. (3) The cortex becomes hypertro- phied during pregnancy. (4) It is ill-developed in sexual deficiency. (5) Changes occur in it during the estrual cycle of many animals. In the frog during the mating season it becomes greatly enlarged at the ex- pense of the medullary tissue and develops^ peculiar pear-shaped ele- ments, the "summer cells" of Stilling. (6) After castration the cortex is said to be hypertrophied. (7) The innermost portion of the cortex, some- 770 THE ADRENAL GLANDS times called the boundary zone, is much hypertrophied in the human fetus, but this hypertrophy entirely disappears after the first year of extrauterine life. The other functions of the cortex are not as yet known, but there is very strong evidence that they are of great importance to the welfare of the animal. It has been suggested that the passage of blood through the cortex before reaching the medulla indicates that some change, which is preparatory to the main change occurring in the medulla, takes place in the blood while it is in the cortex. This view is partly substantiated by the observation that when an excised jportion of cortex is incubated at body temperature, a substance develops in it which has an action like that of the hormone of the medulla epinephrine. It is possible, however, that this action is due to the fact that certain of the decomposition products of protein develop an epinephrine-like action (see page 536). A detoxicating function has been ascribed to the cortex. This possi- bility has been suggested by the fact that cobra venom, to which had been added an emulsion of this portion of the gland, was rendered innoc- uous. (Meyers). 5 The addition of other tissue extracts to the poison was without effect. The weight of evidence favors the view that it is the cortex and not the medulla which is essential to life. Biedl 2 claims to have removed the cortex leaving the medulla intact ; the operation resulted invariably in the death of the animals. Conversely he found that, in cats and dogs, the adrenals could be removed with impunity to the extent of seven eighths of their bulk, provided that the portion remaining consisted of cortex. Wheeler 6 endeavored to remove the medulla, leaving the cortex intact ; though this was not wholly successful, it is a noteworthy fact that it was those animals only, in which the cortex was inadvertently injured, that succumbed to the operation. Evidence for the indispensability of the cortex is also offered from the clinical side. Cases of acute adrenal de- ficiency, followed by rapid death, occur, in which the medulla, postmor- tem, shows few or no lesions or disease of a longstanding nature, whereas acute lesions are observable in the cortex. The Medulla Histologically the medulla is composed of masses of polygonal cells with blood sinuses between them. The blood supply is derived from ves- sels that have proceeded to the medulla through the capsule, and it is extremely rich, being indeed the richest blood supply to any organ in the body, greater even than that to the thyroid gland. The nerves form a dense plexus, extending into and between the secretory cells. The most characteristic feature of the cells composing the medulla is the presence in them of granules which stain readily with chromic acid, and are hence THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS 771 often called chromaffin cells. There are also some cells containing coarser granules that are soluble in water and do not stain with chrome salts. Embryologically, the medulla is developed from tissue common to it and the sym- pathetic nervous system. From that part of the neuroblast in which are laid down the primitive ganglia of the posterior roots, masses of cells are split off which become the common ancestors of the sympathetic ganglia and the chromaffine system. These cell groups wander from their sites of origin and come to lie along the vertebral bodies, ranging themselves, in the case of the abdomen, on either side of the aorta. One mass seeks the adrenal cortex, which has been formed at a prior stage of development, and passes into its interior. Differentiation of these cells then proceeds in two directions, those lying along the aorta form, for the most part, sympathetic ganglia; those within the adrenal cortex develop into chromaffin cells to constitute the medulla of the gland. These embryological considerations will enable us to understand the close functional re- lationship which, as we shall see, exists between the sympathetic nervous system and the adrenal medulla. The intimate anatomical association of the cortex and medulla renders the removal of one part alone, if not an actual impossibility, a procedure at least, of extreme difficulty. On this account, with the exception of the investigations cited above, attention has been paid only to the effects pro- duced by removal, or by the injection of extracts of the whole gland. Adrenalectomy Excision of the adrenal gland in most animals is very quickly fatal, the only well-known exception being in the case of the white rat, in which excision of both adrenals may not be incompatible with life. For some time after recovery from the anesthetic the animal upon which double adrenalectomy has been performed usually behaves in a perfectly normal fashion, although it may be less lively and less inclined to feed than usual. Very soon, however, generally within twenty-four or forty- eight hours, definite symptoms of muscular weakness are apparent. This weakness soon becomes extreme, and is accompanied by a feeble pulse, a depression of body temperature, and, later, by dyspnea. After an interval which is never longer than a few days, death supervenes, being sometimes preceded by convulsions. When only one adrenal is removed, very few animals succumb; and if some time is allowed to elapse so that the immediate shock of the operation has disappeared, it will usually be found that removal of the remaining adrenal, although ultimately fatal, is not so quickly so as when both glands are removed at one operation. The reason for this result is that opportunity is given for a compensatory hypertrophy of accessory adrenal bodies to occur. Such accessory adrenal bodies may be composed of cortical or medullary tissue, and there is a growing belief that the cortical tissue is the more important. Chromaffin tissue is found in most animals along the front of the aorta, between the renal arteries, 772 THE ADRENAL GLANDS where it can usually be recognized by staining the tissue with chromic acid. Sometimes accessory chromaffin tissue is located in distant parts, as in the epididymis of the rat, for example. It is said that life can be maintained if one-eighth of the total amount of the adrenal substance be present in the body. Attempts to prolong life after adrenalectomy by adrenal transplantation have almost invariably met with negative results, because the graft undergoes a rapid process of necrosis and dis- appears; although it is said that transplantation may sometimes be suc- cessfully accomplished if the grafting is done into the kidney. Adminis- tration of suprarenal extract is also without definite benefit after adrenalectomy. Adrenal Disease in Man. Besides the hypertrophy of the cortex, which has already been alluded to, destructive disease (usually tuberculous) of the adrenal gland occurs, which has been recognized to be the cause of a characteristic clinical condition known as Addison's disease. This condition, which runs a more or less protracted course and is almost in- variably fatal, is characterized by muscular weakness, low blood pres- sure, pigmentation of the skin and gastrointestinal disturbances. Injec- tions of epinephrine have little or no influence over the symptoms or over the course of the disease ; administration of extracts of the whole gland are, perhaps, of more benefit. Though it is almost universally accepted that inadequacy of the adrenals is responsible for the disease, the imme- diate cause of the symptoms is obscure, nor is it known whether cortex or medulla is at fault. It might appear that the muscular weakness and ar- terial hypotonus were due to incompetency of the medulla; yet the results of epinephrine administration do not support such a conclusion. Further- more, as we shall see, it has been demonstrated conclusively that epine- phrine is not a factor in the maintenance of normal arterial tone (page 785). The bronzing of the skin, a prominent symptom of Addison's disease, is due to an increase of the normal pigment melanin in the Malpighian layer. To account for the excessive deposition of pigment (chromatosis) Halle 7 has suggested that there is an increase of tyrosine, the precursor of melanin, in the tissues. Part of the tyrosine of the body is believed to be converted, under normal conditions, into epinephrine. This belief is supported by a comparison of the formulae of the two substances (epinephrine C 9 H 13 N0 3 , tyrosine CaH^NOg) and by the following experi- ment: An emulsion of the gland was divided into two halves, to one of which tyrosine was added; the two portions were incubated for six days, after which period analysis showed that the portion to which the amino acid had been added contained from 15 to 33 per cent more epinephrine than the control. That melanin may be produced by the action of the enzymes upon tyrosine is well known, for example, an extract of the THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS 773 tissue forming the wall of the inksac in the cuttle fish will, upon the ad- dition of tyrosine, produce a sepia pigment. Halle's hypothesis, then, implies that the tyrosine which under normal circumstances would be employed for the manufacture of epinephrine, is, in the case of adrenal deficiency converted into pigment. Attractive though this hypothesis may be, it should be pointed out that Ewins and Laidlaw 8 have failed to con- firm it. In addition to this chronic form of adrenal disease cases of acute adrenal insufficiency occur. Death, which very rapidly ensues, may be preceded by symptoms of cerebral hemorrhage, or acute abdominal dis- ease or may follow a short period of extreme myasthenia. In other in- stances death is sudden and unheralded, and that the adrenals are re- sponsible is revealed by finding that they are the seat of extensive disease. This may be manifested by areas of necrosis, by hemorrhage or by ve- nous thrombosis. In a case reported recently by Boyd, 9 the subject, prior to the rapidly fatal attack, was in apparently good health. An autopsy showed the medullary tissue to be entirely consumed by a process of long standing, whereas the cortex was the seat of an acute lesion which apparently was the cause of the fulminating symptoms. Suprarenal Extracts Preparation Injection, particularly intravenous, of extract of the adrenal gland has furnished us with most of the evidence upon which our knowledge regarding the function of this organ depends. Such an extract is best made by grinding the entire gland with fine sand in a mortar and then extracting with a weak (decinormal) solution of hydrochloric acid. The extract may then be boiled, filtered through muslin and nearly neutral- ized, preferably by means of sodium acetate. If kept in this acid reac- tion, the active principle of the extract does not materially deteriorate with time, but if it be neutralized or considerably diluted, destruction due to oxidation occurs, as evidenced by a distinct browning of the solution. The active principle of such extracts has been isolated in a crystalline form (Takamine and Abel). It has been given various names (adrenalin, suprarenin, adrenin, etc.), but the tendency is definitely towards the use of epinephrine. Chemically, epinephrine has been found to be orthodioxyphenylethylolmethylamine. HO ^> -*CH(OH) - CH 2 NHCH 3 . It will be noted that it is closely related to tyrosine (see page 639). It is also closely related to a group of substances (amines) occurring in ^utrid meat and to which the active principles of ergot belong. It 774 THE ADRENAL GLANDS contains an asymmetric carbon atom (asterisked in formula), which indicates that there must be three varieties of epinephrine, differing from one another in the effect which they produce on the plane of polarized light (i.e., a dextro- and a levo-rotatory and a racemic form). Epinephrine can be prepared by synthetic means, the first product of this synthesis being the racemic salt, which can then be split by appro- priate methods into dextro- and levo- varieties. The levo- variety ap- pears to be identical in its pharmacological action with the natural product. The dextro- variety on the other hand has only poorly developed physio- logical activities (about seven per cent that of the levo- variety), while the racemic variety comes in between the two in its action. A valuable assay of the amount of epinephrine in tissue extracts can be made by the method of Cannon, Folin and Denis, 10 in which an acid extract of the gland is treated with phosphotungstic acid, and the blue color thereby developed compared colorimetrically with a standard blue. Physiological Action The physiological effects of the intravenous injection of epinephrine are markedly excitatory and slightly inhibitory in nature. We will consider the excitatory action first. Immediately after the intravenous injection of as small an amount as '00008 milligrams per kilogram of body weight, a distinct rise in arterial blood pressure may be observed. When the rise is distinct, it is accompanied by a slowing of the pulse. This slow- ing is caused by stimulation of the vagus center, as is evidenced by the fact that if the. vagus nerves are cut, or sufficient atropine administered to paralyze them, the same dose of epinephrine produces not a slowing but a quickening of the pulse, and consequently a much greater rise in blood pressure. The vagus action is developed not because of an effect of epinephrine on the vagus center, but secondarily because of the rise in blood pressure. These preliminary experiments indicate that the locus of action of epinephrine, so far as the circulatory system is concerned, is mainly on the small blood vessels, constricting them and thus raising the peripheral resistance. This conclusion can readily be confirmed by applying the epinephrine directly to the blood vessels of the exposed mesentery, or by enclosing a vascular organ such as the kidney in a plethysmo graph during the injection of epinephrine, when a great diminution in volume, accompanying the rise of arterial blood pressure, will be observed. The vasoconstricting effect of epinephrine does not become developed on the large blood vessels near the heart on account of the deficiency in muscu- lar tissue in their walls. Indeed, these vessels may become passively dilated because of the increased blood pressure. The arterioles of dif- THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS 775 ferent parts of the circulation are not equally sensitive to epinephrine ; those of the splanchnic area are most sensitive, whereas those of the heart the coronary vessels do not respond at all in most animals (see page 268). The pulmonary and cerebral vessels have a variable reactivity to epinephrine. The effect on the vessels persists after complete destruction, not only of the central nervous system, but also of the vasomotor nerves; epi- nephrine still acts, for example, on vessels the nerve fibers of which have been allowed to degenerate by cutting them several days before the epinephrine is applied. This would seem to indicate that the epinephrine acts directly on the muscular tissue in the walls of the blood vessels, but this does not appear to be the case, for it has been found that epi- nephrine is incapable of acting on tissues which are devoid of sympathetic nerve fibers, and is also inactive on those tissues in the embryo which have not yet received any nerve supply. In brief, then, although epinephrine acts only on blood vessels that are supplied by the sympathetic nervous system, it is not on the nerve fibers that the epinephrine unfolds its action. We shall see immediately that this conclusion is in conformity with the results of observations made on structures other than the blood vessels. Other muscular structures excited by epinephrine are as follows: (1) the dilator muscle of the pupils, especially after the nerve supply has been destroyed by extirpation of the superior cervical ganglion; (2) the sphincters of the pylorus and of the ileocecal valve; (3) the muscle fibers of the spleen, the vagina, the uterus, the vas deferens, and the retractor penis. Regarding the action on the uterus, however, it should be noted that a different response may be obtained according to whether the uterus is pregnant or not. The plain muscles of the orbit and globe of the eye are sometimes excited by suprarenal extract, causing the eyes to protrude, the palpebral fissure to become large and the third eyelid to be retracted, changes which are very like those which develop as a result of fright. Inhibitory effects of epinephrine on muscle are exhibited by the follow- ing: (1) the muscle of the intestine; (2) the stomach; (3) the esophagus; (4) the gall and urinary bladders. The effect of epinephrine in inhibiting the rhythmic contractions of isolated portion of the intestine in oxygenated Ringer's solution is a rery striking phenomenon, and one which, as we shall see, may be very successfully employed for detecting small quantities of epinephrine. The effects of epinephrine on glandular structures are the same as those rhich would be produced by stimulation of the sympathetic nerve supply of the gland. Thus, the secretions of the lachrymal gland, the salivary 776 THE ADRENAL GLANDS gland (in the cat), the mucous glands of the mouth and pharynx, the gastric but not the pancreatic glands, can readily be shown to be excited. In the case of the kidney the immediate effect is a diminution of the urinary flow, due to constriction of the renal vessels. It has been suggested by Cow 11 that one role of the suprarenal is to act as a regula- tor of the urinary excretion. This observer has demonstrated, by ana- tomical methods, direct vascular communications between the adrenal medulla and the kidney. That epinephrine actually gains the kidney by these channels was shown by collecting and testing the blood after its circulation through them. Blood collected, while the gland was ex- cited reflexly by sciatic stimulation, exhibited marked pressor action. The work of Addis 12 and others shows that the excretion of urea is in- creased under the influence of epinephrine in certain dilutions so that though the urine may be diminished in quantity, its concentration is raised. From these results as a whole, it is evident that the effect of epineph- rine on muscles and glands is exactly the same as that which would be produced by stimulation of their sympathetic nerve supply. This paral- lelism of action between epinephrine and the sympathetic nervous sys- tem becomes still more evident when we consider certain of the changes in metabolism that follow administration of epinephrine. Injection of epinephrine excites glycogenolysis in the liver so that hyperglycemia and glycosuria become established, results which are also obtained by stimulating the great splanchnic nerve. Intravenous injection of epineph- rine causes the clotting time of the blood discharged from the liver to be very materially shortened, an effect also produced by stimulating the splanchnic nerve. 13 As in the case of the blood vessels, the above results are obtained even after the sympathetic nerves to the part have been allowed to undergo degeneration, from which it is concluded that the tissues elaborate some substance which reacts with epinephrine. This substance may be pro- duced either at the junction between the nerve and muscle the myo- neural junction, or perhaps throughout the protoplasm itself. It is called the receptor substance of Langley, and is believed to react not only with epinephrine, but also with various drugs. The receptor sub- stance seems to increase, if not in amount, at least in sensitivity after the removal of the nerve control. Ergotoxin, which is an amine obtained from ergot and also from cer- tain of the products of histidine, has an action on the receptor substance which is inhibitory and therefore antagonistic to that of epinephrine. The antagonistic action of ergotoxin affects the excitatory but not the inhibitory actions of epinephrine. By using this drug we are en- abled to show that, although the main effect of epinephrine on the tissue is THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS 777 excitatory, a less marked inhibitory influence may be simultaneously developed. The inhibitory effect may also sometimes be evoked by doses of epinephrine very much smaller than those used to produce excitatory effects. These facts are well illustrated in the case of the muscle fiber of the blood vessels. With an ordinary dose of epinephrine constriction occurs; after ergotoxin the same dose of epinephrine causes dilatation. This latter result may also be obtained by administer- ing to a normal animal quantities of epinephrine that are very much smaller than the usual quantity. The coexistence of inhibitory and ex- citatory influence is also well noted in the case of the uterus. In some animals the effect of epinephrine on this organ is to augment its rhythmic contractions, in others to inhibit them. In the former case, however, if ergotoxin is first of all administered, epinephrine in its usual dosage will invariably produce an inhibitory effect. The ergotoxin no doubt acts on the receptor substance, and similar effects have also been produced with apocodeine. It was first noted by Moore and Purinton 14 that the usual rise of blood pressure which followed the injection of epinephrine was replaced by a depressor effect when the dose was very small. Later it was shown that this was not an isolated instance of a reversed action of epinephrine when employed in high dilutions; the intestinal tone is augmented by minute doses (1 part in 500 million or more according to Hoskins) 15 aiid the contractions of the pregnant uterus inhibited. The nature of the vasomotor effects differs not only in accordance with the dosage, but it is of dissimilar sign in different vascular areas, though the dilution of the drug be kept constant. Hartman 16 has shown that epinephrine in high dilution causes dilatation of the peripheral vessels simultaneously with vasoconstriction in the splanchnic area. These con- clusions were drawn from blood pressure records taken in two series of experiments in which the splanchnic and the peripheral vessels, respec- tively, were excluded from the circulation. In the former series a fall in pressure was effected, in the latter a pressor response was obtained. Hoskins, Gunning and Berry 17 went further and found, by means of plethysmographic records, that all the vessels of the peripheral circula- tion did not respond alike to a given dose, those of the muscles being dilated, while those of the skin were constricted with high dilutions. The variations in limb volume and of blood pressure would then depend upon which of these effects predominated at the time. Neither are all parts of the splanchnic area affected similarly, for, though the spleen and kidney both show vasoconstriction with all dilutions, the intestinal vessels are constricted by small doses, but dilated by large. (Hartman). The vasodilator responses to epinephrine are not, as is the case with the constrictor effects, mediated by the myoneural junction. The vasodilator 778 THE ADRENAL GLANDS mechanisms for the intestine are located in the sympathetic ganglia, those for the limbs are contained in the sympathetic as well as the posterior root ganglia. These mechanisms are of comparatively late development; in newborn mammals (with the exception of rodents) administration of epinephrine in all dilutions produces universal vasoconstriction, and it is not until the animal is several weeks old that dilator effects can be ob- tained, peripheral dilatation is the first to appear, somewhat later dila- tation of the intestinal vessels may be elicited. Although it is especially on plain muscular fiber having a sympathetic nerve supply that epinephrine unfolds its action, yet, according to Can- non, Gruber, 18 and others, it increases the contracting power of volun- tary muscle and diminishes the tendency to fatigue. CHAPTER LXXXV THE ADRENAL GLANDS (Cont'd) Variations in Physiological Activity Since it is clearly established that the adrenal glands are indispensable to life and that extracts of them have a very pronounced physiological ac- tion, it remains to consider whether the glands produce this internal secre- tion within the body, and if so, whether it is essential for the well-being of the animal or is required only under certain conditions. We must also endeavor to find out upon which of the bodily functions of the intact animal the internal secretion acts. These problems have been attacked by three methods of investigation: (1) by comparing the epinephrine content of similarly prepared extracts of the resting gland and of one removed after a period of supposed increased activity; (2) by collecting the blood as it flows into the vena cava from the adrenal vein and ex- imining it for epinephrine by physiological tests. These consist in observ- ing the behavior of some tissue that is sensitive to the action of epineph- rine, such as the intestine or uterus, after applying the blood or serum to it, or by injecting the blood or serum intravenously into another ani- mal and looking for epinephrine effects; and (3) by allowing the blood of the adrenal vein to be discharged under certain conditions through the vena cava into the blood vessels of the same animal, and observing the effect produced on certain physiological processes which in one way >r another have been sensitized toward the influence of epinephrine. s autoinjection method has recently been used successfully by Stew- art and Rogoff, 19 their favorite structure upon which to observe the epinephrine effect being the denervated pupil. Assaying the Epinephrine Content of the Gland With regard to the first mentioned of the methods, either chemical or physiological means may be employed to assay the strength of the ex- tracts. The best chemical method is that of Cannon, Folin and Denis, 10 the principle of which has already been described. The physiological method yielding most satisfactory results is that of Elliott, 20 which con- sists in injecting a portion of the extract intravenously into animals from which the influence of the nerve centers on the heart and blood vessels has been removed by decapitation. The rise in arterial blood 779 780 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS pressure produced by the injection is then a very fair measure of the amount of epinephrine contained in it. It has been shown that the re- sults obtained by the chemical method agree very closely with those obtained by the physiological, but it should be remarked that it is difficult to see how the physiological method could be accurate in all cases, since it has been shown that with great dilution of epinephrine a reversed effect a vaso- dilatation may be obtained. Attempts to assay the strength of an epinephrine solution by investigating the effects which it produces on other preparations, such as isolated loops of intestine or uterus, or the enucleated eyeball of the frog, are not always successful, since the effects are not alone dependent on the concentration of epinephrine in the extract. When such preparations are used for quantitative purposes, the strength of the extract may be judged by finding the extent to which it can be diluted and still remain active. Quite apart from the foregoing possible sources of error, it must be remembered that the results merely give us an idea of how much epineph- rine may have been contained in the gland at the time of its excision. They can not tell us how much epinephrine the gland was secreting. Prior to excision as much of this hormone might have been undergoing a process of manufacture in the gland as was being discharged from it, so that the assayed amount would represent merely the balance of production and loss of hormone by the gland. We might quite well find that the amount of epinephrine in the excised gland was normal under conditions where there had been an excessive discharge of it into the blood; that is to say, loss and production might have been equal. Where, however, a marked deficiency was found to exist, it would probably indicate that exhaustion of the power of producing epinephrine was taking place. The Epinephrine Content of the Blood, The second method, in which blood from one animal is tested for its epinephrine effect by intravenous injection into another animal or by applying it to some isolated prepara- tion on which epinephrine acts, has yielded important results. Since serum contains all the epinephrine of blood, it can be conveniently used for the tests (Stewart and Eogoff). The isolated physiological prepara- tions that have been used in testing for epinephrine in the animal fluids are as follows: 1. A segment of the small intestine of a rabbit, suspended in oxygen- ated Locke's solution at body temperature. 2. A segment of the uterus of a nonpregnant rabbit similarly prepared. The apparatus used for observing the contractions of either prepara- tion consists of a small glass chamber furnished below with a hook to which one end of the segment is attached, the other end being connected .to a muscle lever, so that the regular rhythmic contractions can be regis- tered on a drum (Fig. 192). THE ADRENAL GLANDS 781 Epinephrine inhibits the contractions of the intestine but stimulates those of the uterus of most animals, the intestine preparation being the more sensitive (Fig. 193). Indeed, it is said that the inhibition in this case may be obtained with a solution containing 1 part of epinephrine in 20,000,000 of solution. In using this method, however, great care and judgment must be exercised in drawing conclusions, because other sub- stances present in the blood are liable to affect the contractions; thus, certain substances in blood serum which have been produced by the act of blood clotting may cause augmentation of the beat in both the intes- Alr vent Mete/ waterbath 38'c. Harvard muscle armer with aduated scale Fig. 192. Arrangement of apparatus for recording contractions of a uterine strip, intestinal strip, or ring, etc. The metal water-bath is made of a cheap metal water-pail with a heating rod soldered through the side at the bottom. A short metal tube is soldered into a 1-inch opening in the bottom to receive a perforated cork for connecting with the Harvard muscle-warmer inside. (From Jackson.) I tinal and the uterine preparations. A certain amount of epinephrine in Locke's solution is consequently more likely to cause inhibition of the intestine than a similar amount added to blood serum, because in the lat- ter case the pressor substance will neutralize the depressor effect of the epinephrine. On the uterine preparation, both the blood serum and the epinephrine have pressor effects. As has been pointed out by G. N. Stewart, 21 if both preparations are employed for testing a solution sup- posed to contain epinephrine, little chance of error is likely to be in- 782 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS curred ; that is, if the solution produces inhibition of the intestine along with augmentation of the uterus, it must contain epiiiephrine. 3'. The fresh carotid artery of the sheep. A ring cut from the artery is suspended in oxygenated Locke's solution and attached below to a small hook and above to a loaded muscle lever, by which the contraction Fig. 193. Tracing showing the effect of epinephrine on the intestinal contractions and on the arterial blood pressure. (The preliminary addition of barium to the nutritive fluid may be disre- garded.) (From Jackson.) of the muscle fibers can be magnified. Epinephrine causes the muscle to contract, but the test is not so sensitive as the foregoing, especially in the presence of blood serum, because the pressor substances therein con- tained also cause contraction. Blood plasma does not contain the pres- sor substances, so that oxalated plasma should be used in place of serum THE ADRENAL GLANDS 783 in applying the test. To increase the sensitiveness of the muscle, the artery ring should be slightly stretched by loading the lever. 4. The blood vessels of a frog. This method depends on the same prin- Funnel, or ll pressure boHle Hook through lower jaw Cannula In one aorta Fig. 194. Arrangement of apparatus for perfusion of the vessels of a -brainless frog. (From Jackson.) ciple as in that just described. The fluid supposed to contain epinephrine is added to Locke's solution, which is meanwhile being perfused under constant pressure through the blood vessels and the rate of outflow 784 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS noted (Fig. 194). If the fluid added to the inflowing fluid contains epi- nephrine, the outflow will become diminished. This is a very satisfactory method, although it is somewhat limited in scope unless large frogs are procurable, because of the difficulty of getting the necessary cannulae into the vessels (aorta and abdominal vein). 5. The pupil of the enucleated eye of the frog. Extremely small traces of epinephrine are observed to cause a dilatation. 6. The denervated iris. The fluid to be tested is placed in the conjunc- tival sac of an animal from which the superior cervical ganglion of the corresponding side has been removed some days previously. Under such conditions, if epinephrine is present in the fluid, dilatation of the pupil occurs. Both of the preceding methods we owe to Meltzer. 22 It should be emphasized that, although each of these methods is in itself very sensitive for the detection of epinephrine without being al- ways specific, yet the result should not be considered conclusive unless definite effects have been secured by at least two methods that are as far as possible independent of each other. As an outcome of investigations by these methods it has been found that when blood was taken from the inferior vena cava at the level of the adrenal veins, i. e., blood with a relatively high concentration of adrenal secretion, the presence of epinephrine could be revealed after splanchnic stimulation or massage of the glands. Such methods which depend upon the removal from the animal of the blood to be tested, are open to the objection that the blood so obtained may become altered in the process of shedding or defib- rination. As a matter of fact it has been shown that shed blood, very rap- idly develops vasoconstrictor substances. On this account conclusions re- garding epinephrine content, based upon the behavior of such samples, are not wholly reliable. The method about to be described is preferable. The Autoinjection Method. Such a method was first of all success- fully used by Asher, who employed an animal from which all the abdom- inal viscera had been removed. On stimulation of the great splanchnic nerve a rise in arterial blood pressure occurred provided the adrenal veins were open, but not so if the adrenal veins were clamped. By re- moving the viscera, the effect of splanchnic stimulation on the abdom- inal blood vessels themselves is eliminated, and any constriction which occurs in the blood vessels of the rest of the body must obviously be due to the action of epinephrine. The most satisfactory modification of this method is that employed more recently by Stewart, Kogoff and Gibson. 23 Blood from the adrenals was col- lected in a pocket of the inferior vena cava, which was made by applying clamps to this vein above and below the level of the adrenal veins. An animal in which the iris had been sensitized towards the action of epineph- rine by prior removal of the superior cervical ganglion was employed THE ADRENAL GLANDS 785 (page 784). It was found that after the pocket had been allowed to fill with blood removal of the upper clamp caused the pupil to dilate. Fur- thermore, the latent period of the response coincided with the time which the wave of concentrated blood was calculated to take in traveling from the pocket to the eye. This reaction time, consequently, varied inversely with the rate of blood flow. Stimulation of the splanchnics or massage of the gland was without effect upon the pupil so long as the upper clamp remained in position, but the usual response ensued when the clamp was removed.* When the adrenals were not artificially excited in any way the con- tents of the pocket, after removal of the upper clamp produced the char- acteristic reaction upon the pupil, thus demonstrating the spontaneous liberation of epinephrine from the glands. The capacity of the pocket, together with its time of filling, having been determined, the rate of flow through the adrenal veins was readily arrived at. The degree of concentration of the pocketed blood was determined indirectly by noting the precise extent of the pupillary response following the removal of the upper clamp, and subsequently reproducing this reaction by the intra- venous injection of an equal quantity of epinephrine solution of known concentration. It is clear that the product of the rate of blood-flow through the veins and the degree of concentration in epinephrine of the blood in the pocket will give the amount of epinephrine liberated from the glands in a given time. This was found to vary between .0003 and .001 mg. per kilo of body weight per minute. The splanchnic fibers concerned in the secretion of epinephrine seem to come from a nerve center situated relatively low down in the spinal cord. Section of the cord at the level of the last cervical segment does not affect the spontaneous secretion, but this disappears when the sec- tion is made below the third thoracic segment. (Stewart and Rogoff). In connection with these observations it is of interest to note that during stimulation of the splanchnic nerve in a normal animal, the conse- quent rise in blood pressure shows two peaks (see Fig. 29, p. 137). The first is no doubt due to direct stimulation of the splanchnic vasoconstric- tors, and the second to the outpouring of epinephrine into the blood, the justification for this conclusion being that the latter rise fails to appear after removal of the adrenal glands. In many cases a well-marked "dip" is seen between the two rises. This is explained as being due to initial minute amounts of epinephrine passing into the blood streamf (see page 777). *It does not seem to be possible to exhaust the adrenal gland of its supply of active material by _ stimulating the splanchnic a fact which would seem to throw considerable doubt on the relia- bility of the conclusions arrived at by the use of those methods in which extracts of the gland are assayed (see page 779). tA great part of the work done by clinical observers purporting to show that in such conditions as nephritis and arteriosclerosis there is an increase of epinephrine in the blood, has been found by Stewart and Rogoff to be unproved. THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS That epinephrine is being constantly liberated in certain minute amounts is probably true, but whether this amount is a factor in the maintenance of normal arterial tone, or is concerned in lowering the resistance of the sympathetic endings, is another question. Experimental investigation does not sustain the so-called "tonus" hypothesis. In the first place, in the experiments already cited (page 785), the amount of epinephrine secreted spontaneously would be, when diluted with the mass of blood of the entire circulation, quite inadequate to have any effect upon blood pressure; in the second place if any effect upon the vessels did occur with these minute doses it would be one not of constriction but of dilatation, on many vessels at least (page 777). No effects were observed on the general health or the blood pressure of animals in which the adrenal of one side was removed, and the nerve control of the opposite gland severed, although under the conditions it is evident that very little epinephrine could have been present in the blood (not more than 1 part in 400 million parts of blood). Experiments performed by Vincent and others gave similar results. Hoskins and McClure 24 at- tacked the problem in a different way, but arrived at a like conclusion. They found that the amount of injected epinephrine necessary for the pro- duction of a certain predetermined response was, after adrenalectomy, but slightly in excess of that required to be injected into the same ani- mal with intact glands. This excess, which represented the tonic secre- tion of the glands, was far below the threshold for vasoconstrictor stim- ulation. Finally, epinephrine is not present in the sera of patients suf- fering from vascular hypertonus in sufficient concentration to be detected by any of the biological tests at our disposal. (Stewart.) 25 Since the "tonus" hypothesis of the adrenal function is untenable, another, or emergency hypothesis has been brought forward. According to this, epinephrine is considered to be secreted into the blood in super- normal amounts when certain emergencies arise, such as asphyxia or con- ditions of extreme emotion such as fright or anger. An experimental hy- persecretion of an analogous character is also said to occur during stimu- lation of the central end of large sensory nerves such as the sciatic. The chief exponent of this hypothesis is Cannon, 26 and he has supported it by a seemingly incontrovertible mass of experimental evidence. In the earlier researches which appeared in 1911 the blood was removed from the vena cava opposite the openings of the adrenal veins, by pushing a cath- eter up to this level through a slit in the femoral vein, and blood was tested for the presence of epinephrine by observing its effect on the beating of an isolated strip of intestine. It was found that whereas the blood of a normal male cat did not give evidence of the presence of epine- phrine, it did so in a cat that had previously been frightened by allowing a to bark at it. Such results were not obtained after the removal of the THE ADRENAL GLANDS 787 adrenal glands, or in a female cat, which is usually indifferent to such a method of frightening. Cannon also thinks that many of the other adap- tations which take place in an animal in this condition are associated with the presence of an excess of epinephrine in the blood. The three most im- portant of these are: (1) increased discharge of sugar from the liver into the blood; (2) increased efficiency of muscular contraction; (3) di- minished clotting time of the blood all of which are adaptations ena- bling the animal either to conquer the source of the fear or to be in a better position to recover from any bodily injury, which he might suffer, involving a loss of blood. It has been pointed out by Stewart and Rogoff, 27 however, that there are several serious sources of error in the methods adopted by Cannon in the earlier investigations, particularly the uncertainty as to the ex- act source of the blood collected through the catheter, the chances for pressor substances developing in the shed blood, the unreliability of using only one test object for the detection of epinephrine in blood, (page 784) and the unknown rate of blood flow through the adrenal glands during the removal of the blood. These authors, as a matter of fact have not been able to secure any results which would confirm Cannon's con- clusions, either by repetition of the catheter method employed by this worker, or in other observations in which blood was collected from a pocket of vena cava (page 784) or in which the pupillary reaction was employed. Neither could Stewart 28 and Rogoff obtain any evidence that an increased secretion of epinephrine bears any relationship to the hyper- glycemia that is induced by ether or by asphyxia or by piqure (page 705). They did not find that animals in which the adrenal had been excised on one side and the nerve supply of the remaining gland cut, responded to emotional conditions in any way differing from normal animals. These criticisms have prompted Cannon 29 to repeat his earlier observa- tions by the use of a method which would not entail the removal of blood, that is, by an autoinjection method. He chose as the test object for ex- cess of epinephrine in the blood, a denervated heart which Levy, 30 Gas- ser 31 and Meek had shown to respond by a quickened beat to extremely small concentrations (for example 0.007 mgm. per kg. of "adrenalin" in- jected intravenously per minute increases the heart rate by as much as 28 beats per minute). The denervation of the heart was effected by section of the vagi and removal of the stellate ganglia, and in such animals it was found that stimulation of the sciatic nerve, asphyxia and emotional states caused decided acceleration. It would be rash to venture a final verdict at the present stage of this most interesting controversy, but it appears to the author that Cannon's evidence is very strong, provided it can be proved that the heart is really thoroughly denervated and that sub- 788 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS stances in the blood other than epinephrine may not be responsible for the cardiac changes. Of interest with regard to the action of epinephrine as part of a pro- tective mechanism, is the fact that the contractile pigment cells pos- sessed by certain types of lizards and whereby the hue of these creatures is varied in accordance with the shade of the surroundings, are stimu- lated by epinephrine ; the latter contracts such cells, and nervous excite- ment in these animals has a similar influence (Redfield 32 ). It is scarcely necessary to point out that, until it is definitely established by experi- mental investigation that epinephrine may be discharged in excessive amounts under certain conditions, it is irrational to assume that such may occur in disease. The surgical removal of the adrenal gland is certainly not warranted under any circumstances. The Association of the Adrenal with Other Endocrine Organs We have at present very little accurate and reliable information on the association of the adrenal with other endocrine organs. That epinephrine has an influence on many diverse organs and glands is an undoubted fact, but this is more probably to be attributed to an activating influence on sympathetic nerve endings than to any specific relationship between the adrenal glands and the particular gland in question. The most impor- tant of the results that have been obtained are the following : 1. With the Thyroid and Parathyroid. Cannon and Cattell, after con- firming Bradford's discovery that an electric current of action is set up in the salivary gland when it is excited to activity, proceeded to investigate the occurrence of such a current in the thyroid gland. 33 By placing one nonpolarizable electrode on the gland itself and the other on the neigh- boring subcutaneous tissues or on the trachea, a current was found to be set up by stimulation of the sympathetic nerve supply of the thyroid, by intravenous injection of epinephrine, or by stimulation of the great splanchnic nerve before it reaches the adrenal gland. This last result, which is the most important in the present connection, was, however, not observed when the blood of the inferior vena cava was prevented by the application of a clamp from getting to the heart, but immediately ap- peared, after stimulation, when the clamp was removed. This experiment taken alone does not, however, justify the conclusion that there is any direct relationship between the adrenal glands and the thyroid, because there are in the thyroid gland structures such as the muscle fibers in the blood vessels, which a hypersecretion of epinephrine might affect. Before any direct relationship between the two glands could be claimed to exist, it would be necessary to show that the thyroid action current is obtained with a concentration of epinephrine in the blood lower than that affecting the blood vessels. THE ADRENAL GLANDS 789 2. With the Sexual Glands. As mentioned above (page 768), a very direct relationship exists between the development of the sexual glands and that of the suprarenals, particularly the cortex of the glands. In ad- dition to the evidence already furnished, it may be mentioned that, in hyper- plasia of the adrenals changes occur in the testicles, particularly in their interstitial cells. 3. With the Liver. Of the many functions of this gland that which is most directly associated with epinephrine is the production of glucose from glycogen the glycogenolytic process (see page 701). The injection of epinephrine causes an immediate discharge of such an excess of glucose into the blood that hyperglycemia and glycosuria immediately follow. This result is most striking when the injection is made in glyco gen-rich animals. In animals from which all the glycogen of the liver has been removed by starvation, the injection of large amounts of epinephrine causes glycogen to accumulate in the -liver cells a result which it is difficult to interpret. In the light of the fact that stimulation of the great splanchnic nerve causes a demonstrable increase of epinephrine in the blood, a natural con- clusion is that the glycosuria and hyperglycemia which are known to re- sult from stimulation of the splanchnic nerve or of its center in the medulla, must be dependent upon a hypersecretion of epinephrine. Evidence supporting this hypothesis seemed to be furnished by the obser- vation that, after the removal of the adrenal glands, stimulation of the splanchnic or of the so-called "diabetic" center in the fourth ventricle no longer produced glycosuria even in a glycogen-rich animal. But it is difficult to see how such an important physiological process as that of the nerve control of the production of sugar by the liver should be dependent on the hypersecretion of the adrenal gland, especially since the epineph- rine would have to be carried by the blood around a considerable part of the circulation before it arrived at the place on which it was to act. More- over, it has been shown that stimulation of the previously cut hepatic nerve plexus (around the hepatic pedicle) in a normal animal produces hyperglycogenolysis, in which case there can be no question of a hyper- secretion of epinephrine. No doubt the adrenal glands have some important relationship to the nerve control of the glycogenolytic process, for, in animals from which the adrenal glands have been removed, stimulation of the hepatic plexus does not produce hyperglycemia. From this result it would appear that the presence of a certain amount of epinephrine in the blood i& necessary for the proper transmission of the nerve impulse from the sympathetic nerve fibers to the liver cell. When the nervous system is stimulated in such a way as to excite the glycogenolytic process, two effects both operat- 790 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS ing in the same direction with regard to the glycogenic function are developed: the one, a hypersecretion of epinephrine, which activates the sympathetic nerve endings, the other, the transmission of the nerve impulse to the liver cell (Macleod and E. G. Pearce). 34 4. With the Pancreas. The function of the pancreas here concerned is that of its supposed internal secretion from the Isles of Langerhans. Since epinephrine readily produces glycosuria, and since excision of the pancreas has the same effect, it has been natural to inquire whether any relationship exists between the two glands, and some observers have obtained results which they interpret as indicating that there does. Certain observers even state that glycosuria does not occur after the injection if at the same time extract of pancreas is injected. It is al- most certain, however, that these results are not trustworthy. Thus, removal of the adrenal glands in an animal suffering from pancreatic diabetes does not restore any of the lost power of utilizing glucose during the few hours that the animal remains alive. 34 That some rela- tionship may, however, exist is indicated by the fact that epinephrine causes dilatation of the pupil when it is dropped into the eye of a per- son suffering from diabetes, whereas it has no such effect in the normal individual. CHAPTER LXXXVI THE THYROID AND PARATHYROID GLANDS Structural Relationships The thyroid and parathyroid glands are intimately associated, anatomically, in most animals. The thyroid is present in all the vertebrates, but the parathyroids do not occur below the amphibia. The thyroid exists as two lateral lobes joined over the trachea by the so-called isthmus. The parathyroids are very much smaller, being four in num- ber and located in pairs on the posterior aspect of the thyroid lobes. The two upper parathyroids are usually more or less embedded in the thyroid tissue, where as lower ones are much more loosely attached to the thyroid; indeed, in some animals, particularly the herbivora, they are quite separate from it and may be located at a distance, as in the mediastinum. Accessory thyroid and parathyroid glands are sometimes present in the tissues of the neck, or in the anterior mediastinum, accessory parathyroids being common in the rabbit and rat, and parathyroid tissue being present in the thymus in 5 per cent of dogs (Marine 35 ). Before these anatomical relationships were thoroughly worked out, there was much confusion in the interpretation of the results following removal of one or the other gland. In their histologic structure and embryological derivation, the two glands are very different. The parathyroids are developed as an outgrowth from the third and fourth branchial pouches, and they are composed of masses of epithelial-like cells, sometimes more or less divided up into lobules or trabeculse by bands of connective tissue. The cells contain granules, some of which are of a fatty nature. Sometimes colloid-like ma- terial is found between the cells, or it may be enclosed in small vesicles not unlike those of the thyroid, although usually considerably smaller. The blood vessels are extremely numerous, and form sinus-like capillaries, which come into close relationship with the epithelial cells of the glands. Nerves also are abundant and pass both to the vessels and to the secreting cells. The blood vessels are derived from the inferior thyroid artery. The thyroid is developed by immediate outgrowth from the entoderm lining the floor of the pharynx, at a level between the first and second branchial pouches. ^Represented at first by a solid column of cells, there very soon occurs a division at the lower end into two lateral portions, and the original solid column becomes hollowed out. The two lateral branches of the original column divide again and again so as to form a system of hollow tubes lined with epithelium. These afterward become cut up. so as to form the closed vesicles characteristic of the gland. Each vesicle is more or less spheroidal in shape, and has no basement membrane, but its walls are formed by a layer of epithelial cells, which may be columnar, cubical, or flattened in shape. Each vesicle is filled with the so-called colloid material, which is peculiar in containing iodine, and between the vesicles is a layer of connective tissue often containing small cells, some of which are not unlike those of the parathyroid. The connective tissue also contains the blood ves- sels, which are very numerous indeed, the thyroid, in proportion to its size, receives more than five times as much blood as the kidneys, the only tissue that surpasses it in this regard being the medulla of the adrenal gland (see page 211). The nerves arise from both the vagus and the sympathetic systems and have been traced to the secreting epithelial cells. The above description applies to a strictly normal gland. 791 792 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS THE THYROID GLAND Condition of the Gland In the crowded communities of the Great Lakes Basin of this conti- nent, it has been found that in most animals the thyroid gland is more or less abnormal. In Cleveland, for example, Marine has found this to be the case in well over 90 per cent of the dogs brought to the laboratory. 36 The condition usually goes under the name of simple goiter, which in- cludes all thyroid enlargements except those of exophthalmic goiter. In man the goiter originates usually about the age of adolescence and more frequently in girls than in boys. It may sometimes pass over into the exophthalmic type. The exact pathological changes in the goitrous gland vary with the species of animal and with the duration of the dis- ease. In man, besides the cystic or colloid goiter an adenomatous type is very common although rare in other animals. From the numerous observations that have been made on the glands of domestic animals, it has been clearly established that the very earliest sign of goiter is a diminution in the iodine content of the gland; fol- lowed by an increase in the epithelial cells and in the blood supply and a decrease in the colloid. Such hyperplasia may be induced in what re- mains after removal of a large part of a normal gland (compensatory hyperplasia), or if a similar operation be performed early in pregnancy, the young when born will be found to have hyperplastic thyroids. A certain degree of hyperplasia exists as an accompaniment of pregnancy, and it can be produced in certain normal animals (particularly rats) by placing them on an excessive meat diet. Important observations bearing on this point have been made by Marine 37 on brook trout, in which it has been found that the so-called carcinoma that develops when the fish kept in hatcheries are fed with unsuitable food and overcrowded, is really a typical hyperplasia. In its second stage this develops into what is known as colloid goiter which is produced by a deposition of colloid material between the rows of cells so as to cause an opening out again of the vesicles (Fig. 195), with a consequent tendency to a reversion to the normal histological structure, so far as this is possible. The vesicles in such a gland are of enormous size, and the lining epithelium, low cubical, or almost flat in shape. The outstanding characteristic feature of the colloid material is that it contains iodine, which exists in combination with a nonprotein nitrog- enous base, and is usually called iodothyrin. In the gland itself the iodothyrin may be in combination with protein, forming iodothyro- globulin. E. C. Kendall 38 has recently succeeded in isolating a pure crys- talline substance of perfectly constant composition and containing over 60 THE THYROID AND PARATHYROID GLANDS 793 per cent of iodine. It is called thyroxin and has been identified as an indole compound and has been made synthetically. In extremely minute dosage it greatly affects the energy metabolism, and is said to induce symptoms like exophthalmic goiter. Its therapeutic value in cases of thyroid deficiency is remarkable. Kendall believes this substance to be the active constituent of the thyroid and to be associated with the metabolism of amino acids. For S. B. Fig. 195. Microphotographs of thyroid gland of dog. A, normal gland; B, active hyperplasia- C colloid goiter. (From Marine and Lenhart.) instance when given combined with amino acids its effects are increased several fold. The importance of the relationship between the function of the thyroid and the iodine-containing material is indicated by the changes which occur in the percentage of iodine in the glands under varying condi- tions of activity. Marine observed that the amount of iodine is inversely proportional to the degree of hyperplasia of the gland, and when the 794 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS hyperplastic condition becomes fully developed, scarcely a trace of iodine is contained in the gland. Later, when the hyperplasia gives place to colloid goiter, the iodine increases again, both absolutely and relatively. Moreover, it has been found that if iodide is administered to an animal suffering from hyperplasia, the hyperplastic condition very quickly disappears and the animal becomes normal. Thus, in brook trout, the poor nutritive condition of the fish when hyperplasia has developed can be immediately remedied by placing them in larger quan- tities of running water or by adding small traces of iodide to the water. The administration of small amounts of iodine as in ordinary salt from salt deposits also prevents goiter in farm stock, this having been first noted in the State of Michigan, where prior to the discovery of salt deposits sheep breeding was an entire failure. The importance of admin- istering small doses of iodides to school children living in goitrous dis- tricts has recently been emphasized by Marine and Kimball. 39 As small a dose as 0.001 gin. at weekly intervals prevents goiter in puppies sus- ceptible to it. Feeding experiments carried out by Gudernatsch 40 and subsequently by Kogoff and Marine, 41 indicate that the thyroid hormone has a powerful influence upon the development of the body. Tadpoles fed upon thyroid substance showed a striking acceleration of the normal metamorphosis. Those fed with the glandular material grew less rapidly than the con- trols fed upon ordinary diet, but the tails of the former showed more rapid involution and the arm buds developed prematurely. Experimental Thyroidectomy A correct interpretation of the functional changes and symptoms which follow upon partial or complete removal of the thyroid gland, or from its disease, has proved a very difficult problem, partly because sufficient care has not been taken to note how much parathyroid tissue was re- moved along with the thyroid, and partly because the fact has been over- looked that the effects produced by thyroidectomy and parathyroid- ectomy are often very different in animals of the same kind at dif- ferent ages. Speaking generally, it may be said that the influence of the parathyroid is focused mainly on the nerve centers and only to a second- ary degree on the metabolic functions, whereas the reverse is the case with the thyroid, its main effect being on metabolism, although it prob- ably also exercises a secondary effect on the nerve centers. More so than in the case of any other endocrine organ, our knowledge concerning the function of the thyroid has been gained by clinical experience, and it is difficult to say whether the clinical or the experimental method has contributed the greater amount of information. THE THYROID AND PARATHYROID GLANDS 795 The results of experimental extirpation of the thyroid vary accord- ing to the age of the animal, and frequently they are by no means marked, provided sufficient parathyroid tissue has been undamaged. The symptoms are in general thickening and drying of the skin, with a tendency to adiposity and a loss of tone of the muscles. The body tem- perature is low and the sexual functions become subnormal. Nervous symptoms in the direction of mental dullness and lethargy are also usually present. Surgical removal of the thyroid in man produces the condition known as cachexia strumipriva. The symptoms may first of all become apparent a few days after the operation, or they may remain latent for years, and then develop so as to produce the condition known as myxedema. When nervous symptoms are prominent in cachexia strumipriva, it is usually taken as evidence that an excessive amount of parathyroid tissue has been destroyed. Kocher states that after com- plete loss of the thyroid, life is impossible for more than seven years, and that to prevent ultimate ill effects, at least one-fourth of the organ should be left intact. Disease of the Thyroid The symptoms of diseased conditions of the thyroid may be inter- preted as the consequence of increased or diminished functioning of the gland. Sometimes, however, the less active gland is really increased in bulk, this increase being caused by the accumulation in it of very large quantities of colloid material accompanied by an attenuated condition of the vesicular cells (see page 793). When the gland is atrophied at birth, the condition of cretinism soon becomes developed (Fig. 196). The characteristic features of cretinism are: (1) An arrest of growth, espe- cially of the skeleton, accompanied by incomplete ossification of the long bones and a delay, often of several years, in the closure of the fontanelles. The disturbance in growth of the long bones occurs along the epiphyseal line, but the deposition of new bone beneath the periosteum proceeds, more or less, normally. The consequence is that the bones increase in thickness but fail to develop properly in length. (2) Poor development of the muscular system. (3) An unhealthy, swollen condition of the skin, so that it is yellowish in color, the face being pale and puffy. (4) An abnormal development of the connective tissues causing a shapeless con- dition of the surface; the abdomen is always swollen, the hands and feet are shapeless, and the root of the nose depressed. (5) The nerv- ous system also fails to develop properly, so that at the age of puberty or over, the child remains like an infant in his mental behavior, idiocy being common. Indeed, the whole clinical picture is so character- istic that once having seen a case no one can fail afterward to 796 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS recognize the disease. Besides being due to congenital absence of the thyroid (sporadic type), cretinism may also occur as a result of goitrous degeneration of the gland. This forms the so-called endemic variety of the disease, and is more commonly seen in goitrous districts, being not infrequently associated with disease of the parathyroid, in which case the nervous symptoms are very prominent. The occurrence of thyroid deficiency in adults produces clinical mani- Fig. 196. Cretin, nineteen years old. The treatment with thyroid extract started too late to be of benefit. (Patient of Dr. S. J. Webster.) festations allied to those of cretinism, but necessarily modified by the fact that the patient has attained full stature and sexual maturity. Though the skeletal changes are absent, the metabolic disturbances are if anything more pronounced. This adult form of the disease, which is known as myxe- dema, may occur spontaneously from atrophy of the gland, or follow the sur- gical removal of the latter, when the term cachexia strumipriva or operative myxedema is applied to the condition. The symptoms are very character- istic (Fig. 197). The skin is dry and thick, with a deposition of connective THE THYROID AND PARATHYROID GLANDS 797 tissue often containing fat in its deeper layers ; the hands and feet become unshapely; the lips are thick and the tongue somewhat enlarged, so that when the person attempts to speak, it appears as if the tongue were too large for the mouth ; the hair falls out ; there is a low body temperature, and it can be shown that the basal metabolism is greatly depressed, as can be shown by the measurement of oxygen consumption. It is said the person can take a larger quantity of sugar than an ordinary individual without the development of glycosuria, but the depression of the metabolic function causes the patient to take sparingly of food, in spite of which, however, the body weight may steadily increase. The sexual function be- comes depressed, and there is involvement of the nervous system as shown by mental dullness and lethargy. Although the thyroid gland is much atrophied in myxedema, symptoms A. B. Fig. 197. A, Case of myxedema; B, Same after seven months' treatment. (From Tigerstedt.) that are very similar may also occur when the gland is enormously en- larged. As already explained, however, this enlargement is due merely to an accumulation of colloidal material and is really an atrophic con- dition. A patient suffering from endemic goiter may at first exhibit symptoms which are usually attributed to a hypersecretion of thyroid material into the blood (the symptoms will be described immediately), but later these give place to symptoms not unlike those of myxedema. It is concluded that the above conditions are due to deficiency of thyroid function, or hypothyroidism, because: (1) the gland is atrophied, and (2) similar symptoms to those exhibited by the clinical conditions 798 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS can be produced experimentally by the removal of the gland in animals. By observations on the effect of administration of thyroid extract to cretinous or myxedematous patients, prompt amelioration of the symp- toms occurs, which certainly suggests that the real cause is the absence of an internal secretion. There is probably nothing more striking in the whole domain of therapeutics than this effect from the administration of thyroid extract or, more so still, of thyroxin. If the treatment is started early enough, the cretinous child from being an ill-developed idiot quickly catches up with children of his own age and becomes in every respect normal. Even if this treatment is not undertaken until the child is several years of age, it is remarkable how quickly the benefit may show itself. In myxedema and cachexia strumipriva also, the symptoms very quickly disappear and the person becomes perfectly nor- mal by the treatment. In all these conditions, however, the thyroid extract must be administered continuously in order to prevent the reap- pearance of symptoms. Quite distinct from the above described conditions of hypothyroidism are those produced by an excess of thyroid autacoid in the blood, namely, hyperthyroidism. Such a condition can be produced experimentally in normal animals by the administration of thyroid extract or of thyroxin (Kendall). In man its continuous administration is soon followed by great quickening of the pulse with some irregularity, flushing of the skin, in- creased perspiration, tremor in the limbs, emaciation, and marked nervous excitability. Along with these symptoms, metabolic investigations have shown that the energy output per square meter of surface is greatly in- creased, being sometimes nearly doubled ; that the nitrogen excretion is ex- cessive; and that alimentary glycosuria is very commonly present. The body temperature is not, however, as a rule increased, because although metabolism is excited, yet heat loss is correspondingly increased. Ex- ophthalmos is said to develop very occasionally after such administra- tion, but this is doubtful. Lastly, there are usually digestive disturb- ances, although the appetite is likely to be increased. The pulse is quick- ened after administration of thyroxin only when protein food is also taken. This is believed by Kendall to be due to the association between th^fhyroid hormone and the metabolism of the amino acids. It has been shown that a single large dose of thyroxin has little demonstrable effect, whereas minute doses administered over a prolonged period produce decided, toxic manifestations, and, if the administration is persisted in, death results. These facts are explained on the hypothesis that thyroxin acts in the body as a catalyst and hastens certain metabolic processes which are responsi- ble for the symptoms, and that they are not due to the action of thyroxin per se. (Kendall.) 42 The symptoms following the injection of the extract are very similar THE THYROID AND PARATHYROID GLANDS 799 to those of the disease known as exophthalmic goiter. Indeed, the symp- toms are so much alike in the two conditions that it is scarcely neces- sary to describe them specially for the disease except to mention that in the former exophthalmos generally does not occur. Like simple goiter this variety is from three to four times more fre- quent in women than in men, a fact of significance when we recall the evidence of association between the thyroid gland and the generative organs. It is said that the disease is usually coupled with persistence of the thymus gland. The thyroid gland in exophthalmic goiter is enlarged, sometimes in one lobe; it is hard and pulpy, and on auscultation a mur- mur is heard. Histologically the gland presents a picture very like that which lias been described above as hyperplasia ; that is to say, the vesicles have a deficiency of colloid material; their epithelium is colum- nar and folded up into the vesicles; and the interstitial tissue between the vesicles is very markedly increased. Exophthalmic goiter is generally claimed to be due to hyper- secretion of the thyroid, because: (1) the symptoms of the disease are not unlike those produced by excessive administration of thyroid to a normal individual; and (2) they are in general opposite in character to the symp- toms found in cases where the thyroid gland is atrophied. The blood of a person with exophthalmic goiter when injected into mice increases their resistance to the toxic action of acetonitrile, which is also the case after thyroid extract has been injected. In many cases of exophthalmic goiter partial removal of the gland is said to ameliorate the symptoms. Other clinicians, however, state that if the patient is given proper medical treatment, rest, and diet, equally beneficial results can be obtained. Certain investigators, however, deny that it has yet been conclusively demonstrated that exophthalmic goiter is due to hypersecretion of the thy- roid (Marine). 43 It is pointed out that, if hypersecretion were the cause of the disease, one would expect that the injection into animals of the blood of patients suffering from it would produce symptoms similar to those following the injection of thyroid extract. The results of such experi- ments, however, have been extremely confusing and very indecisive, since it is difficult to recognize in laboratory animals many of the characteristic symptoms, especially those affecting the skin and eyes and the general bodily nutrition. Another difficulty in accepting the hypersecretion hypoth- esis is the fact that an extract of a gland removed from an exophthalmic patient has no different physiological action on a normal animal from an extract of a normal gland containing the same percentage of iodine. The evidence is by no means conclusive one way or the other, and it may well be that the observed changes in the thyroid gland are not the cause of the symptoms of exophthalmic goiter, but merely, like the other symp- 800 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS toms of this disease, a result of some condition elsewhere. In this con- nection it is of interest to note that degenerative and pigmentary changes in the ganglion cells of the cervical sympathetic have been found by Wil- son 44 in cases of exophthalmic goiter and which are believed to be dis- tinctive of this condition. On this account disease of the sympathetic is suggested, by some, as the possible cause of the cardiac and ocular symptoms, as well as of the thyroid hyperactivity, the latter producing secondarily, it is supposed, the metabolic phenomena characteristic of the disease. The Relationship of the Thyroid with Other Endocrine Organs 1. With the Generative Organs. Evidence of an association between the female generative organs and the thyroid is very strong; thus, the thyroid becomes enlarged at puberty, during the menses, and during pregnancy, and in thyroidectomized young animals the sexual glands fail to develop properly. 2. With the Adrenal Glands. (See page 788.) 3. With the Pituitary Body. After removal of the thyroid, the pitu- itary becomes greatly altered and enlarged, particularly the pars an- terior, in which it is not uncommon to find that a certain amount of vesicles containing colloid, not unlike those of the thyroid, become devel- oped. This colloid material, however, does not contain iodine. It is said that this increase of the pituitary after thyroidectomy does not occur if thyroid extract be administered. Increased activity of the pars inter- media of the pituitary is also quite plain. These facts would at first sight seem to indicate that the pituitary and the thyroid can act vica- riously, but this is very doubtful, for it has not been found that pitu- itary extract has any beneficial effect in the treatment of goiter and myx- edema. Nevertheless the association in function of the two glands must be more or less close, not alone for the above reasons, but also because they are both associated to much the same degree with the sexual organs, and both act on the higher functions of the nervous system in much the same manner. 4. With the Thymus Gland. The persistence of the thymus in ex- ophthalmic goiter, as well as the anatomical and embryological relationship between thymus and thyroid, is taken to indicate some close relationship. THE PARATHYROIDS Experimental Parathyroidectomy Experimental parathyroidectomy yields results which vary in dif- ferent groups of animals, undoubtedly because of the fact that in some, such as the rat and rabbit, accessory parathyroids may exist. In gen- THE THYROID AND PARATHYROID GLANDS 801 eral, however, it has been found that if more than two of the four parathyroids be removed, very definite and pronounced nervous symp- toms soon supervene and if all four glands be removed, a quickly fatal result is inevitable. The most acute symptoms are exhibited by the carnivora. They may not be apparent for a day or two after the opera- tion, although during the period the animal is in a depressed state, re- fusing food and losing weight rapidly. The muscles are also more or less stiff during this stage. When more definite symptoms appear, they con- sist of a marked abnormality of muscular contraction, leading to the occurrence of fibrillar contractions, or tremors and, later, to cramp-like and clonic contractions. When spontaneous movements are made, a peculiar shaking of the foot, like that made by a normal animal to shake water off its pads, is a characteristic symptom. The slightest stimulation of the peripheral nerves is sufficient to induce one of these attacks, which recur with ever increasing frequency, becoming at the same time more pronounced and accompanied by other disturbances, such as diarrhea, profuse salivation and rapid pulse. In addition to the clonic seizures, there appears a tonic contraction of the extensor muscles of the limbs and in a certain percentage of dogs (but not of cats) spasm of the adductor muscles of the larynx (laryngismus stridulus) occurs. In this latter con- dition owing to the consequent narrowing of the rima glottidis the res- pirations are noisy, difficult and high pitched in tone. In cases that are not quickly fatal the hair tends to be shed and the teeth to be improperly calcined (in young animals). Where a certain amount of parathyroid tis- sue has been left for example, one of the four lobes the symptoms may not appear except under conditions of special strain to the animal econ- omy, such as pregnancy or improper diet. Thus, in a bitch from which three of the four glands had been removed, no symptoms of tetany oc- curred until she became pregnant. Under the same conditions it has been found that a diet of flesh is much more apt to bring about the con- dition than one of vegetables or milk. Injury or Disease of the Parathyroids in Man Tetania parathyreopriva, as the condition described in the foregoing paragraph is called, may become developed also in man as the result of surgical removal of the parathyroids. This was a common enough sequela to operations for goiter a few years ago, before the significance of these bodies was recognized or, indeed, even their existence known. It was the result of accident rather than of design, were the parathyroids not removed along with the thyroid. A similar condition (idiopathic tetany) occurs spontaneously, in children more particularly, but also in adults when it may be associated with gastrointestinal disorders, infec- tious diseases, or pregnancy. The clinical phenomena resemble closely 802 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS those described above as appearing in laboratory animals, with the dif- ference that the clonic movements and the extensor spasm are more or less replaced by a tonic spasm of the flexor muscles, which produces charac- teristic attitudes of the hands and feet. Associated with the carpopedal spasm generalized convulsions or laryngismus stridulus may occur. Laryn- geal spasm may, indeed, be the sole manifestation of parathyroid de- ficiency and it is also believed that many cases of convulsions occurring in young children really depend upon a deranged function of these glands. In brief, tetany, infantile convulsions, and laryngismus stridulus are proba- bly but different manifestations of the same condition. It has been suggested that certain obscure nervous diseases of adults such as paralysis agitans and Thomsen's disease are dependent upon lesions of the parathyroids. Chorea, epilepsy, and eclampsia likewise have been ascribed to disease of these bodies, but no cogent evidence has been adduced to connect the parathyroids with any of these conditions. That, on the other hand, the belief in the association of idiopathic tetany and parathyroid disease is well founded is evidenced by the close resemblance between the nervous symptoms of the two conditions. In the case of monkeys especially, are the symptoms of the experimental condition often strikingly similar to those of the idiopathic disease. In these animals the muscular spasms following parathyroidectomy may imitate almost unerringly those of infantile tetany, so that tonic flexor spasms pro- ducing the characteristic carpopedal attitudes of the idiopathic condi- tion, appear. With regard to the nature of the disturbances set up ~by parathyroid de- ficiency. Noel Paton, Findlay and Watson 45 have recently shown that the clonic spasms are not primarily dependent upon the cerebrum or cere- bellum, since they persist after ablation of these parts of the nervous system ; in fact, removal of the hemispheres or suppression of their usual functions by light anesthesia increases the severity of the clonus. This does not imply that secondary involvement of the cerebrum may not occur; on the contrary, the epileptiform convulsions, observed in the se- verer types of tetany, indicate considerable cerebral mischief. Division of the cord does not remove the spasms below the site of section, nor have section and degeneration of the posterior roots any influence upon them; but they disappear after division of the anterior roots. These observations show that the seat of origin of the spasms cannot lie in the peripheral nerves or in the muscles. This indicates that the efferent neurone within the cord is the structure affected. The other nervous disturbance following parathyroidectomy, namely, tonic spasm of the extensor muscles has been shown to depend upon the cerebellar arc. The hypertonus is uninfluenced by decerebration, whereas the removal of cerebellar impulses by severance of the spinal cord abol- THE THYROID AND PARATHYROID GLANDS 803 ishes this symptom below the transection. In the severer cases other more marked evidences of cerebellar involvement may appear, e. g., dis- turbances of equilibrium and forced movements of a circling or rotatory nature. Though the foregoing ascribes a central origin to the eminent nervous symptoms the peripheral nerves are not entirely unaffected as has been shown by Noel, Paton and his co-workers. These investigators compared the response of muscle and nerve to electrical stimulation in nor- mal and in parathyroidectomized animals, and found that though there were considerable variations in the responses of a normal animal, they were very definitely exaggerated in experimental tetany when either the motor nerve or the muscle itself was stimulated. This increased electrical excitability is uninfluenced by the central nervous system, since it persists after section of the nerves, and though it is manifested by direct stimulation of the muscle it does not depend upon any change in the myal structures, for degeneration of the nerves or the administra- tion of curare in sufficient dosage to block nervous impulses, abolishes it ; it is concluded that the nerve ending is the part of the neural structure affected. Idiopathic tetany shows a similar exaggeration of electrical excitability, the excitability to mechanical stimuli is increased to an even greater degree in this condition, a fact most valuable in the diagnosis of latent disease ; tapping over the facial nerve is the common method em- ployed for its elicitation. Though these changes in the excitability of the peripheral nerves are useful in diagnosis, neither in the experimental nor in the idiopathic condition can they be taken as a measure of the se- verity of the process, for they may be no more marked in instances where there is involvement of the cerebral hemispheres (causing epileptiform convulsions) than in milder cases. The parathyroid gland, besides influencing the nerve centers, has also an influence on metabolism. The symptoms produced by parathyroidec- tomy are; (1) rapid emaciation and failure to grow; (2) a tendency to the production of glycosuria, often detected by finding that the assimi- lation limit for carbohydrate is lowered (page 685) ; and (3) most defi- nitely of all, an interference with calcium metabolism, as illustrated by the failure of the teeth and bones to calcify properly. When the tetany is the result of a complete extirpation of all parathyroid tissue, the symptoms can be combated by a successful transplantation or graft of parathyroid tissue made from an animal of the same species. In- deed, it has been found that the success of a graft of parathyroid is assured only when the graft is derived from the same kind of animal as that from which the parathyroid has been removed. Implantation into the subcutaneous tissue of a tetany patient of parathyroid tissue ob- tained fresh from the deadhouse has been performed with beneficial out- come. 804. THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS As to the cause of the symptoms, many possibilities have to be considered, in the first place, no direct relationship exists between the thyroid and parathyroid in this connection. One cause might be the absence of some substance which checks the activity of the nervous system, some chalone in Schafer's sense. It was previously thought by W. G. Macal- lum 46 that, since, nervous symptoms like those of tetany could be pro- duced by deficiency of calcium in the body and the symptoms of parathy- roidectomy were relieved by the administration of this cation, calcium deficiency was the cause of the symptoms. The defective calcification of the bones and teeth following parathyroid deprivation together with the frequent association of rickets (a condition characterized by a modified calcium metabolism) enhanced the plausibility of such an hypothesis. On the other hand, that no view can be correct which takes as its basis the absence or deficiency of some one or other substance which is sup- posed, normally, to influence the activity of nervous tissues is indicated by the fact that blood-letting or the transfusion of normal saline imme- diately removes the symptoms, and keeps them in abeyance for some time. Moreover, such a view does not adequately explain the metabolic dis- turbances, which may continue when the nervous symptoms are slight as, for example, after the administration of calcium. The most probable ex- planation of the beneficial effect of calcium upon the nervous symptoms is that it behaves merely as a sedative, reducing the excitability of the nervous system, an action which it is known to possess. While not denying that calcium ions may have some minor relation- ship to the symptoms, Noel Paton ascribes them chiefly to intoxication by guanidine (page 640). The evidence is as follows: (1) Guanidine or methyl guanidine administered to normal animals produces symp- toms that are identical with those following parathyroidectomy. (2) No drug, other than guanidine, which can effect a decided increase in the excitability of the motor nerve endings to the constant current, has been found. (3) There is a marked increase in the amount of these sub- stances in the blood and urine of parathyroidectomized dogs, and in the urine of children suffering from idiopathic tetany. It is also true that while creatine (which contains the guanidine nucleus and is a prob- able source of this substance) is absent from the urine of healthy adults, it is normally present in the urines of children between the ages of 12 and 15 years, a period during which the incidence of tetany is most frequent. Under 6 months creatinuria does not usually occur, tetany also is extremely rare at this age. These facts taken in conjunction with the other evidence, seem to have more than a coincidental bearing upon the genesis of tetany. (4) In certain cases the serum of parathyroidecto- mized dogs acts upon the muscles of the frog similarly to weak solutions of guanidine. (5) There is a striking similarity in the relative amounts of the nitrogenous metabolites in the urine of parathyroidectomized dogs THE THYROID AND PARATHYROID GLANDS 805 and normal animals injected with guanidine, in either instance the total nitrogen is increased and the proportion of urea diminished. It is concluded that the parathyroids control the metabolism of guani- dine "by preventing its development in undue amounts. In this way they probably exercise a regulative action upon the tone of the skeletal muscles." Though the evidence of these observers is most formidable, it seems that the question has not yet reached finality, for Howland and Mar- riott 47 still insist that a lowered calcium content of the blood is responsi- ble for idiopathic tetany. This contention is supported by a mass of analytical data from which the fact is brought out that the blood of chil- dren suffering from tetany shows a reduction of calcium, to the extent of 40 per cent in many instances. The question whether this deficiency is merely an accompaniment of the condition or the causative factor does not appear to have been fully investigated. It is possible that neither of these factors guanidine formation or calcium deficiency is the pri- mary cause of tetany, but that one or perhaps both may be secondary to some condition as yet unrevealed. BIBLIOGRAPHY Grant and Goldman : A Study of Forced Respiration : Experimental Production of Tetany. Am. Jour. Physiol., Hi, 209. CHAPTER LXXXVII THE PITUITARY BODY Structural Relationships Situated at the base of the brain and lying in the sella turcica, the pituitary body in man does not weigh much more than half a gram. It is connected with the brain by a funnel-shaped stalk, the infundibulum. On account of a natural cleft, which runs across the gland in an oblique plane, it is an easy matter to split it into two portions, an anterior, or pars glandularis, and a posterior, or pars nervosa. This cleft in the case of man is usually found to be more or less broken up into isolated cysts containing a colloid-like material, and it represents the remains of the original tubular structure from which the pars glandularis is developed; namely, a pouch growing out from the buccal ectoderm. On histologic examination it will be found that the pars glandularis consists of masses of epithelial cells with large sinus-like blood capillaries lying between them. These blood vessels are very numerous, so that in an injected gland this portion of the pituitary stands out very prominently. The vessels are derived from about twenty small arterioles that converge toward the pituitary from the circle of Willis, and enter the gland by the infundibulum or stalk by which the gland is connected with the base of the brain. Three types of cell can be differentiated: nonstaining (chromophobe) and granular (chroma- phil), of which latter there are cells with acid-staining and others with base-staining granules, the former being by far the more numerous (Schafer). In some animals such as the cat, the cells of the pars anterior are arranged around the blood sinuses in rows as in a columnar epithelium. The cells with acid-staining granules are said to become much increased in number in pregnancy and also in the enlarged gland of acromegaly (see page 816). After thyroidectomy it has been observed that colloid-like masses ac- cumulate in the pars glandularis, the cells sometimes arranging themselves around these masses as in the thyroid gland. The colloid, however, contains no iodine. The posterior part of the gland, or pars nervosa, is composed almost entirely of neuroglia, cells, and fibers, usually with some hyaline or granular material lying be- tween them, particularly in the neighborhood of the infundibulum, into which it may be traced. It is believed that the active principle of the gland is represented by this ma- terial. The blood supply of the pars nervosa is relatively scanty. Between the pars nervosa and the intraglandular cleft above referred to is a layer of cells differing from those of either the anterior or the posterior lobe. This layer of cells constitutes the so-called pars intermedia. The cells are somewhat like those of the pars glandularis, except that they are distinctly granular, the granules being of the neu- trophile variety, that is to say, they stain with neither basic nor acid dyes. Well-defined vesicles, containing an oxyphile colloid material which is believed to furnish the active principle of the posterior lobe, are often found between them. Although well separated by the cleft from the pars glandularis, the pars intermedia is not well separated from the pars nervosa, because many of its cells extend for some distance into the latter between the neuroglial fibers. Certain of the cells in the pars intermedia may be seen in various stages of conversion into globular hyaline bodies, or a granular mass of ma- 806 THE PITUITARY BODY 807 terial may appear in them. In either case, the cells ultimately break down, setting free the hyaline or granular material, which is believed to be the origin of the similar ma- terial already described as existing between the neuroglial fibers of the pars nervosa and therefore ultimately finding its way by the infundibulum into the third ventricle of the brain. As evidence that the pituitary secretion takes this course are the facts, that in the first place, the active principle of the gland may be detected in the cerebrospinal fluid, and secondly, if in the living animal the infundibulum be severed close to its junc- tion with the ventricular floor, a subsequent examination of the gland shows an unusual accumulation of hyaline material in the stalk and adjacent portion of the posterior lobe. It should be mentioned, finally, that at the margin of the intraglandular cleft the inter- mediary and anterior portions of the pituitary come together, although the cells of each can readily be distinguished on account of their staining properties. This pars glandu- Fig. 198. Drawing from a photograph of a mesial sagittal section through the pituitary gland of a human fetus (5th month): a, optic chiasma; c, third ventricle; d, pars glandularis; e, iniun- dibulum surrounded by epithelial cells; f, pars intermedia; g, intraglandular cleft; h, pars nervosa. (Herring, from Howell's Physiology.) laris et intermedia also extends as a thin layer over part of the pars nervosa and around the neck of the gland at the infundibulum. These relationships are well shown in the accompanying diagram (Fig. 198). Functions Concerning the functions of the pituitary, it may be said in general that the anterior lobe has an important relationship to the nutritive con- dition of the body during growth, especially of the skeletal structures, and that the posterior lobe produces a very active autacoid having to do with the physiological activity of unstriped muscle fiber. The pars inter- media seems to be associated with the posterior lobe in the production of 808 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS this autacoid. The function of these two parts will therefore be con- sidered together. Function of the Anterior Lobe. The facts concerning the function of the pars glandularis have been gleaned largely by observing the ef- fects produced by partial or complete removal of the entire pituitary. Justification for ascribing the results of this operation to removal of the anterior lobe, rather than to removal of the posterior is furnished by control experiments in which removal of the posterior lobe alone failed to produce similar effects. Complete removal of the pituitary is almost invariably fatal, the con- dition being called apituitarism. Two operative procedures have been employed for the removal of the gland. One of these, originated by Paulesco and elaborated by Gushing and his pupils, 48 consists in trephin- ing the skull and elevating the temporal lobe of the cerebrum so as to expose the gland. The other, elaborated by Horsley, 49 consists in ap- proaching the gland through the orbital cavity. Although there is some danger of injury to nervous tissues by the intracranial method, its re- sults are more dependable since the gland is actually exposed to view before being removed. Most hypophysectomized animals die within two or three days, unless they are very young. This longer survival of young animals is ascribed to the presence of accessory pituitary material situated in the dura mater lining the sella turcica. The most extensive observations have been made on dogs. On the day following the operation the animal appears about normal, but it gradually becomes less active, refusing food and respond- ing slowly to stimulation. It gradually gets weaker and weaker; muscu- lar tremors may appear, the respiration and pulse become slow, the back arched, the temperature subnormal; and, usually within about forty- eight hours, coma develops and the animal dies in this condition. Symp- toms of equal severity ensue if the anterior lobe alone is removed. When the symptoms are less acute and death does not occur so early, it is be- lieved by Gushing either that small portions of the gland have been left behind or that some vicarious activity of other organs has developed to replace that of the pituitary. When only a part of the pituitary is removed either unintentionally or intentionally, the symptoms are not nearly so acute, provided that the portion remaining includes tissue of the anterior lobe. The condition is known as hypopituitarism. It is by a study of this condition that most facts concerning the function of the anterior lobe have been learned. When the operation is performed on young animals, they fail to grow properly; the milk teeth and the lanugo are retained; the epiphyses do not ankylose; the thyroid and thymus glands are en- larged; and the cortex of the suprarenal and the sexual organs fails to THE PITUITARY BODY 809 develop. The animal, though small, becomes very fat and may therefore in- crease in weight. There is distinct evidence of mental dullness. From these results it is concluded that the anterior lobe of the pituitary produces autacoids having to do with the development of the skeletal and other structures of the growing animal. That this autacoid is not derived from the posterior lobe is indicated by the fact that partial injury of this lobe, or indeed its entire removal, is not followed by similar symptoms. Closer examination of the metabolic function in hypophysectomized animals has shown that there is a marked depression in the respiratory exchange of oxygen and carbon dioxide, and that the ability to metabo- lize carbohydrate becomes heightened ; that is to say, the animal can tolerate a larger quantity of sugar than the normal animal without develop- ing glycosuria. This effect on carbohydrate metabolism may how- ever be associated not so much with the function of the anterior lobe as with that of the posterior, for, as we shall see later, Gushing and his pupils have found that extract of the posterior lobe has a marked influence on the assimilation limit of carbohydrate. Attempts have been made to graft the pituitary, especially the anterior lobe, into various parts of the body. It has been found, however, that within a few days the grafts atrophy and disappear unless there has been complete removal of the pituitary itself, in which case the graft may remain for a month or so and the otherwise fatal outcome of hypophy- sectomy be warded off. Sometimes, where the graft has remained for a longer time, it is said that a temporary increase in the growth of the animal has been noticed. Other observers have investigated the effects in normal animals of continuous oral administration of pituitary substance or of subcutaneous injection of extract. The earlier results were indefinite and confusing, but recently Brailsford Robertson 50 has succeeded in isolating from the anterior lobe a substance called tethelin, which accelerates growth in young animals and is thought to have a possible value in hastening the healing process in wounds. Tethelin is precipitated by dry ether from an alcoholic extract of the carefully isolated anterior lobes. It contains 1.4 per cent of phosphorus, and nitrogen in the proportion of four atoms for every atom of phos- phorus, two of the nitrogen atoms being present as amino groups and one in an imino group. The effects on growth of mice are in every par- ticular like those of the administration of anterior lobes, and consist in retardation of the first portion of the third growth cycle,* followed by , * Robertson has contributed valuable and very extensive data on the normal curve of growth of white mice' kept under carefully controlled conditions. Three growth cycles are present: the first attains its maximum velocity between seven and fourteen days after birth; the second, between twenty-one and twenty-eight days; and the third about six weeks, after which the velocity decreases progressively, until further growth ceases between the fiftieth and sixtieth weeks succeeding birth. 810 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS acceleration of the latter portion of this cycle. When fully grown, tethelin-fed mice also differ from normal animals in being smaller in size but of greater weight, with a distinct difference in the condition of the coat. Normal animals at fourteen months of age have " shaggy, staring and discolored coats," whereas tethelin-fed animals have the glossy and silky appearance of young animals. During growth, nor- mal animals display a greater variability in weight than tethelin-fed animals. Extraordinary effects have been observed by Clark 51 to be produced by feeding laying hens with pituitary gland. Thus, by giving to one- year-old hens, in addition to their usual food, 20 milligrams of fresh pituitary substance for four days, it was found that the average daily number of eggs laid by a batch of 655 hens was raised from 273 during the four days preceding the pituitary feeding to 352 during the four days of the administration, these results being obtained at a time of year when the natural egg-production of the hens was diminishing. It was further observed that not only is the output of eggs greatly increased as a result of the pituitary feeding, but likewise their fertility, for in another experiment in which 35 hens were kept along with two cockerels of the same breed, not only was the output of eggs increased (from 18 up to 33), but the fertility of the eggs was greatly enhanced. Functions of the Posterior Lobe (and Pars Intermedia). As already mentioned, excision of this part of the pituitary can be tolerably well with- stood by the animal, so much so indeed that from its behavior after the operation we can conclude little as to the function of the lobe. On the other hand, extracts of the posterior lobe injected into normal animals produce effects that are very striking, indicating that the main function of this lobe is the production of an autacoid. The extracts have more or less an epinephrine-like action. Such extracts, rendered protein-free and steril- ized, are obtainable on the market under the various names of pituitrin, hypophysin, etc. From them a crystallizable material has been obtained, but this is probably a mixture of various substances. In discussing the functions of these various extracts, it must be remembered that the inter- mediary part (pars intermedia) is included with the posterior lobe in their preparation. Although the effect of pituitary extract on plain muscle fiber (and on glandular tissue) appears, on first sight, to be very like that produced by epinephrine, it has been found on closer examination that the two substances really act in different ways. The rise in blood pressure pro- duced by pituitary autacoid is likely to be more prolonged than that produced by epinephrine. It stimulates increased cardiac activity, but after the vagi have been cut or sufficient atropine administered to para- THE PITUITARY BODY 811 lyze them, the pituitary autacoid continues to stimulate the strength of the heartbeat without producing the acceleration noted with epinephrine. Whereas epinephrine has little or no action on the coronary vessels (page 268) or on those of the lungs, pituitary autacoid usually produces constric- tion of both types of vessel ; and on the renal arteries the actions of the two autacoids are entirely different, for epinephrine has a marked constric- ing effect, while the pituitary autacoid produces dilatation. Another striking difference in the extracts from the two glands is re- vealed by repeating the injection after the effect of a previous one has completely passed off. With epinephrine the original effect is repro- duced; with pituitrin, on the other hand, the effect of the second injec- tion is very often the reverse of that of the first; that is to say, the blood pressure, instead of rising, may fall, or the rise be very much less marked. Whether this effect of the second dose is caused by the action of an autacoid having a chalonic rather than a hormonic influence, or whether it is due to a reversed effect of the same hormone, it is impos- sible at present to say. The chalonic effect in any case is much more evanescent than the hormonic, and it is not caused by cholin, as some have suggested. The effect of epinephrine, it will be remembered, is abolished by ergotoxin and apocodeine. These drugs, on the other hand, have no influence on the action of pituitrin. The difference in action between the two autacoids is usually explained by assuming that the epinephrine acts on the receptor substance associated in some way with terminations of the sympathetic nerve fibers in involuntary muscle, whereas pituitrin acts directly on the involuntary muscle fibers themselves. Other types of involuntary fiber are also acted upon by pituitrin. The uterine contractions, for example, are stimulated (Fig. 199), this effect being unconditioned by the state of the uterus. A similar effect is produced upon the musculature of the intestine and bladder (in contrast to the inhibitory effect of epinephrine) and upon the muscle of the ureter. Pupillary dilatation of the excised frog's eye, but not of the mammal's, is produced by pituitrin. The effect of this substance upon the bronchioles is shown in Fig. 200. The glands upon which pituitrin has the most pronounced action are the mammary glands and the kidneys. It has no influence upon the salivary secretion. The effect on the kidney is evidenced by the remarkable increase in the urinary flow following injection of the pituitrin. This diuresis might of course be due merely to the vasodilatation that we have seen such extracts produce a vasodilatation which is all the more marked because the vessels elsewhere in the body undergo constriction. But pituitrin continues to cause increased urinary outflow in the absence of any demonstrable vascular change; it also acts after the administration of atropine, so that it is con- sidered by some observers to act on the excretory epithelium of the convo- 812 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS luted tubules and to constitute a renal hormone which affects the epi- thelium of the kidney in a manner analogous to that by which the pancreatic cells are stimulated by secretin. Another reason for believ- Fig. 199. Tracing showing the action of pituitrin on the uterine contractions and blood pressure in a dog. Made by Barbour's method. (From Jackson.) ing that the secretory hormone is independent of that producing vaso- dilatation of the renal vessels is the fact that a repeated dose of pituitrin, although, as we have seen, usually has a depressor action on the blood THE PITUITARY BODY 813 vessels, still produces a stimulating effect on the excretion of urine. The work of Knowlton and Silverman/' 8 on the gaseous exchange of the kidney during pituitrin diuresis is against this view. These observers could detect no increased oxygen consumption accompanying the in- creased urinary flow, and ascribe the latter effect purely to augmented blood flow. The value of pituitriii as a diuretic in clinical practice is now well recognized. The work of Cow 52 upon the secretory activity of the kidney in rela- tion to pituitrin administration indicates that the hypophysis normally, Fig. 200. Tracing showing the constricting action of pituitrin on the bronchioles and its effect on blood pressure in a spinal dog. (From Jackson.) is part of a mechanism for the control of the urinary flow its action being opposed to that of the adrenal medulla. It is asserted that ingested fluid taken up by the gastrointestinal mucosa absorbs a substance of a hormonic nature contained therein, which passing into the general blood stream calls forth the diuretic principle from the pituitary body. The effect on milk secretion is best demonstrated by placing a cannula in the mammary ducts so that the milk may freely flow out. By observ- ing the rate of outflow during the injection of pituitrin, it will be found that a remarkable increase occurs. After this increased secretion has ceased, however, the injection of more pituitrin has no further effect, indicating that the influence of the first injection must have been, not so 814 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS much to stimulate the secretion of milk, as to accelerate the outflow of that which previously had been secreted and had collected in the alveoli and ducts. This effect explains why the pituitary galactagogue should have very little if any effect on the total production of milk or on the total amount of fat and other constituents contained in it. Histological examination of sections of a resting mammary gland and of the same gland after administration of the pituitrin, bears out the above interpre- tation of the action. Alveoli in the resting state will be found largely distended with milk and the epithelium flattened against the basal mem- brane, whereas alveoli from the gland after pituitary activity show small shriveled-up alveoli, containing little milk, and with epithelium that is well marked and stands out prominently from the basal membrane. These facts taken together indicate that pituitrin stimulates the mus- cular fibers of the ducts of the mammary glands, thus squeezing out the milk contained in them. Muscular fibers have been described as existing between the basal membrane and epithelial cells, much in the same way as they do in the case of the sweat glands. At least Schafer has suc- ceeded in demonstrating in this position rod-shaped nuclei which prob- ably belong to muscular fibers. 3 By their contraction, the milk in the alveoli is expelled into the ducts. The observation of Maxwell, 53 namely, that the alveolar contour of a lactating gland failed to show any change when irrigated with pituitrin, is opposed to the foregoing view. It has also been found that pituitrin stimulates the secretion of cerebrospinal fluid and that this stimulation is independent of a rise in blood pressure. Eecently Abel and Kuboto 54 have brought forward evidence to show that the depressor effect of pituitrin is not specific, but is due to the presence of histamine (/?-iminazolethylamine) a substance we have seen to be produced by the decarboxylation of histidine (page 536). These authors have isolated the depressor and the plain-muscle stimulant principle from the posterior lobe of the pituitary in the form of a di-picrate. With regard to crystalline form, solubility, melting point and the method of its isolation, this sub- stance is identical with the di-picrate of histamine ; since the physiological actions of the two substances are also the same their unity is believed to be established. Pituitrin has a distinct effect on carbohydrate metabolism. After its intravenous or subcutaneous injection, a marked lowering in the toler- ance for sugar is observed (page 685), usually to such an extent that glycosuria becomes established. Gushing and his pupils have concluded that the posterior lobe contributes an autacoid which influences the utili- zation of sugar in the body. Confirmatory evidence for this view is fur- nished by the observation that mechanical stimulation of the posterior lobe, such as is produced by puncturing it with a needle, is followed by THE PITUITARY BODY 815 a temporary glycosuria, which is said to be as pronounced as -that fol- lowing puncture of the diabetic center (page 704), provided glycogen is present in the liver. The production of this carbohydrate autacoid would appear to be under the control of the sympathetic nervous system, for it has been found by Gushing and others that stimulation of the superior cervical ganglion, which has been known for many years to be fre- quently followed by glycosuria, has this effect only provided the posterior lobe of the pituitary is intact. Even surgical manipulation of the pitui- tary may excite a hypersecretion of pituitrin, which would account for the glycosuria often observed after experimental excision or partial A. B. Fig. 201. A, To show the appearance before the onset of acromegalic symptoms; B, The ap- pearance after seventeen years of the disease. (After Campbell Geddes.) destruction of the pituitary. A similar irritation may be set up in disease of the gland. The glycosuria which is usually observed after partial hypophysectomy soon passes off, to be followed by a permanent condition of increased tolerance for sugar, because now less pituitrin is being produced. It is said that during the stage of increased tolerance diabetes can not be pro- duced even by excision of the pancreas. The glycosuria produced by irritation of the posterior lobe is accompanied by a marked polyuria (dia- betes insipidus), which may outlast the glycosuria. The extract of the pars intermedia has an action similar to but not identical with that of the posterior lobe, some of the effects of which it lacks, for example, it possesses no pressor action or influence upon the kidney, and, though it exhibits galactogogue and oxytocic qualities, these 816 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS are considerably less potent than in the case of extracts of the pars nervosa. The active principle of the pars intermedia, on account of these differences, is looked upon by Herring 55 as representing an immature stage in the manu- facture of the active principle of the gland, the final product of which is represented by the extract of the posterior lobe. The fact that the cells of the pars intermedia are, apparently, the ultimate source of the pituitary autacoid (see page 806) supports this view. Clinical Manifestations of Deranged Pituitary Function Because of their importance from a physiological standpoint, we shall now proceed to review briefly some of the more important facts that have so far been brought to light by clinical observations. The pathological condition most frequently observed affecting the pituitary is an adenom- atous growth particularly located in the anterior lobe. Besides pro- ducing general symptoms of pressure, such as diminution of the visual field and, perhaps, headache, a shadow can usually be observed when the patient is examined by means of the x-rays. General symptoms, com- monly ascribed to a hypersecretion of the autacoid of the anterior lobe of the pituitary hyperpituitarism begin sooner or later to show them- selves. These symptoms are almost exactly opposite in character to those observed in animals after removal of this portion of the gland. Thus, the bones of the extremities become stimulated to increased growth, so that if the patient is young, and the epiphyses therefore not ossified, remarkable elongation of the long bones occurs, pro- ducing the condition known as gigantism. On the other hand, if the dis- ease does not develop until after ossification is complete, its effects be- come most marked in the bones of the face, the lower jaw becoming enormously hypertrophied and the supraorbital ridges very prominent. The long bones also become enlarged at their extremities, and there may be some increase in length of the vertebral column, although the stature does not increase because of kyphosis (bowing of the spine). The condition is called acromegaly. Nutritive disturbances of the skin and hairs also become marked, causing the skin to become dry and yellowish, and the hairs to undergo abnormal increase over the body. An early symptom of the condition is a failure of the sexual power (Figs. 201 and 202.) After a time the disease begins to affect the pars intermedia et nervosa, and disturbances in carbohydrate metabolism come to be observed, con- sisting usually in a diminished tolerance accompanied by glycosuria, in the early stages of the disease, followed by increased tolerance in the later stages. The glycosuria is usually accompanied by marked polyuria. It should be observed that sometimes tumor of the pituitary has been THE PITUITARY BODY 817 found to exist postmortem though none of the above symptoms had been recorded during life. In these cases it is probable that the disease from the start had been of such a nature as to produce a tendency to hypo- pituitarism rather than hyperpituitarism, for the symptoms are very like those observed in animals after partial or complete removal of the gland. If the condition commences before adolescence, the body fails to grow, although the child may continue to increase in weight because of the Fig. 202. Hand of a person affected with acromegaly. remarkable deposition of fat in the tissues. Sexual development is strik- ingly interfered with, and the secondary sexual characteristics fail to show themselves. In boys, for example, the pubic hairs fail to extend up to the umbilicus; and the hairs on the chin do not develop, whereas the hair of the scalp grows profusely. The bones remain of the female type, and a broad pelvis, rounded limbs, small feet and hands are often ob- served. In these cases there is usually excessive tolerance for carbohy- drates, which may explain the adiposity, sugar being converted into fat. In the light of the experimental results, the effect on carbohydrate 818 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS metabolism may be explained as due to involvement of the posterioi lobe. Mental development is retarded, and psychic derangements ar< sometimes observed. Where the hypopituitarism does not develop until after adolescence, some of the above symptoms will of course be missed, but many will observed, such as dryness of the skin, loss of hair, and the tendency ri the male to adopt certain of the female characteristics, particularly witl regard to the growth of hair. Obesity and increased tolerance for sugai are also evident, and pigmentation of the skin, something like that ol Addison's disease, is said often to be a prominent feature. These clini< types of hypopituitarism (both the preadolescent and the postadolescent] were first described as entities by Frohlich, and are grouped under th( term dystrophia adiposo-genitalis. They are due undoubtedly to involv( ment of both lobes of the pituitary. In contrast to the foregoing, a form of infantilism associated with hypopi- tuitarism occurs, in which the subjects are not obese but rather the revers( There is a markedly retarded development of the skeleton and the sexual organs, while the patient presents to the casual observer the appearam of an ill-nourished child. When stripped, however, it is seen that he 01 she, is in reality, except for the sexual immaturity, a man or a woman ii miniature. The form possesses the lineaments of the adult, the relative bodily proportions of the child being absent. This type, which is usually known as that of Lorain, is probably due to disease affecting chiefly th( anterior lobe. Operative interference in the early stages in many cas( of hypopituitarism as well as of hyperpituitarism is of undoubted benefit, as is shown by the brilliant work of Harvey Gushing, to which the reader referred for further information. The Relationship of the Pituitary Gland with Other Endocrine Organs The relationship of the pituitary gland with other endocrine organs seems to be an intimate one. 1. With the Thyroid and Parathyroid Glands. That enlargement oJ the pituitary occurs after thyroidectomy in man has been known for considerable number of years. The enlargement affects more particu- larly the pars anterior, although changes are also described in the pai intermedia et nervosa. Accompanying the enlargement of the anterioi lobe, vesicles containing colloid-like material often become developed ii it, but even after the hypertrophy has proceeded to a considerable d< gree, this colloid does not contain iodine, nor does an extract have th< same physiological effect as one of the thyroid gland. It can not replac< thyroid extract in the treatment of patients with goiter or myxedema, THE PITUITARY BODY 819 or ameliorate the symptoms produced in animals by the removal of the thyroid gland. Deposition of colloid-like material in the pars anterior also occurs in myxedema. Histological changes in the pars intermedia et nervosa, although less pronounced than in the pars anterior, are never- theless said to be perfectly distinct following thyroidectomy, and to con- sist in an increase in the hyaline and granular masses which have already been described as present to a certain extent in the normal gland. Less direct evidence of an association in function between the pituitary and the thyroid is furnished by the similarity of the effects produced on the sexual functions and on the general development of young animals by the removal of either gland. In both cases the animals fail to grow properly; the sexual organs remain undeveloped; and the mental func- tions are infantile in type. In hypophysial deficiency, however, extreme adiposity is likely to be more marked than is the case in cretinism. 2. With the Sexual Organs. That the pituitary gland has much to do with the development of the sexual organs has already been shown. Fur- ther evidence of a relationship between the sexual glands and the pitui- tary is furnished by the following observations. After castration en- largement occurs in the pituitary, and on histological examination the gland is found to contain a large number of oxyphile cells, particularly in the pars anterior. This influence of the sexual glands on the pituitary is believed to depend on the interstitial cells present in them, for it has been found that if the ovary or testis is transplanted into other parts of the body after the castration, the changes in the pituitary do not occur, although, as we shall see, the transplanted gland becomes entirely atrophied except for the interstitial cells. The enlargement of the pitui- tary during pregnancy an enlargement which often brings it to two or three times its normal weight is further evidence of its association with the ovary. 3. With the Suprarenals. Association of function is suggested in this case by the fact that extracts of suprarenal and pituitary have very much the same effects on involuntary muscular fiber and glandular structures, and it is said that the two extracts mutually facilitate each other's action in this regard. It should be remembered, however, that pituitrin and epinephrine do not appear to act on exactly the same peripheral mechanism (see page 811). 4. With the Isles of Langerhans. Since pituitrin affects carbohydrate metabolism, which is thought to be primarily controlled by the Isles of Langerhans, it is claimed by some observers that a relationship also exists between the pituitary and these structures. Injections of duodenal extracts are also said to cause a hypersecretion of pituitrin into the- cerebrospinal fluid. CHAPTER LXXXVIII THE PINEAL GLAND, THE GONADS, AND THE THYMUS THE PINEAL GLAND This peculiar structure lies between the anterior corpora quadrigem- ina, and weighs about two-tenths of a gram. It is largest in the early years of life, and undergoes retrogressive changes after puberty. Micro- scopically it consists of epithelial cells arranged loosely in trabeculae, with large sinus-like capillaries between them; neuroglia and sometimes muscle-fiber cells are also present. Curious globules of calcareous mat- ter (brain-sand) are also found, especially in the pineal gland of man. The gland is developed from an evagination of the third ventricle, and it is homologous with the so-called median eye of reptiles. The functions of the pineal gland are obscure. In cases where its extirpation has been successfully accomplished (in the fowl), it has been found that the body growth is stimulated and that the sexual characteris- tics develop more quickly. This result would seem to indicate that the clinical observation that tumors of the pineal gland associated in young boys with abnormal growth of the skeleton and with early development of the secondary sexual characteristics, depends on the fact that a new growth produces destruction of the gland with consequent hypopinealism. The immediate effects of the injection of extract of pineal gland are not characteristic, consisting merely of a fall in blood pressure, which is, however, obtainable when an extract of practically any cellular organ is injected. Prolonged administration of an extract to growing ani- mals is said to accelerate the growth and to bring about a precocious develop- ment of the sexual organs; but this result is somewhat difficult to inter- pret, for, as we have just seen, similar changes occur after experimental removal of the gland. It is stated by McCord and Allen 56 that the pig- ment cells (melanophores) of tadpoles become contracted after pineal feeding. As the receptors of the reflex governing such color reactions of various animals are situated in the retina, these investigators give signi- ficance to their observations by correlating them with the fact that the pineal gland, as stated above, is the representation of a reptilian eye. 820 THE PINEAL GLAND AND THE GONADS 821 THE GONADS OR GENERATIVE ORGANS The Generative Glands of the Male The structures which are responsible for the well-known influence of the testicles on the development of the male sexual characteristics are the so-called interstitial cells of Leydig, which consist of polygonal- shaped epithelial-like cells, with well-marked nuclei and nucleoli. Lipoid granules, staining black with osmic acid, are also present in the cyto- plasm. The degree of development of the interstitial cells varies in dif- ferent animals, being marked in the cat and man and ill-marked in the rat and rabbit. In animals which show seasonal changes in sexual activ- ity, the cells are most prominent between the periods of sexual activity, when the semeniferous epithelium is less evident. They also become prominent in cases where the semeniferous epithelium is atrophied, either as a result of disease or following ligation of the vas def erens done in such a way that the artery and nerves to the testicles are not included in the ligature. When the testicle or a portion of it is grafted into another part of the body, the semeniferous epithelium degenerates, but the interstitial cells remain alive and become quite prominent. It is believed that the interstitial cells are responsible for the production of an autacoid that has to do with the development of accessory sexual characteristics. T~he effects of castration are not significant in animals below the verte- brata. In all of these, however, they are very pronounced. The cas- trated male frog fails to show development of the thumb pad, but this development immediately ensues if portions of testis from another frog be placed in the dorsal lymph sac. In birds the results are more pro- nounced; m the castrated male chick the comb, spurs, wattles, etc., fail to develop, but will usually do so if some testis from another bird is trans- planted into its tissues. In mammals the effects are most striking in animals that develop marked male characteristics, such as the growth of antlers in stags. These fail to develop properly and are prematurely shed after castration. In man also, as is well-known from a study of eunuchs, castration has a very profound effect. Hair fails to grow on the face; the larynx remains undeveloped; the epiphyses are a long time in ossifying, so that the stature may become great, but at the same time the limb bones may be more delicate than usual ; the sutures of the skull are slow in closing ; and the whole architecture of a castrated male comes to be very like that of the female. Confirmatory evidence of the influ- ence of the testicles on the development of secondary sexual character- istics is afforded by the observation that malignant tumors of the testes in boys are associated with the premature development of the secondary 822 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS sexual characteristics, and that these may recede after the removal oJ the tumor. As a result of castration, interesting changes have also been observe* in other ductless glands. Thus, the suprarenal cortex and the thymuj become enlarged, whereas the thyroid and pituitary become atrophie< The metabolic functions also become tardy, as is evidenced by a tendency to the deposition of fat. When the castration is performed on an adult man, the above chang< in the sexual characteristics are of course not so evident, although the prostate, etc., atrophy. The effect on the metabolic functions is, how- ever, very marked, there being a striking tendency to increased form* tion of fat. It is interesting that accompanying this there should usual] occur a lowering of the assimilation limit for carbohydrate, so that glyc< suria is very readily induced. We can not assume, therefore, as Gush- ing has done in the case of hypopituitarism, that the fat deposition if attendant upon an improper combustion of carbohydrate. These remarkable effects of castration have naturally prompted ol servers to study the influence of injection of testicular extract on th( development of sexual characteristics in different animals, but the re- sults have in general been considered to be of a negative character. The Female Generative Organs It is well known that, besides their function in producing ova, th< ovaries also produce autacoids that have to do not only with the fix* tion of the embryo in utero, but also with the changes that occur during pregnancy in the maternal organism. It is however at present' uncertain as to where these autacoids are produced in the ovary. The two mos likely sources are the stroma cells and the corpus luteum. In the stroma of the ovary of certain animals, groups of cells have been describe( having a different appearance from those of ordinary stroma cell They have been called the interstitial cells of the ovary, and are believe< to be analogous with the similar structures found in the testicle. It i* possible, however, that these interstitial cells are nothing more thai cells derived from previous corpora lutea. The latter are formed proliferation of the follicular epithelium which remains after extrusioi of the ovum, and by the ingrowing into the follicle of the so-called theci cells and blood vessels. The fully ' developed corpus luteum in most animals consists of cells arranged in trabeculse converging toward th< scar which formed at the place where the follicle had burst. The luteal THE PINEAL GLAND AND THE GONADS 823 cells, as they are called, are characterized by containing considerable quantities of lipoid material. That the ovary produces some autacoid is evidenced by both clinical and experimental observations. Thus, if both ovaries are removed in a young animal (oophorectomy or spaying), it is well known that not only does the uterus fail to develop properly, but the external changes characteristic of puberty in the female fail to materialize, although act- ually the general effects are not so pronounced as they are in the male after castration. Menstruation does not set in ; the mammary glands fail to develop ; and there is a tendency for the hair to grow as in the male. When the operation is performed in adult life, the changes are not very pronounced, except that menstruation ceases and the uterus and mam- mary glands atrophy. Metabolism also becomes altered, causing a tendency to the deposition of fat, and in the case of the human animal at least, there is frequently evidence of mental disturbance. Attempts to acquire more definite information regarding the physio- logical effects of the ovarian autacoid have recently been made by Schafer and Itagaki. 3 Extracts were prepared from the corpus luteum or Graafian follicles or from the hilum ovariae, and observations were made on the effect produced on the behavior of the chief forms of unstriated muscle by adding the extracts to isolated preparations of uterus or intestine or by injecting the extracts into animals. Applied to the isolated prepa- rations, extract of follicular tissue or of liquor folliculi was found to increase the force and rate of the rhythmic contractions of the uterus as well as its tone, whereas inhibition was produced when extract of the hilum was used. Extract of corpus luteum, when injected into the veins, was found to cause the uterus to increase its contraction or if quiescent to begin contracting. It was further noted that extracts of the hilum caused a fall in arterial blood pressure, whereas those of the corpus luteum had little or no effect. It would appear from these observations that the extracts contain two different autacoids, one having a hormonic and the other a chalonic action on plain muscular fiber. Extract of corpus luteum when intravenously injected also stimulates the outpouring of the milk from the mammary glands, although not so markedly so as extract of pituitary gland. This pituitary-like action is not obtained with extracts of ovary that do not contain corpora lutea. Besides being concerned in the outpouring of milk, corpus luteum has also been shown to be related in some way to the development of the mammary gland during pregnancy. These glands become developed in young virgin rabbits after the continuous administration for a month or so of extract of corpus luteum, and they also develop in unimpreg- 824 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS nated animals when the corpus luteum is made to develop by artificial means such as puncturing the Graafian follicle. Furthermore, destruc- tion of the corpora lutea in a pregnant rabbit arrests development of the mammary glands. The corpus luteum has also an important func- tion in connection with the formation of the uterine decidua and the fixation of the embryo. Thus, after destruction of the corpus luteum at an early period in pregnancy, the embryo fails to become adherent to the uterus. THE THYMUS* The structure of the thymus is of a lymphoid nature consisting of a cortex composed of closely packed masses of lymphocytes and a medulla made up of a cellular reticulum, in the meshes of which are seen large bodies possessing a concentric configuration the corpuscles of Hassel. The gland is developed from outgrowths of the third branchial pouch on either side, which, meeting in the midline, unite to form a solid block of cells, this later, becoming hollowed out and branched. In the walls of the tubules, so formed, lymph nodes appear which ultimately form the cortex. The walls of the tubules themselves break up, their cellular elements subsequently forming the reticulum and concentric corpuscles of the medulla. Though prominent in early childhood the thymus un- dergoes progressive involution with advancing years, until in adult life it is more or less vestigial in character. It is still a question whether this body should be included with the organs of internal secretion, and though many views, mostly of a more or less speculative nature, have been ad- vanced to justify its consideration as an endocrine organ, the proof that it possesses an internal secretion, in the generally accepted sense, is wanting. It is wiser perhaps to state that its functions are obscure, and that we do not know what role it plays in the animal economy. The re- sults of feeding the gland to animals or of ablation experiments are con- flicting and of little help in arriving at any conclusion in this regard. Attention, however, should be drawn to certain significant facts regarding its behavior. First, its involution is arrested or retarded after castration, a fact suggestive of an endocrine function ; secondly, it is believed to be a source of the lymphocytes, and possibly also of the granular leucocytes. Examinations of the blood, with a view to the lymphocytic counts, show, from infancy to puberty, a declining curve, the gradient of which follows closely that of thymic involution. On this account it is believed by some that "the thymus functions as a lymphoid organ in infancy and child- hood when a large number of lymphoid cells and leucocytes are needed to combat infection." (Hoskins, E. R.). 'For a review of the literature, the reader is directed to a recent article by Blatz. 57 THE PINEAL GLAND AND THE GONADS 825 DUCTLESS GLANDS REFERENCES (Monographs) i Vincent, Swale: Internal Secretions and the Ductless Glands, Ed. Arnold, London. sBiedl: The Internal Secretory Organs, Wm. Wood & Co., 1913. sSchafer, Sir E. A.: The Endocrine Organs, Longman's, Green & Co., New York and London, 1916. (Original Papers) *Guthrie, L., and Emery, W. d' E.: Trans. Clin. Soc., London, 1907, xl, 175; also Bul- lock, W., and Segueira, J.: Trans Path. Soc., London, 1905, Ivi, 189. sMyers: Trans. Path, Soc., London, 1898, xlix, 368. e Wheeler: Quoted by Swale, Vincent: Endocrin, 1917, i, 140. 7Halle, W. L. : Quoted by Schaf er, E. A. : Brit. Med. Jour., June 6, 1908. sEwins, A. J., and Laidlaw, P. P.: Jour. Physiol., 1910, xl, 275. sBoyd, W. : Jour. Lab. and Clin. Med., 1918, iv, 133. loFolin, O., Cannon, W. B., and Denis, W. : Jour. Biol. Chem., 1913, xiii, 447. nCow, D.: Jour. Physiol., 1915, xlix, 441. 32Addis, T., Barnett, G. D., and Shevky, A. E.: Am. Jour. Physiol., 1918, xlvi, 39. isCannon, W. B., and Gray, H.: Am. Jour. Physiol., 1914, xxxiv, 232; also with Men- delhall, W. L. : Ibid., 243 and 251. i^Moore, B., and Purinton, C. : Am. Jour. Physiol., 1900, iii, Proc. Am. Physiol. Soc. XV. isHoskins, B. G.: Am. Jour. Physiol., 1912, xxix, 363. leHartman, F. A., and others: Am. Jour. Physiol., 1915, xxxviii, 433; ibid., 1917, xliii, 311; ibid., xliv, 353; ibid., 1918, xlv, 111; ibid., xlvi, 168, 502 and 521; Endocrin., 1918, ii, 122; ibid., 1919, iii, 321; Jour. Pharm. and Exper. Therap., 1919, xiii, 417. i7Hoskins, R. G., Gunning, R. E. L., and Berry, E. L.: Am. Jour. Physiol., 1916, xli, 513. isGruber, C. M., and Fellows, A. P.: Am. Jour. Physiol., 1918, xlvi, 472; and Gruber, C. M.: ibid., 1914, xxxiii, 335; Gruber, C. M., and Kretschmer, O. S.: ibid., 1918, xlvii, 179. isStewart, G. N., and Kogoff, J. M.: Jour. Lab. and Clin. Med., 1918, iii, 209. See full bibliography by Rogoff in this paper. soElliott, T. R. : Jour. Physiol., 1912, xliv, 374. 2iStewart, G. N. : Jour. Exper. Med., 1911, xiv, 377; ibid., 1912, xv, 547; ibid., xvi, 502. 22Meltzer, S. J.: Deutsch. Med. Wchnschr., 1909, xiii. 2^Stewart, G. N. : Rogoff, J. M., and Gibson, F. S. : Jour. Pharm. and Exper. Therap., 1916, viii, 205. 24Hoskins, R. G., and McClure, C. W. : Arch. Int. Med., 1912, x, 343. 25Stewart, G. N.: Jour. Exper. Med., 1911, xiv, 377. "~mnon, W. B., et al: Am. Jour. Physiol., 1911, xviii, 64; ibid., 1914, xxxiii, 356; also Bodily Changes in Pain, Hunger, Fear and Rage, D. Appleton & Co., 1915. ^Stewart, G. N., and Rogoff, J. M.: Jour. Exper. Med., 1917, xxvi, 637; Jour. Pharm. and Exper. Therap., 1917, x, 49; Am. Jour. Physiol., 1917, xliv, 543. Stewart, G. N., and Rogoff, J. M.: Am. Jour. Physiol., 1920, li, 366. ^Cannon, W. B.: Am. Jour. Physiol., 1919, 1, 399. vy, A. G.: Heart, 1913, iv, 342. ^Gasser, H. S., and Meek, W. J.: Am. Jour. Physiol., 1914, xxxiv, 63. '2Redfield, A. C.: Jour. Exper. Zool., 1918, xxvi, 275. Cannon, W. B., and Cattell, McK.: Am. Jour. Physiol., 1916, xli, 74. 'Macleod, J. J. R., and Pearce, R. G.: Am. Jour. Physiol., 1912, xxix, 419 ^Marine, D. : Personal communication. 3 Marine, D. : Jour. Exper. Med v 1914, xix, 89. '^Marine, D., and Lenhart, C. H.: Jour. Exper. Med., 1910, xii, 211; ibid., 1911, xiii, 455; also Bull. Johns Hopkins Hosp., 1910, xxi, 95. ssKendall, E. C.: Boston Med. and Surg. Jour., 1916, clxxv, 557; also Proc. Am. Phys. Soc., 1918, xliv. ^Marine, D., and Kimball, O. P.: Jour. Lab. and Clin. Med., 1917, iii, 41. 3 Gudernatsch, J. F.: Am. Jour. Anat., 1913, xv, 431. ll Rogoff, J. M., and Marine, D. : Jour. Pharm. and Exper. Therap., 1916, ix, 57. ^Kendall: Enderin, 1918, ii, 81; ibid., 1919, 3, 156. Also Plummer, H. S-: Am. Jour. Physiol., 1918, Proc. Am. Soc. Phys., 1918. 826 THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS 43Marine, D., and Lenhart, C. H.: Arch. Int. Med., 1911, viii, 265. 44 Wilson, L. B.: Am. Jour. Med. Soe., 1916,. elii, 799 5 ibid., 1913, cxlvi, 731; also Wilson, L. B., and Durante, L. : Jour. Med. Kesearch, 1916, xxxiv, 273. 45p a ton, Noel, and Finlay, J.: Quart. Jour. Exper. Physiol., 1916, x, 203; also Paton, Noel, Finlay, J., and Watson, A.: Ibid., 233, 243, 315 and 377. 46MacCallum, W. G., etc.: Jour. Exper. Med., 1909, xi, 118; ibid., 1913, xviii, 646; Jour. Pharm. and Exper. Therap., 1911, ii, 421. 47Howland, J., and Marriott, W. McK. : Bull. Johns Hopkins Hosp., 1918, xxix, 235. 48Cushing, Harvey: Pituitary Body and Its Disorders, J. B. Lippincott Co., 1912. 49Horsley, V.: Brit. Med. Jour., 1885, i, 111. soKobertson, Brailsford, and Kay, L. A.: Jour. Biol. Chem., 1916, xxiv, 347, 363, 385, 397, 409. siClark, L. N.: Jour. Biol. Chem., 1915, xxii, 485. 52Cow, D.: Jour. Physiol., 1914, xlviii, 443. ssMaxwell, A. L. J.: Jour. Physiol., 1915, xlix, 483. 54Abel, J. J., and Kuboto, S. : Jour. Pharm. and Exper. Therap., 1919, xiii, 243. See also Cow, D.: ibid, 1919, xiv, 275; Abel, J. J., and Nazayama, T.: ibid., 1920, xv, 347; and Dudley: ibid., 1919, xiv, 295. soHerring, P. T. : Quart. Jour. Exper. Physiol., 1914, viii, 267. 56McCord, C. P., and Allen, F. P.: Jour. Exper. Zool., xxiii, 207. ."Blatz, W. E.: Jour. Lab. and Clin. Med., 1919, v, 3. ssKnowlton, J. P., and Silverman, A. C.: Am. Jour. Physiol., 1918, xlvil, 1. PART IX THE CENTRAL NERVOUS SYSTEM AND THE CONTROL OF MUSCULAR ACTIVITY (Contributed by A. C. REDFIELD) CHAPTER LXXXIX THE EVOLUTION OF THE NEUROMUSCULAR MECHANISM Disease of the nervous system confronts the physician with a complex group of symptoms, a syndrome due to more or less sharply localized disturbances in its function. It is the province of physiology to analyze the fundamental activities of the nervous system and assign to various parts a functional significance, so that the complicated picture presented by disease may be recognized as the logical result of a lesion of definite nature, location, and extent. We find the symptoms of nervous dis- ease grouping themselves as disturbances (1) of sensation (anesthesia, etc.) (2) of movement, both volitional and reflex (paralysis, etc.) (3) of postural coordination (muscular spasms and flaccidity, ataxia, etc.) (4) of the mechanisms of integration in the nervous system including the higher mental functions of association, memory, and attention, etc. Corresponding to each of these groups we can recognize a definite as- pect of nervous activity, the successful performance of which depends on the continuity of more or less precisely known groups of cells within the nervous system, and disturbances of any of these aspects can be as- signed to lesions affecting some part of the group of nerve cells on which they depend. The fundamental function of the nervous system is to correlate the activities of the body so that its many parts may act harmoniously and as a unit in preserving the welfare of the individual. What the basis of this integration of activity which manifests itself so remarkably in the behavior of the higher animals, is, can best be illustrated by a consid- eration of its evolutionary development. Primitive Neuromuscular Mechanisms In the unicellular organisms three processes occur when a response is brought about by a stimulus. These are (1) excitation, or the setting up of a physiological disturbance; (2) conduction, or the spread of the dis- 827 828 CENTRAL NERVOUS SYSTEM turbance to parts of the cell remote from the point of excitation; and (3) response on the part of the protoplasm to which the disturbance has spread. Thus when a paramecium swims against a hard object, an excitation is set up by the contact at its anterior end ; the disturbance so produced spreads to other parts of the cell and causes the reversal of the direction of the stroke of the cilia in relatively remote regions, with the result that the paramecium backs away from the obstacle, and then starts off in another direction. The three fundamental processes of excitation, conduction, and response occur whenever the neuromuscular system of an animal is brought into play. The case of multicellular animals differs from that cited above only in that the conduction is intercellular, so that the disturbance set up by an excitation spreads from one cell to another. There exist in multicellular animals many examples of cells independent of the influence of nervous tissue which respond directly to stimuli. These are known as independent effectors. The most primitive muscle cells known are those which close the terminal pores of sponges, thus regulating the flow of water through these animals. Parker 1 has shown that these muscles respond to a variety of stimuli in spite of the fact that no nerve cells can be found in association with them. Ciliated epithelial cells, such as occur in certain ducts and passageways in man are also independent ef- fectors, acting independently of nervous control. The ameboid white blood corpuscles also must be quite comparable in their mechanisms of response to the protozoa. The forerunner of nervous conduction is seen in the activities of these independent effectors. If a field of ciliated epithelium is examined micro- scopically, it is found that the cilia are not beating in a disorderly way, but that definite waves are passing across the field, each cilium beating a mo- ment later than the preceding one. The appearance is quite like that pro- duced by gusts of wind sweeping across a field of grain. In the sponges, also, a stimulus applied several centimeters from the terminal pore will cause its muscles to contract in spite of the absence of nervous tissue con- necting the muscles with the point of stimulation. This type of intercel- lular conduction by nonnervous tissue is called neuroid transmission. It illustrates the significant fact that conductivity is not a specific property of nervous tissue, but is displayed by many other tissues as well. Nervous tissue makes its first appearance in the Coelenterates (Fig. 203). A most primitive condition is found in the tentacles which sur- round the mouth of the sea anemone. At the base of certain epithelial cells fibrous processes are developed which connect with underlying mus- EVOLUTION OF THE NEUROMUSCULAR MECHANISM 829 cles. The epithelial cell serves as a receptor for stimuli, and transmits the disturbances set up by them to the muscle or effector. Such a mecha- nism is called a receptor-effector system. The responses which it brings about are purely local, since there is no provision for conducting the disturbance to remote parts of the animal, but the introduction of the receptor serves a valuable purpose in increasing the sensitivity of the system. Moreover, the arrangement is adequate for the purpose for Fig. 203. The evolution of the nervous system. A, the independent effector as illustrated by muscle cells of the sponge; B, a receptor-effector system such as occurs in the tentacles of sea anemones; C, the neuromuscular mechanism of the trunk of the sea anemone, in which a network of nerve cells is interposed between receptor and effector; D, the nerve net surrounding a small blood vessel of the frog; E, the nervous system of the earthworm, illustrating a typical reflex arc and the occurrence of association neurons; a, within the ganglion; r, receptor; nn, nerve net; an, ifferent neuron; en, efferent neuron. (Modified after Parker, Prentiss and Bayliss.) it is employed, which is to cause the tentacle to bend toward a irticle of food situated between it and the mouth, so that the currents up by the cilia with which the tentacle is covered may sweep the food ito the mouth. An arrangement quite similar to the receptor effector Astern of the Coelenterates is found in the axon reflexes of mammals, in which disturbances set up in certain sensory neurons spread directly through collateral fibers to the vasomotor muscles of the skin (page 898). 830 CENTRAL NERVOUS SYSTEM The Nerve Net In the trunk of the sea anemone nervous tissue assumes a much more important role. Between the receptors of the epithelium and the muscles there is interposed a layer of nerve cells arranged as a network. This network can transmit disturbances set up in the receptors to quite dis- tant muscles, with the result that a local stimulus may bring about a gen- eralized contraction of the trunk. The primitive nervous system is thus seen to consist of a network of nerve cells or nerve net. Its charac- teristic consists in the fact that the nerve cells are joined together by continuous fibers so that the structure is essentially a syncytium. No cell membranes can be distinguished separating the fibers of the constit- uent nerve cells. The result of this structure is that nerve impulses set up in one region can spread at random to all parts of the nerve net. The responses produced by such a mechanism are necessarily diffuse in char- acter, since large groups of muscles will be brought into play at one time. Since conduction is not limited to definite paths in the nerve net, local in- jury will have little effect on the reactions of the organism because the nerve impulses can pass around the injured region through other parts of the nervous network. The rapidity with which impulses are conducted by the nerve net is not great, being only 146 centimeters a second, or about two hundred times less than the velocity of the nerve impulse in the motor nerves of the frog. The nerve net is retained in many parts of the higher animals. Wher- ever it occurs it is usually very closely associated with the muscles which it innervates. The most important nerve net in man is found in the intestine (myenteric plexus). In this structure an important modifica- tion in behavior has developed. While .the reactions of the intestine maintain the sluggish, diffuse character seen in the eoelenterates, con- duction no longer proceeds as readily in all directions. Excitation pro- duces a contraction of the muscles above the point stimulated and relax- ation below it (the myenteric reflex page 501). Waves of contraction travel along the intestine usually only in one direction, from pyloric end downward. The nerve net has developed a definite polarity, a property which is fundamental in the central nervous system of the higher animals. The Central Nervous System A typical central nervous system appears in simple form in the seg- mented worms. The nerve cells of which it is composed do not form a syncytium, as in the nerve net, but are separated from one another at the points at which their fibers meet by a specialized structure called a synapse. A definite membrane may be seen at this point separating the protoplasm of one fiber from that of the other. .The nerve cell con- EVOLUTION OF THE NEUROMUSCULAR MECHANISM 831 sequently acquires a certain individuality, and can become specialized for special kinds of activity, and is called a neuron. The neuron is the fund- amental unit of structure and function in the central nervous system. It consists typically of a nerve cell body containing the nucleus, from which extend numerous short fibers, or dendrites, and one long fiber, or axon. The axon may be branched, such branches being called collaterals. Fig. 204. Normal cell from the ventral horn, stained to show Nissl's granules, a, the axon. (From Howell.) The arrangement of neurons in the central nervous system of the rorms is characteristic. Most of the nerve cell bodies are collected to- gether in centrally located masses or ganglia. Fibers pass from the 'eceptors in the epithelium to the ganglion of the same segment in defi- nite nerve trunks, while other fibers originating from nerve cell bodies within the ganglion pass in other trunks to the muscles. In addition 832 CENTRAL NERVOUS SYSTEM fibers pass from one ganglion to the next in trunks known as intergan- glionic connectives. In order that a muscular response may result from a stimulus applied to the skin of a worm, a disturbance must pass through the following structures: (1) the receptor with its afferent fiber, (2) a motor neuron with its efferent fiber, to (3) the muscle (or other effector). This group of structures is called a reflex arc, It is the simplest anatomical arrange- ment in the central nervous system, capable of bringing about a signi- Fig. 205. Arborization of collaterals from the dorsal root fibers around the cells of the posterior horn. A, ascending fiber in dorsal columns; B, collaterals; C, cells of dorsal horn; E, synapses. (From Ramon y Cajal.) ficant motor response. The activity produced by such a mechanism is called a reflex. The reflex arc as we have described it lies frequently within a single seg- ment of the worm. Consequently it can provide each segment with a great degree of autonomy, so that many of its activities are undisturbed by the removal of other parts of the animal. No provision is made by such an arrangement for correlating the activities af adjacent segments. The ganglia of worms contain, as a matter of fact, an additional type of neuron which makes the correlation of the activities of different seg- ments possible. These are neurons which lie entirely within the central nervous system. Their axons lie in the interganglionic connectives and EVOLUTION OF THE NEUROMUSCULAR MECHANISM 833 Fig. 206. Part of a ventral cornual cell from the calf's spinal cord, stained to show neurofibrils. ax, axon; a, b, c, dendrites. (From Bethe.) Fig. 207. Schema of simple reflex arc; r, receptor in an epithelial membrane; a, afferent fiber; s, synapses; c, nerve cell of motor neuron; e, efferent fiber; m, effector organ. 834 CENTRAL NERVOUS SYSTEM serve to connect afferent fibers in one segment with motor neurons in some distant segment. They are called association neurons because they asso- ciate the activities of remote parts of the body. The fundamental characteristics of the central nervous system as it appears in the worms consists in (1) the individuality of the component nerve cells and their specialization for certain functions, (2) the arrangement of the neurons in such a way that the impulses must pass over definite paths or reflex arcs in order to reach an effector, (3) the introduction of neurons specialized to associate the activities of reflex arcs in remote parts of the body, and (4) the segregation of the greater part of the nervous tissue into a centrally located chain of ganglia, from which fibers pass to peripheral structures in definite nerve trunks. These characteristics persist in the central nervous system in the higher animals, in- cluding man. Certain modifications of the simple arrangement found in the worms have been introduced into the nervous sys- tem as the motor activities of organisms became more precise and complex. Of minor importance may be men- tioned the introduction of an afferent nerve cell distinct from the receptor between the latter and the central ganglionic mass, (see the Evolution of Receptors page 854) and the introduction of an outlying neuron between the termination of the primary motor neuron and the effector organ (see the autonomic system page 894). Of far greater importance is the great increase in the num- ber of association neurons which become the salient fea- ture of the vertebrate nervous system. In the worms the inter-ganglionic connectives are scarcely more bulky than the peripheral nerves from a single ganglion, whereas in the central nervous system of man the num- ber of the fibers extending from segment to segment ex- ceeds by many times the number of fibers in any single pair of spinal nerves. This increase in the mass of the central portion of the nervous system is not distributed equally in all parts, but tends to concentrate itself par- ticularly at the anterior end. Even in the worms the most anterior ganglion, the supraesophageal ganglion, is larger than the others, while this tendency finds its culmination in the great bulk of the brain in man. This condition is obviously correlated with the development Fig. 208. Dia- gram of nervous system of seg- mented inverte- brate; a, supra- esophageal g a n- glion; b, subeso- phageal ganglion; oe, esophagus or gullet. EVOLUTION OF THE NEUROMUSCULAR MECHANISM 835 of special sense organs at the anterior end of the animal: the sense of taste at the mouth, the tactile organs found on the tentacles which adorn the head of many invertebrates, and of special receptors for stim- uli originating at a distance, such as the eye and ear. The development of association neurons for the correlation of activities initiated by the stimulation of these receptors has made this portion of the central nervous system the natural place for the seat of the higher mental functions as they evolved. CHAPTER XC THE CONDUCTION OF THE NERVOUS IMPULSE We have seen that the activity of the neuromuscular mechanism in- volves the three processes of excitation, conduction, and response. These processes may be regarded as fundamental properties of protoplasm which may all be exhibited by a single cell. The evolution of the cen- tral nervous system is the history of a gradual specialization of cells each for the execution of one of these processes. Thus in a reflex arc the receptor is a cell primarily fitted to react to slight changes in its environment, the neurons are especially adapted to the conduction of im- pulses, and the effector is constructed for the sole purpose of carrying out some motor response or producing some specialized secretion. It would appear as though a single cell could not develop the ability to perform all of these functions with perfection and as a consequence a high degree of division of labor has been established. It should be remembered, however, that this specialization for one process has deprived the cells to only a limited degree of the ability to be the seat of the other processes. Thus although excitability is pri- marily a property of the receptors, nerve cells must also retain the abil- ity to become excited by disturbances set up in the receptors, and muscles, must be able to be excited by impulses reaching them from motor neurons Similarly conduction must occur not only in nerve cells, but in receptors, so that a disturbance set up at the distal end of the receptor cell may reach the nerve fibers which terminate about its proximal end. Muscle cells also must be able to conduct disturbances set up in the myoneural junction to all parts of the muscle fiber, so that they may contribute to the response. It has been seen that the conductivity of the cardiac mus- cle is of great importance in the co-ordination of the action of the heart (page 182). Even contractility which would appear at first sight to be a function of the effector alone is displayed occasionally by the other parts of the reflex arc. Thus embryonic nerve fibers have been observed to perform ameboid movements and the rods and cones of the retina, which are the receptors of light, become shorter or longer in response to changes in illumination. While excitation, conduction, and response are seen to occur in all parts of the reflex arc, when we come to study these processes it is con- venient to examine each in that tissue in which it is most highly developed and where the other processes will introduce a minimal complicating ele- 836 THE CONDUCTION OF THE NERVOUS IMPULSE 837 ment. Thus excitation is best considered in connection with the recep- tors and a consideration of it may be postponed until we are ready to take up the aspect of central nervous activity which leads to sensation and the phenomenon of consciousness. Response must be studied in mus- cular tissue and its treatment may be put off until we have seen what the nervous mechanism initiating muscular activity consists of. Con- ductivity is readily studied in nerve, and since this process forms the base of all nervous activity, it must be treated before the study of the activity of the nervous system as a whole can be undertaken. Conduction in the Nerve Fiber When a nerve fiber supplying a muscle is stimulated, the response of the muscle is so prompt that for many years physiologists despaired of determining the rate at which the nerve impulse traveled along the nerve. Helmholtz succeeded, however, in devising a simple method by which the velocity of the impulse could be measured and found that in the sciatic nerve of the frog the rate of propagation was about 30 meters a second. Later determinations have shown that in the nerves of man the velocity of the impulses is three or four times as great, a dif- ference which may be attributed to the higher temperature of the human body. It is seen from this that very little time is lost in the transmission of impulses along nerve trunks, and rapid responses to stimulation are thus insured. Although nerve fibers are closely bound together within a nerve trunk, impulses cannot spread in a lateral direction from one fiber to its neighbor. Consequently if a branch of the lumbar plexus is stim- ulated, contraction occurs in a more limited group of muscles than if the nerve trunk is stimulated below the junction of the various parts of the plexus. The response of muscles to the artificial stimulation of a nerve trunk also differs in its distribution from responses induced reflexly or volitionally. These facts show that the constituent fibers of a nerve trunk are completely isolated from one another. Within the single nerve fiber nervous impulses may travel in either direction along its length irrespective of the direction in which con- duction normally takes place. Thus if a collateral of a motor neuron is stimulated close to its termination in a muscle, the impulse will travel up the collateral to its point of junction with a second collateral, and down the latter to cause a contraction of the muscles which this in- nervates. The polarity which is such an important feature of the activ- ity of the reflex arc as a whole is not exhibited by the conduction within a single neuron, which thus preserves one of the fundamental proper- ties of the primitive nerve net. The All or None Nature of Conduction. Activity cannot occur in the body without the expenditure of energy. It is pertinent to inquire what 838 CENTRAL NERVOUS SYSTEM is the source of the energy expended in the conduction of a nerve im- pulse. It was perhaps natural for physiologists to assume that this energy was derived from the stimulus, much as the energy of a pro- jectile is derived from an explosion. That this is not true has been shown by important experiments by Adrian. 2 ' 3 The intangible nature of the nerve impulse makes a measurement of its strength difficult. One cannot use the muscular contraction produced by it as a measure of its strength, because the degree of muscular response will be determined by the number of nerve fibers brought into action as well as by any variation which may occur in the intensity of the nerve impulse in each fiber. Adrian chose as a criterion of the strength of the nerve impulse its ability to traverse a region of nerve fiber in which conduction had been ren- dered difficult by the application of a narcotic. Whether or not a nerve im- pulse can emerge from such a region depends on the distance which the im- pulse must travel before coming to the normal part of the fiber. This indicates that in passing through the narcotized area the impulse becomes weaker and weaker, and may become completely extinguished if it fails to emerge in time. If the nerve impulse derives its energy from the stimulus, it should be impossible for it to regain its strength after dissipating its energy in a region in which conduction is difficult. This point can be tested by passing the impulse through two equal narcotized areas separated by a region of normal nerve. If the length of each of these areas be not quite great enough to extinguish an impulse, but if their combined length is sufficient for this purpose, and if no recovery occurs in the intervening region of normal tissue, then the impulse will be unable to pass through both areas. In passing the first area the intensity will be reduced in part ; on passing through the normal region there will be no recovery, and in the second area of narcosis the strength of the impulse will be reduced to extinction. As a matter of fact Adrian found that the nerve impulse does not behave in this man- ner. Impulses which would be complete^ extinguished by a given area of narcosis could pass through two areas one half as long if these were separated by a short length of normal tissue. In passing through the normal tissue the strength of the impulse recovered so that it was as great on entering the second region as it was when it came to the first, and consequently it was able to pass through one as well as the other (Fig. 209). From this observation several important conclusions can be drawn. The energy of the nerve impulse is derived not from the stimulus, but from the nervous tissue through which it is passing. Consequently the strength of the impulse depends on the condition of the tissue, and it will vary only as the condition of the tissue varies. If a nerve conducts an im- pulse at all, it will be of the maximum strength possible for the -con- THE CONDUCTION OF THE NERVOUS IMPULSE 839 dition of the nerve at that time. This conception is known as the all or none law of conduction, because the tissue acts with all its power or not at all. It is quite comparable to the all or none action of cardiac muscle with which we have already become familiar (page 177). The importance of the conclusion that the energy of the nerve im- pulse is derived from the nerve fiber itself is this. No matter in how complex a fashion a neuron may be subdivided into collaterals or den- drites an impulse set up in it may pass with undiminished intensity along each of these subdivisions. Since each collateral may be united with other neurons, the impulse may spread from the one neuron to several others without becoming attenuated by the multiplicity of the paths A B Fig. 209. Diagram illustrating the effect of areas of narcosis (n) on the strength of the nerve impulse. A indicates the gradual decline in the strength of the impulse as it penetrates an area just long enough to cause its complete extinction. B and C illustrate the two possible results of- sending an impulse through two shorter areas, separated by a length of normal nerve. If no re- covery occurs in the normal tissue, the impulse may be extinguished in the second narcotized area, as B indicates. If recovery occurs in this area, the impulse may reach the muscle in undiminished strength. Adrain showed the latter alternative to be true. in which it is traveling. Hence the conception of the older physiology of the presence of special reinforcement centers in the central nervous system for the purpose of reinforcing the strength of the impulse as it spreads may be dispensed with, since every part of every nerve fiber contributes the energy necessary to keep the impulse going as it travels along. The Refractory Period. Although the nerve impulses induced by ar- tificial stimulation may be momentary in duration the activity of the reflex arc brought about by normal conditions in life is usually main- tained for some time in order to achieve continuous contraction of the 840 CENTRAL NERVOUS SYSTEM appropriate groups of muscles. Such continuous activity on the parl of nerve cells might be due to either (1) the passage of a series of dis- tinct impulses along the nerve fiber, or (2) to a continuously main- tained activity. Experiment has shown that the first conception is th< correct one. Two impulses set up in rapid succession in the nerv( fibers of a nerve muscle preparation from the frog may cause a greater contraction in the muscle than a single impulse. If the interval betweei these stimuli is decreased to less than 0.0025 of a second the effect due to the second stimulus disappears and it has evidently failed to initi- ate a second nerve impulse. This result occurs whether the two stimuli are applied to the same, or to different parts of the nerve fiber, showing that it is not due to the impossibility of setting up the process of ex- 100 50 01 -02 Time since previous stimulus (seconds) 03 Fig. 210. The recovery of excitability in the nerve fiber after the passage of a nerve impulse. After a brief time in which no second excitation is effective, the excitability gradually returns to normal, and then becomes temporarily greater than normal. The changes in conductivity following the passage of a nerve impulse over the nerve follow a similar course. (From Adrain and Lucas.) citation in one place twice in such rapid succession, but that the nerve fiber is incapable of conducting a second nerve impulse until a sufficient period of recovery has intervened. Moreover by cooling the nerve be- tween the point of stimulation and the muscle, without affecting the temperature in the place at which the process of excitation occurs, the period of recovery may be increased three fold, because at a low tem- perature it takes longer for the nerve fiber to regain its power to con- duct. This period of recovery during which a second impulse cannot pass along a nerve fiber is called the refractory period of conduction. It is quite analogous to the refractory period which occurs in the heart (page 179) and like it imparts certain characteristics to the tissue in which it occurs. THE CONDUCTION OF THPJ NERVOUS IMPULSE 841 Because of the refractory period the continuous activity of nerve must consist of a series of nerve impulses occurring at brief intervals. Nerv- ous discharge consequently has a discontinuous or rhythmic character. The actual rate of this rhythm has not been determined precisely for the voluntary movements of man, but the length of the refractory period, which is shorter in the nerves of warm-blooded animals than in the frog, renders it unlikely that it is greater than 3000 impulses per second, while certain experiments indicate that it is at least 300 impulses per second. 22 The nerve fiber has been found to be unfatigued even by continuous stim- ulation for twenty-four hours, a fact which is explained by the discovery that the nerve will not conduct an impulse until it has recovered from the fatiguing effects of the preceding impulse. The rhythmic character of nervous activity also suggests a way in which nerve impulses set up by different stimuli may differ from one another and consequently bring about different reactions. Although the all-or-none law of conduction may preclude the possibility of nerve impulses differing in strength or inten- sity, there is no reason why the rate at which impulses follow one another in normal nervous activity may not vary greatly, and this difference may be a determining factor in the spread of the impulses through the spinal cord and brain. Conduction between Neurons So far conduction has been considered only with regard to the spread of a nerve impulse through a single nerve cell. In passing from one neuron to another the nerve impulse must pass through the synapse which is the structure uniting their respective fibers. The presence of synapses in the path of conduction imposes certain characteristics on the activities of the reflex arc which do not appear in conduction within a single nerve fiber. The key to an understanding of reflex activity and the higher mental processes by which reflex acts are controlled undoubt- edly lies in the physiology of the synapse. The Polarity of the Reflex Arc. In nerve fibers it has been seen that impulses may spread in either direction along the axon. In the reflex arc as a whole the passage of the impulse is in one direction only, from the receptor to the effector organ, because a nerve impulse can pass in one direction only through the synapse, which acts as a valve to prevent impulses passing back in the opposite direction. This fact is demon- strated by the classic experiments of Bell and Magendie, who found that no muscular response followed stimulating the central end of the cut ventral root of a spinal nerve. The motor neurons whose axons compose the ventral root can transmit impulses only to the muscles which they innervate, and in this experiment they were separated by the operation from the point of stimulation. That impulses do not 842 CENTRAL NERVOUS SYSTEM spread backward from the motor neurons to the afferent neurons can be demonstrated by attaching a galvanometer to the afferent nerve, when it will be seen that no action current is set up in the afferent neuron if the stump of the ventral root is stimulated. Impulses set up in these fibers can not pass to other neurons within the spinal cord, be- cause the synapses will not allow them to spread in that direction. Thus it is seen that the polarity which is characteristic of the central nervous system is imposed upon it by the synapse. In the primitive nerve net, in which synapses can not be demonstrated, polarity does not exist. Resistance to Conduction due to Synapse. The synapse is also a re- gion which offers some resistance to the passage of the nerve impulse and may prevent its passage altogether. It is usually impossible to cause reflex response by applying a single induction shock to a sensory nerve, although the stimulus may be quite strong enough to excite a nerve muscle preparation. Not until the stimulus is repeated several times, setting up a series of nerve impulses, will the resistance of the synapses in the reflex arc be overcome so that an impulse can pass through to the muscle. The synapses in which the collaterals of a single neuron terminate differ considerably in the resistance which they offer to the passage of the nerve impulse. If a series of weak stimuli are applied to the foot of a frog from which the brain has been removed a flexion of the leg may be induced. If the stimulus is increased in strength movements of the opposite leg will occur, while still stronger stimuli will cause the excitation to spread to the muscles of the trunk and forelimbs. This observation indicates that the impulses set up by a weak stimulus can pass only through those synapses which connect the afferent neurons with the motor neurons of the same limb, while stronger stimuli are required to set up impulses which can pass to the synapses leading to the motor paths to more remote muscles. This graded synaptic resistance is con- sequently an important mechanism in determining what paths an im- pulse shall follow in its course through the greatly branching systems of nerve fibers which occur in the nervous system. Summation in Reflex Conduction. The resistance presented to the passage of the impulse by the synapse suggested to Lucas that con- duction in a synapse is comparable to that in a narcotized area of nerve. A second point of resemblance is that the impulse travels slower through both synapse and through a length of nerve treated with alcohol. He consequently studied with care the conditions of conduction through narcotized nerve and discovered several facts which are of value in understanding the peculiarities which the synapses give to reflex conduc- tioii. If an area of narcosis is just deep enough to check the passage THE CONDUCTION OF THE NERVOUS IMPULSE 843 of a single nerve impulse, it is found that a second impulse can pass through it, provided it is produced immediately after the termina- tion of the refractory period. For a short interval after the passage of one impulse into a region in which conduction is difficult the nar- cotized nerve becomes better able to conduct a second impulse. The effects of two impulses added together is thus able to produce a re- sponse which a single impulse alone cannot accomplish. This phenom- enon is known as summation in conduction. Such summation is a characteristic of reflex conduction, as we have seen in the experiment in which stimulation with an induction shock fails to bring about a reflex act unless it is repeated several times. In some cases as many as 40 or 50 stimuli must be applied before reflexes are established. Two characteristic phenomena of reflex conduction closely related to summation which may be explained on the assumption that one im- pulse can alter the ease with which a second impulse can overcome the resistance at the synapse are Induction and Facilitation. Induction is the production of a reflex response by the application to different afferent nerves of two stimuli each of which alone is incapable of setting up impulses which can break through the resistance of the reflex arc. Induction may occur when the stimuli are applied at the same time or when one stimulus is commenced after the other has been discontinued. Facilitation resembles induction except that the stimuli are each capable of producing the response when applied independently. Their combined effect is to produce a greater response than either can elicit when acting alone. The underlying assumption in these cases is that the afferent paths over which the impulses travel in to the nervous system impinge upon a common motor path in the synapses of which summation in conduction occurs. Inhibition. In studying the heart we have seen that nerve impulses traveling over the vagi may depress or inhibit the action of the cardiac muscle. Inhibition is also an important process in the action of the central nervous system, and is of great importance because when cer- tain groups of muscles are made to contract the activity of opposing groups must be depressed in order that movement may be made without opposition. Coordination in the nervous system depends 011 the in- hibition of certain reflex activities in order that other reactions may be carried out without confusion. Certain forms of inhibition can be explained by considerations quite similar to those employed in the ex- planation of summation. We have seen that immediately after the pas- sage of a nerve impulse along a nerve a period occurs during which a second impulse cannot be conducted by the nerve fiber. This is the refractory period of conduction. Later still there is a period during 844 CENTRAL NERVOUS SYSTEM which the conductivity of the fiber is supranormal, and although the first impulse has failed to pass through a region of resistance such as a narcotized area or a synapse, the second impulse falling in this period of supranormal conductivity may be powerful enough to pass through and excite the muscle. This is the phenomenon of summation. Between the refractory period and the period of supranormal conduction the nerve fiber is recovering its ability to conduct. Impulses set up at this period will be of subnormal strength and will be less able to pene- trate regions in which conduction is difficult. Consequently if the rhythm of nervous discharge is such that each impulse falls in the period of subnormal conductivity which follows the passage of the preceding im- pulse, the discharge will be of impulses of subnormal strength. Such a series of impulses may be unable to pass through the resistance of the synapse and no activity can result. Whether a series of impulses will produce summation or inhibition depends on the relation between their frequency and the time required for the conducting tissue to recover from the effects of each impulse, that is, on whether each impulse falls in the period of supranormal or of subnormal conduction set up by its predecessor. If the synapses connecting a single afferent path with two motor neurons have different rates of recovery, the impulses might fall in the period of supranormal conductivity of one synapse and be summated and cause a contraction of the corresponding muscle, while they might fall in the period of subnormal conductivity of the other synapse and be inhibited, the corresponding muscle remaining inactive. In this way we obtain a picture of how the reciprocal inhibition of antagonistic groups of muscles may be accomplished. It must be remembered that physiologists have just made a beginning in analyzing nervous activity from this point of view, and that our present ideas are no doubt crude and subject to revision. Also the facts on which our conception of the nature of nervous conduction is based have been made out chiefly by a study of the motor nerves. It is conceivable that the im- pulses conducted by sensory nerves are of a different nature from those motor impulses, but so far as they can be studied they appear to be the same. It is also possible that the impulses set up by electrical stimulation of motor nerve trunks are different from those arising through voli- tional or reflex activity. Canalization. The frequent use of a path through the nervous system appears to lower permanently the resistance of the synapses along its course. This process is known as canalization, and results in a greater facility in bringing about movements of the muscles to which the path leads. The bearing of canalization on the development of skill in mechanical manipulation and in habit formation is obvious. THE CONDUCTION OF THE NERVOUS IMPULSE 845 The conductivity of the synapse can be altered not only by the pas- sage of nerve impulses through it, but by many other agents. Certain chemical substances such as strychnine and tetanus toxine have a selec- tive action upon the synapse, lowering the synaptic resistance, and con- verting inhibition into active contraction (see page 941). In those invertebrates which possess an asynaptic nervous system, or nerve net, strychnine is without such effect. Nicotine has a selective action upon the synapses of the sympathetic nervous system, increasing the resist- ance so that impulses are unable to pass (page 895). Eeflex activity is very easily abolished by lack of oxygen, as will be indicated in the next chapter. In this regard it differs from nerve trunk conduction to a marked degree. Since conduction can be shown to be quite independent of the nerve cell body, it must be the synapses which are rendered impassable by asphyxia. Fatigue occurs readily in re- flex conduction, and must also have its seat in the synapse, since the nerve trunk itself is quite indefatigable. The Myoneural Junction. The synapse is a region of tissue at the junction of two nerve fibers having properties quite distinct from those of the nerve fibers themselves. The myoneural junction, interposed be- tween nerve fiber and muscle is an analogous structure, the properties of which are different from both muscle and nerve. Certain drugs have a selective action upon it, such as curare, which decreases the conductiv- ity of the myoneural junctions of skeletal muscles and thus results in their paralysis. Curare is a poison which is used by certain savages on their arrow heads. Its fatal effects are due to its action in paralyzing the respiratory muscles by blocking the passage of nerve impulses across the myoneural junction. Epinephrine, the secretion of the adrenal glands, has a specific affinity for the myoneural junctions of certain autonomic nerves, exciting the junctional tissue to action similar to that produced by the nerve impulses (see page 776). The most prominent characteristic of the myoneural junction is its resemblance to the synapse. Like it, it is a region in which conductivity is difficult and readily modified, so that it may be the seat of summation, inhibition, and fatigue. CHAPTER XCI THE NUTRITION OF NERVOUS TISSUE The Function of the Nerve Cell Body In the preceding chapter we considered the physiology of the nerve fiber and the synapses in which it terminates. We must now inquire what part the nerve cell body, containing the nucleus of the neuron, takes in the conduction of the nervous impulse. In the crayfish it is possible to remove the cell bodies from the motor neurons of the antenna without disturbing the reflex connections of the nerve fibers. This can be done because the motor neuron possesses a single axon which divides at some distance from the cell body into two collaterals, one connecting with the afferent neurons of the reflex arcs which control the movements of the antennas, the other passing directly to the muscles of that organ. Cutting away the part of the cephalic ganglion which contains the cell bodies of these neurons does not interfere with the reflex excitation of the muscles of the antenna?, provided that the continuity of the collaterals is not destroyed. The nerve fibers, deprived of their cell bodies are able to function normally for two or three days. Conduction does not then depend primarily upon the presence of the nerve cell body. On the days following the operation the reflex is elicited with greater and greater difficulty, and fails altogether on the third or fourth day. The nerve cell body is consequently necessary for maintaining the conductivity of its nerve fibers, that is, it is concerned with the nutrition of the outlying parts of the neuron. Degeneration and Regeneration of Nerve Fibers. 4 The nutritive func- tion of the nerve cell is also illustrated by the phenomena w r hich follow the section of a peripheral nerve trunk or of the tracts of fibers in the central nervous system. "When a man's motor nerve is severed, the excitability of the peripheral part may be increased for one or two days, but will then decline rapidly and disappear completely by the end of the second week. Microscopic examination reveals the fact that such a nerve, the fibers of which are cut off from their cell bodies, has under- gone degeneration. The fibers have broken up into ellipsoid segments of myelin, each containing a piece of the axis cylinder, and these seg- ments later fragment very irregularly into smaller pieces which are eventually absorbed. In animals such as the rabbit, in which the process occurs slowly, it can be seen that the degenerative changes begin at the 846 THE NUTRITION OF NHRVOUS TISSUE 847 wound and proceed peripherally, although in the dog, in which the degen- eration is rapid, it appears as though the process occurred synchro- nously in all parts of the fiber. In the fibers on the central side of the wound degeneration also oc- curs, but it is limited to the several internodal segments which lie just above the point of injury. Nearer to the nerve cell body the fibers re- main for the most part unchanged. An influence, howeyer, is exerted - I H II i II i| *\\ IV VI Fig. 211. Degeneration and regeneration of a sectioned nerve fiber. I. Fibrillation of the axis- cylinder and swelling of the myelin. II. Segmentation of the axis-cylinder, swelling and displace- ment of the myelin. Proliferation of neurilemma cells. III. Disappearance of the axis-cylinder; myelin bulbs; proliferated connective tissue cells. Retrograde degeneration. IV. Formation of granular bodies; elimination of degenerated myelin by phagocytes. Soldering of fragments by proliferated connectve tissue cells. Retrograde degeneration. V. Beginning of regeneration in the central end. VI. Progression of the regenerated axis-cylinder in the empty sheath of the peripheral end. VII. Regeneration of the peripheral segment. Commencement of myelin reconstruction. (After Tinel.) on the cell body by the peripheral injury. The chromatic substance of Nissl's granules becomes modified so that they lose their staining power (chromatolysis), the cell becomes swollen, and the nucleus may assume an eccentric position. Peripheral to the wound the destruction of the tissue is complete, a fiber separated from its cell body being unable to continue to live. The changes which occur on the central side of the injury are usually of a temporary nature, and are restored by the process 848 CENTRAL NERVOUS SYSTEM of regeneration, by means of which the nerve cells reestablish a func- tional connection with the muscles. If such reestablishment is impos- sible, as in the case of amputation, disuse may be accompanied by an atrophy of the neurons so that the number of cells in the ventral horn of the cord decrease and their fibers degenerate. While degeneration of the nerve fiber is doubtless influenced greatly by the presence of the nerve cell body, a major part in the process is attributed to the cells of the neurilemmal sheath. The process of re- generation occurs coincidently with that of degeneration. It commences with activity of the nuclei of the neurilemma, which divide rapidly and form about themselves strands of protoplasm which replace the frag- ments of the degenerated fiber as it is absorbed. While it has been claimed that the fibers of the new nerve are actually formed by the neu- rilemma, it seems more probable that they grow out along the strands of this tissue from the central stump of the nerve, the neurilemma fur- nishing them with guidance, support, and perhaps nutrition as they grow. The importance of the neurilemma in the process is supported also by the fact that in the spinal cord and brain, where the neurilemma is absent, nerve fibers do not display an ability to regenerate. And herein lies an important principle in prognosis, for injuries to peripheral nerves may be repaired by regeneration, whereas the severance of fibers in the central nervous system results in a permanent injury which can be corrected only by developing the use of new paths for the control of the functions which are disturbed. It should be obvious that these facts form the basis for understanding the clinical treatment of peripheral nerve injuries. Although the periph- eral end of a severed nerve is doomed, its neurilemma will form the guiding path in the establishment of a connection between the new fiber and the muscle. It is consequently desirable to reunite the ends of the fibers in order to facilitate the regeneration by allowing the nerve fibers to establish a connection with the neurilemma of the peripheral stump. Even when the stumps are not brought into direct contact it is very remarkable to observe with what surety the new fibers grow out into the intervening cicatrix and establish a relation with the neurilemmse of the peripheral trunk. The phenomenon of degeneration has provided an invaluable tool for the study of the anatomical relationships within the nervous system and the function of its parts. By destroying the cells of a nerve center and observing which fibers degenerate, one can ascertain the distribu- tion of the fibers which arise from the center, while a study of the disturbances in the activity of the animal produced by the lesion tells us what functions are dependent on the neurons which are involved. Since degeneration does not extend beyond the synapses of the neuron THE NUTRITION OF NERVOUS TISSUE 849 affected by the lesion, it is possible to learn by this method just how far the axons from any group of nerve cells extend. The atrophic changes which occur in the nerve cell bodies after section of their nerve fiber may also be used to show from what nucleus any tract of fibers originates. The fact that regeneration does not occur in the brain or cord after degeneration forms the basis of a method known as suc- cessive degeneration by which the anatomical paths followed by reflexes may be made out. For example the scratch reflex, by means of which the dog relieves itself of irritation set up by parasites on the skin may be initiated through afferent impulses entering the cord in the thoracic region. The problem is to discover what neurons conduct these impulses to the motor neurons of the leg which lie in the lower segments of the cord. Section of the lateral column of the cord abolishes the reflex, but we know that in this column lie also fibers descending from centers in the cerebrum, midbrain, and medulla, and it is desirable to differentiate these from the propriospinal fibers whose cell bodies lie within the cord itself. By making a transection of the cord, above the afferent path of the reflex, all these fibers will be caused to de- generate. After a year the degenerated fibers will have disappeared and their paths will be occupied by a neuroglial scar. A section is now made of the lateral column just below the entrance into the cord of the afferent path of the reflex. Degeneration will now set in in those de- scending fibers whose cell bodies lie .between the primary and secondary lesion, and the course of these fibers may be traced to their junction with the motor neurons. By methods such as these the paths followed by the nerve impulses involved in the various functions of the nervous system, with which we are to become familiar, have been made out. The nutrition of the nervous system is a subject of great importance because of the fact that many manifestations of nervous disease are the result of nervous lesions, secondary to some primary disturbance in the circulation of the spinal cord or brain. Thrombosis or hemorrhage may produce a mechanical block in the course of vessels supplying the nervous tissue, or pathological modifications of the blood vessel walls may interfere with the metabolic exchange between blood and tissue. In myelitis and poliomyelitis the nervous lesions may have a close relation to the distribution of the blood vessels. In this connection the section on the circulation of the brain may be reviewed with profit (page 254). Certain regions of the central nervous system are supplied with blood from two sets of arteries so that occlusion of one does not interfere with the nutrition of the part. We will see that this is a characteristic of the foveal part of the visual center, which consequently is rarely affected by the vascular lesions of civil life (page 882). The importance 850 * CENTRAL NERVOUS SYSTEM of the nervous system to the existence of animals and its limited power of regeneration is correlated with the fact that in prolonged starvation its metabolism is maintained at the expense of other organs of the body with the result that, excepting the heart, it is the organ which least and last undergoes a diminution in Aveight. The Metabolism of the Nerve Fiber The all-or-none nature of the nerve impulse has given reason to be- lieve that the energy of the nerve impulse is derived from processes going on in each part of the fiber which it traverses. The refractory period in conduction is occupied with processes which restore the nerve fiber to its original state of conductivity. These processes constitute the metabolism of activity in the nerve fiber ; in addition to them we may expect a certain amount of metabolism concerned with the maintenance of the normal condition of the tissue. With regard to the chemical exchange in nervous tissue, practically nothing is known except regarding the consumption of oxygen, the output of carbon dioxide, and the effects of disturbances in these proc- esses produced by abnormal conditions of circulation and respiration. Some uncertainty exists regarding the production of carbon dioxide during the activity of the nerve fiber. It is maintained by Tashiro, 5 who has used a very sensitive method for its detection, that the carbon dioxide production of nerve is distinctly increased while it is being stimulated. On the other hand A. V. Hill 6 has failed to detect any heat production occasioned by the passage of nerve impulses through a nerve, although he used a method capable of detecting a change of one hundred- millionth of a degree in the temperature of the tissue. If any oxidative process had occurred in the nerve, such as might have resulted in the liberation of carbon dioxide, it should have been accompanied by a heat production readily detected by this method. The presence of dis- agreement on this point at least emphasizes one point, that the carbon dioxide production in nerve fibers, both at rest and in activity, is small and must contribute a minor share to the metabolic requirements of the body as a whole. The nerve fiber is dependent on a supply of oxygen to only a limited degree, its requirements being apparently small. A nerve trunk of the frog may retain its ability to conduct for three to five hours in an atmosphere of pure nitrogen, but at the end of that time its ability to transmit an impulse will come to an end. On supplying it with oxy- gen its conductivity is restored again in a few minutes. Oxygen is consequently necessary for the continued function of the nerve fiber, which can, however, be deprived of its oxygen supply for long periods without losing its ability to recover. THE NUTRITION OF NERVOUS TISSUE *851 Metabolism of the Central Nervous System The synapses are regions in which conductivity is modified by a va- riety of conditions. It is to be expected consequently that nutritional defects will influence the ease with which impulses may pass through these regions. The cell body is the nutritive center for the neuron as a whole. In the reflex arc, of which the synapses and nerve cell bodies form a part it is logical to expect that deficiencies in nutrition would show themselves more promptly than in the isolated nerve trunk. This is indeed, the case. While a nerve trunk will retain its conductivity in an atmosphere of nitrogen for several hours, the reflexes of the frog, deprived of oxygen supply, disappear in thirty minutes. In the warm- blooded mammal all reflexes disappear within a few minutes after fail- ure of the blood supply, and in man unconsciousness may be the im- mediate result of disturbance in the cranial circulation or of asphyxia. We have already seen how dependent the activity of bulbar centers, which control the heart beat, the blood pressure, and the movements of respiration, are on certain conditions, such as the hydrogen-ion concen- tration of the blood, which must be profoundly altered by disturbances in the circulation of the brain. In the same way the excitability of the spinal centers concerned with reflex conduction is modified by asphyx- ial conditions. Spasmodic contractions of skeletal muscles, or convul- sions, commonly precede death from asphyxia. These occur in animals in which the brain has been removed and are consequently due to the activity of the spinal cord. In spinal animals in which reflexes may be elicited with difficulty a mild degree of asphyxia frequently causes a reflex to appear, which could not be produced previously. Deeper as- phyxia will cause certain reflexes, i. e., the scratch reflex, to take place spontaneously. When death is threatened, general convulsions of the skeletal muscles of the trunk and limbs are invariably set up. These effects show that asphyxia may increase the excitability of the spinal centers of skeletal muscle and may result in the spontaneous contraction of the muscles which they supply. The heightened excitability is of short duration, however, if the asphyxia is complete, and passes into a condition of depression, followed by the complete failure of the re- flexes, which may be restored, however, if the respiration of air or oxygen is established in time (Mathison 7 ). The responses of the spinal centers for movement of skeletal muscle do not differ fundamentally from those of the medulla, which are con- cerned with the regulation of the circulation and respiration. The dif- ference in their behavior consists in the fact that the bulbar centers react to smaller changes in the condition of circulation. Thus the vaso- motor center reacts to thirty seconds of oxygen lack or to breathing 5 per cent of C0 2 whereas the spinal centers require two minutes of oxygen 852 CENTRAL NERVOUS SYSTEM lack or 30 per cent of C0 2 . The significance of the difference in sensi- tivity of the spinal and bulbar centers to changes in the composition or flow of the blood lies in the fact that stimulation of the latter tends to bring about automatically reactions which restore the blood to its proper condition. As a result the spinal centers are rarely confronted in healthy life with a circulatory condition which might modify their activity. After all activity of the central nervous system has been suppressed by anemia, or asphyxia, complete recovery may result if the circulation is restored to a normal condition soon enough. The respiratory reflexes are the first to reappear, then the excitability of spinal reflexes is regained, and finally cerebral function is restored. Less prompt recovery occurs if the circulation remains inadequate for a longer period, manifesting itself chiefly in a failure of the cerebrum to regain its normal function completely. A pregnant cat, which had been subjected to anemia of the brain and upper cord for 10 minutes, regained her ability to walk, to clean her paws, and to lap up water or milk, but her movements were poorly controlled. On the twelfth day she gave birth to kittens. To these she paid no attention unless one of them came in contact with her nose when she would lick it with her tongue. She allowed the kittens to suckle and fondled them with her paws when nursing very much as a normal cat would do, but if one of them wandered away she would make no attempt to bring it back. The picture was one of the automatic, reflex aspects of motherhood deprived of the discriminative attributes which depended on cerebral processes. The central nervous system exhibits a difference in the nutritive re- quirements of its different parts. It is interesting to see how long dif- ferent groups of nerve cells will resist complete anemia without losing their ability to revive. Considerable variations occur in different ani- mals, but the following figures may be taken as typical. They represent the time beyond which anemia cannot be extended without producing changes in the nerve cells which cannot be recovered from. Cerebrum, small pyramidal cells 8 minutes Cerebellum, Purkinje cells 13 minutes Medullary centers 20-30 minutes Spinal Cord 45-60 minutes Sympathetic ganglia 3-3% hours Myenteric plexus 7-8 hours The great susceptibility of the cerebral cells explains the ease with which consciousness is lost as the result of circulatory failure or asyphyxia. A prolonged condition of low blood pressure may result in the failure of the medullary centers because of the insufficiency of the blood supply. THE NUTRITION OF NERVOUS TISSUE 853 This is perhaps one of the limiting factors in the resuscitation of patients suffering from low blood pressure induced by hemorrhage or secondary traumatic shock. Bayliss has found that if the blood pressure of the cat is maintained at a low level for an hour or two the vasomotor center loses its reflex excitability and the respiratory center fails to maintain a ventilation of the lungs sufficient to support life. Restoration of the blood pressure by transfusion may come too late to cause these centers to recover. In the cat the respiratory center loses its power of revival before the vasomotor center, but the relative susceptibility no doubt varies in different species of animals. The cells of the outlying ganglia of the sympathetic system and, particularly, those of the myenteric plexus are able to withstand a prolonged disturbance in their blood sup- ply much better than the neurons of the central nervous system. For this reason a strangulated loop of intestine which appears hopelessly damaged, by the prolonged circulatory statis to which it has been sub- jected, may recover its normal function with remarkable success. 8 A close relation probably exists between the distribution of the cap- illaries in the nervous system and the nutritional requirements of its various parts. Measurements indicate that the grey matter, in which the nerve cell bodies lie, is much more richly supplied with capillaries than the white matter. The grey matter is more adequately supplied in the medulla than in the cord, and the same relation holds true between the white matter in these regions. Among the nuclei of the medulla it appears that sensory nuclei are, in general, more richly vascularized than motor nuclei. This relation is perhaps explained by the almost con- tinuous activity of the sensory neurons as contrasted with the more inter- mittent activity of the neurons concerned with motor acts. CHAPTER XCII THE RECEPTORS Having reviewed the fundamental conditions of conduction in nerve fibers and in the reflex arc, we are now in a position to consider the arrangements of neurons which form the basis of the principal aspects of central nervous activity. We will consider the afferent part of the reflex arc, the activity of which gives rise to the discharge of impulses over motor neurons in reflex activity, and to the phenomena of sensa- tion and discrimination which determine the nature of volitional acts. Disturbances in these arrangements give rise to the sensory symptoms of nervous disease. Such a consideration must start with the study of the receptors or sense organs. The Evolution of Specialized Receptors The receptor is a cell specialized in such a way as to be excited by minute changes in the condition of its environment. As a result of such excitation reflex actitity is set up which causes the animal to re- spond, usually in a way which is to its advantage, and sensations are produced which give information concerning the nature and position of the stimulus and its probable consequences. The primitive type of re- ceptor, which appears in the coelenterates and occurs generally in most invertebrates consists of an epithelial cell from which a fiber extends into the central nervous system. Such an arrangement persists in the olfactory sense organs in man. With the development of longer nerve trunks in the vertebrate nervous system the cell body is no longer found at the termination of the afferent fiber, but has taken up a position nearer to the central nervous system, in the spinal ganglion. The fibers of such an afferent neuron terminate peripherally in a number of fine branches which extend along the cells of the epithelium, forming a sense organ known as a free nerve termination. The sense of pain in man can be definitely associated with receptors of this type. The highest degree of sensitivity cannot be attained by such an arrangement. For this purpose special cells in the skin become modified into receptors, and about these terminate the fibers of the sensory nerves. Thus one cell serves as a receptor, while the other is concerned primarily with con- ducting the disturbance set up in the receptor to the central nervous system. The receptors of the ear, the eye, the sense of taste and probably other cutaneous sensations are of this type. (Fig. 212.) 854 THE RECEPTORS 855 Just as a division of labor has been necessary between the receptor and afferent fiber in order that the former may become as sensitive as possible to weak stimuli, so we find a specialization among the receptors, each being adapted to respond to some particular kind of physical or chemical change in its surroundings. Thus we may classify receptors with respect to the stimuli to which they respond most readily. We may speak of chemo-receptors which respond to chemical changes (taste, smell), tango-receptors which respond to pressure (touch), photo- receptors which respond to light (sight), phono-receptors which respond to sound (hearing), and caloro-receptors which respond to temperature changes (heat and cold). This does not mean, however, that these sense organs respond only to the type of stimulus to which they are especially Fig. 212. Evolution of the sense organs. I. Receptor of coelenterate. II. Type of receptor found in many invertebrates and in gustatory epithelium of vertebrates. III. Simple afferent neuron of vertebrate with free nerve terminations in skin. IV. Specialized sensory mechanism of vertebrate, typified by the organ 'of the sense of taste. It consists of specialized receptor cells in the integument, about which terminates the axon of an afferent neuron. (After Parker.) attuned. Pressure upon the eyeball may stimulate the retina, an elec- tric current may give rise to the sensation of taste, a temperature of 45 C. will excite the sense organs not only of heat but of cold and pain. But in the latter case, for example, the heat receptors alone are responsive to temperatures between 45 C. and the temperature of the body. The characteristic of the specialized receptor is that its threshold or lower limit of stimulation, is much lower for one type of stimulus than for any other. The Quality of Sensation and Its Local Sign Whatever the form of stimulus which excites a given receptor, the quality of the sensation is always the same. A temperature of 45 C. 856 CENTRAL NERVOUS SYSTEM produces a sensation, not of warmth, but of cold when applied to a re- ceptor for cold, and of pain when applied to a pain receptor. Pressure applied to the eyeball gives similarly a sensation of light. This is be- cause the quality of a sensation depends on the part of the brain to which the nerve impulses set up by the stimulus are conducted. All the fibers from a given type of receptor group themselves together in the spinal cord or brain stem and lead to a common group of cells, or sensory area, in the brain from which the sensation arises. The im- pulses traveling over these fibers are apparently the same in quality no matter what form of physical or chemical disturbance has set them up. Consequently on reaching the brain centers, no difference can be recog- nized between them, and they all give rise to a common quality of sen- sation. The quality of a sensation depends on two factors: (1) the low threshold of a special group of sense organs for the particular stimulus, and (2) the anatomical arrangement by which the impulses from such a group are conducted to a common region in the brain. From whatever source impulses reach this region the character of the sensation will be the same. These considerations give rise to the law of the specific properties of nerve, which is to the effect that, however excited, each nerve of special sense gives rise to its own peculiar sensation. The sensations which are set up by the stimulation of receptors not only have a definite quality, but are recognized as coming from a defi- nite region of space. They are said to have a local sign. Certain sensa- tions are referred to parts of our body, as we recognize when we say our feet are cold, our tooth aches, or our skin itches. Others are referred to objects recognized to be in contact with the body, as when we assert that a bed is hard, or a piece of ice cold. Still others are referred to distant objects, as when we recognize the color of a picture, the sound of a bell, or the odor of a flower. In all these cases the process of ex- citation is located actually in a sense organ within or at the surface of the body, and the phenomenon of sensation is set up somewhere within the brain. The reference of sensation is a psychological phenomenon depending on the past experience of the individual. We have learned, for example, that whenever impulses arrive in certain regions of the brain, giving rise to characteristic sensations, that they have come from stimuli set up in some particular group of receptors located in some par- ticular part of the body and excited by a disturbance which we have discovered can come only from some particular region in space. Thus experience tells us that a certain sensation is associated with an object in contact with the foot. Whenever the afferent paths from the foot are brought into play, the same sensation results, and we learn to asso- ciate the resulting sensation with the foot and refer all such sensations with accuracy to the foot. If the sensation is one commonly associated THE RECEPTORS 857 only with the obvious contact of some object with the foot, i. e., if it results from a combination of the sense of touch and sensations of other quality, the sensation as a whole is referred to the external object, and we speak of the object being cold, hard, etc. When components of such a sensation occur without the obvious contact of some object, the sensa- tion is referred to the part of the body in which the stimulation arises, and we speak of the foot being cold or in pain. Similarly we have learned to associate sensations resulting from stimuli arising in certain sense organs with objects at a distance from the body. We have learned for example to associate sensations which arise from stimuli falling upon particular parts of the retina with the particular regions in space from which light may come which can stimulate that particular retinal area. Unconscious of the mechanism of the optical system, or of the arrange- ment of the afferent paths of vision, we have simply learned that cer- tain visual sensations can always be attributed to objects occupying a certain position in space, and we can consequently assert with assurance that the upper part of a picture is blue. The local sign of sensation is con- sequently largely a matter of experience or learning. Reflex acts also bear an accurate local sign, although they may be carried out by parts of the nervous system apparently devoid of consciousness or of the ability to learn. Thus a frog, from which the brain has been re- moved will raise its hind leg and attempt to sweep away an irritating ob- ject placed on any part of the body, directing its foot to the point of stimula- tion with the utmost accuracy. A spinal dog will carry out a scratch reflex which differs considerably in its objective point, depending on the part of the back to which the stimulus is applied. How the mechanism has come about which enables stimulation of receptors in one part of the body to bring about these reflex movements of an appropriate sort is a fundamental problem in evolution which we cannot take up. The basis of the recognition of quality and local sign in sensation is of the utmost importance in interpreting the sensory manifestation of disease in the central nervous system. It rests on the fact that sensation of definite quality and local sign depends on the arrival at a certain sta- tion in the brain of impulses which are set up ordinarily in a group of sense organs specialized for the reception of one particular type of stimulus and located in a definite region of the body. Loss of sensation may result from the interruption of the path of conduction anywhere between, and including, the receptor and the sensory center in the brain. Sensations of definite quality may be set up by stimulation of the nerve fibers at any point along this path, or of the sensory center itself. One must exercise great care before accepting the location to which the patient attributes the source of sensation, for he has only the experience of healthy life to guide him. Consequently he may be misguided into 858 CENTRAL NERVOUS SYSTEM ascribing sensations to definite parts of the body, which in fact are hallucinations arising from some functional disorder of the brain which is affecting directly the sensory centers of the cortex. Referred Pain, The accuracy of sensory location seems to be corre- lated with the abundance of sense organs in different parts of the body and with the frequency of their employment. On the hands, the lips, and tongue localization is extremely accurate, for these organs are in frequent use in examining the nature and position of various objects. Localization is good on the soles of the feet, which are used to sound out the ground in walking. On the other parts of the limbs, the trunk, and particularly the back, stimuli can be localized only in a rather rough way. Particularly interesting is the phenomena of localization of sen- sations arising from the viscera. In healthy life these organs do not give rise to sensory manifestations, but in disease of the viscera acute pains may be set up. Because visceral sensations arise from organs within our body, we have no way of knowing just where the trouble lies and consequently have no basis for localizing the disturbance accu- rately. In the case of stationary organs such as the heart, the pain may be referred at times to the proper internal region, but in the case of movable organs such as the intestine, the reference is most inexact. DISTRIBUTION OF REFERRED PAIN FROM VISCERAL ORGANS (AFTER POTTENGER) The eight cervical segments are indicated by Cl, C2-C8; the twelve dorsal or thoracic segments by Dl, D2-D12; the five lumbar segments by LI, L2-L5; and the four sacral segments by Sac. 1, Sac. 2, Sac. 4. The areas of the head are indicated as follows: N nasal or rostral area; FN f ronto-nasal area ; MO medio-orbitalarea; FT fronto- temporal area ; T temporal area ; V vertical area ; P prietal area ; O occipital area ; NL naso-labial area; Max. maxillary area; Man. mandibular area; M mental area; L. S. Superior laryiigeal area; LI inferior laryiigeal area; TO hyoid area. AREA IN THE TRUNK AND LIMBS AREA IN THE HEAD Heart Lungs Stomach Intestine Rectum Liver Gall bladder Kidney and urethra Bladder (mucous membrane and neck) Detrusor vesicac Prostate Epididymis Testicle Ovary Ovarian appendix Uterus Neck of uterus Mammae Spleen Ventricles and aorta, N, FN, MO, FT 03, C Aur i c j cs p T T, V, P 03, C4 D4 D9 Y.'.NJ FN, MO, FT,' T,' V,' P D7 D9 FN, MO, T, V, P D9 D12 V, P, O Sac. 2 Sac. 4 C3, C4 D7 DIG FN, MO, T, V, P, D8 D9 T, V Dll LI Sac. 3 Sac. 4 Dll L2 D10 D12 Sac. 1 Sac. 3 1)11 D12 DIG 1)10 Dll LI D10 LI "9.c. 2 Sac. 4 D4 D5 D6 THE RECEPTORS 859 Very commonly pain arising in the internal organs is referred to some remote region on the surface of the body. This phenomena is known as referred pain, Such pains are sharp or aching in character, whereas pain which seems to come from the internal organs is dull or heavy (Head 13 ). The afferent nerves from the viscera apparently terminate in the cord in close association with afferent nerves from certain skin areas. Just how one group of afferents affects the other is not known, but the fact is that the sensation from the viscus is referred to the peripheral distribu- tion of those fibers which terminate in the same segment of the spinal cord. The nerves of cutaneous sensation have a definite segmental dis- tribution (Fig. 214) and consequently the reference of internal pain will be to one or another of these segmental regions. Not only is the visceral pain referred to these segmental areas, but these parts of the skin become hypersensitive, so that the sensation of pain and sometimes of heat and cold arising from them is greatly exaggerated. Thus the location of the referred pain and of hypersensitivity may be taken as an accurate indication of the internal situation of the source of irritation. The table on page 858 indicates the segmental distribution of referred pain from the major viscera. The sensitivity of the viscera is of a low order. When the sensitivity of a region of the skin is reduced to a similar condition by disease, sen- sations arising therefrom may be referred to other parts, just as the vis- ceral pains ordinarily are. This phenomenon is known as allocheiria, If the hyposensitive area is limited to one side of the body, the sensation arising from it is referred to the corresponding part of the other side of the body. If both sides are hyposensitive, the reference is to the next segment above or below. Like the reference of visceral pain, this condition must be attributed to the central relationship between the tracts carrying afferent impulses from symmetrical points on the skin and from neighboring segments to the sensory centers. When the mecha- nism of sensation for one part is depressed, the sensation is referred to the most closely associated normal region. Cutaneous and Deep Sensibility The physiology of receptors may be illustrated by a consideration of sense organs which have a general distribution throughout the body. These comprise the receptors of chief interest in practical neurology, and will serve to illustrate principles which apply as well to the spe- cial sense organs of the head (of the eye, ear, gustatory and olfactory epithelia), the consideration of which space will not allow. They may be divided into organs of cutaneous and of deep sensibility, depending on whether the receptors lie in the skin or in the internal parts of the body. We can recognize four primary qualities in the sensations to 860 CENTRAL NERVOUS SYSTEM which they give rise. These are touch, heat, cold and pain. When an area of skin is examined carefully it is found that these sensations are not elicited with equal readiness from all parts, but that definite spots exist which may give rise to one or another of these sensations. Some give rise to touch alone, others to the sensation of warmth, others to that of cold, and still others to a feeling of pain. Each spot evidently marks the location of one or more receptors for the stimulus in ques- tion. The spots giving rise to the four different qualities of sensation frequently do not coincide, nor do their numbers correspond, i.e., cold spots are more numerous than heat spots. In many regions of the body one quality of sensation may be lacking altogether. Thus pain is absent from the inner surface of the cheek opposite the second molar; it .alone occurs on the cornea. These facts furnish proof that the qual- A. Fig. 213. Cold spots {A) and heat spots (5) of an area of skin of the right hand. In each case the most intense sensations were experienced in the black areas, less intense in the lined, and least in the dotted. The blank areas represent parts where no special sensation of either kind was experienced. (From Goldseheider.) ity of the cutaneous sensations depends on the stimulation of sense or- gans which are specialized each for a different type of stimulus. Touch. Touch spots are stimulated by pressure which deforms the tissue. This may be seen to be so by dipping a finger into mercury when it will be found that the sensation arises only from a band of skin at the surface which is bent by the pressure of the mercury. The skin deeper down is pressed upon uniformly and consequently is not deformed and no sensation is set up. Touch spots are not uniformly distributed on all parts of the body, as we have already indicated. On the hairy regions it is found that they are most numerous at the base of the hairs, especially on the "windward" side. These spots are stim- ulated when the hair is bent, and since the hair acts as a lever, very THE RECEPTORS 861 light pressures will excite them. Consequently the threshold for touch is lowered on the hairy parts of the body. The examination of the sense of touch in the skin is important in the study of nervous disease. The presence or absence of the sensation is tested by touching the skin with a pledget of cotton or a soft paint brush. If the sensation is impaired but. not destroyed, more exact in- formation can be obtained by measuring the threshold, i. e., the smallest pressure which will stimulate the touch spots by repeating the test with a series of hairs or brushes of graded stiffness. The ability to localize the spot touched is known as spot finding 1 or one dimensional localiza- tion. Two dimensional localization is the process of recognizing that stimulation is being applied to two spots at the same time. If the points of a pair of compasses are applied to the skin at once they will be recog- nized as distinct points only if separated by a certain distance. This distance becomes greater as the number of touch spots becomes fewer, it apparently being necessary that a certain number of unstimulated spots should separate them before the points of stimulation are recog- nized as distinct. This process of recognizing the distinctness of two spots is called two dimensional localization. The compass test is use- ful in detecting deficiencies in the process which may result from de- struction of the afferent paths for the sense of touch. Very closely associated with the sense of touch are the sense organs, which give us knowledge of the position and movements of the parts of our body. These are located in the muscles, tendons and joints, and are doubtless stimulated by deforming pressures arising from the contrac- tion of muscle and the movement of the bones at the joints. The senses are tested by the ability of a patient to touch one part of the body with another, the eyes being shut and the affected member being moved into various positions. Quantitative measurements can also be obtained by determining through how many degrees a joint may be bent before the patient recognizes that the limb is being moved. The ability to recog- nize the position and movement of the parts of the body is called three dimensional localization. Heat and Cold. The heat and cold spots are stimulated by abnormal temperatures, heat spots being excited by temperatures above and cold by temperatures below that previously existing in the skin. Whether one or the other sensation will be felt depends not so much on the ab- solute temperature to which the heat and cold spots are brought as to the relation of this temperature to that of the skin. If the skin is chilled luke warm water will feel warm to it, whereas the same water will feel cool if the skin has previously been brought to a high tempera- ture. Thus if one finger is placed in cold water and the other in hot water for a few minutes and then both are thrust into water at an 862 CENTRAL NERVOUS SYSTEM intermediate temperature the sensation of warmth will be felt in the former finger and of cold in the latter. The sense organs thus become adapted to the conditions to which they are exposed, and become ex- cited again only by a change in these conditions. Adaptation is a quite general sensory phenomenon. It occurs prom- inently in the retina, with the result that an eye which has become used to the dark is dazzled momentarily by the ordinary daylight. Adapta- tion is also a marked feature of the touch sense, as is the experience of every one who has worn flannel underclothing or a plate of false teeth. It serves to protect the organism from the fatiguing effects of over- stimulation from conditions to which it is continually exposed. Sensory adaptation must be borne in mind not only when examining the senses of others, but in making judgments with our own sense organs. Thus if the hands are cold, the skin of another may feel warm and feverish, even though its temperature is not above normal. Pain is of particular importance to the physician, whose services are judged by the community largely by his skill in suppressing this sen- sation. Unlike the other receptors, the pain spots are not specialized for the detection of any particular form of stimulus, but may become excited by any condition which threatens to harm the tissues. Pain may be pro- duced by excessive pressure, the caustic action of chemicals, excessive ex- posure to light as in sunburn, temperatures above 45 C. and the effects of extreme cold, as in frost bite. The threshold for these stimuli is high, so that in the strengths at which they are ordinarily experienced the sensation of pain does not arise. All stimuli of sufficient intensity to threaten the welfare of the tissues and give rise to pain are called noxious stimuli. It was thought at one time that pain resulted from the overstimulation of any type of receptor. This conclusion was natural when one considered the great variety of conditions which might give rise to painful sensations. Undoubtedly the overstimulation of any sense organ may produce sensations of unpleasant character, but that specific sense organs for pain exist can no longer be doubted. This is shown by the presence of pain spots in regions from which other sense organs are absent, as the cornea, the absence of pain in regions in which other sense organs occur, as the inside of the cheek opposite the sec- ond molar, by the existence of special tracts in the central nervous sys- tem for the conduction of the impulses which give rise to pain, and by the fact that certain drugs such as cocaine may abolish the excitability of the receptors for pain without disturbing the reception of other sensations. Pure sensations, arising from the stimulation of a single type of re- ceptor, rarely occur in life except under the artificial conditions which we employ in the laboratory. The sensations which we experience are the composite result of the simultaneous combination of a variety of THE RECEPTORS 863 stimuli, acting together on sense organs which give rise to sensations of different quality. The nature of the resulting sensation is consequently modified. Water heated to 40 C. feels warm to the hand arid stimulates only the heat spots. At 45 C. the cold spots and pain spots are also excited, and the resulting sensation takes on a distinctly different qual- ity which we call "hot." Since pain is excited only by extreme in- tensities of stimuli which can set up other qualities of sensation, it is natural to find painful sensations varying considerably in their quality, depending on the sensations which occur in association with them. Thus a throbbing pain is due to the simultaneous pressure produced by dilated blood vessels. It is interesting to find that certain qualities of sensation are incompa- tible with one another. When stimuli capable of setting up two such incom- patible sensations occur at once, one sensation is suppressed or inhibited by the other. An interesting experiment is described by Head which illustrates this. The tip of the glans penis is supplied with receptors for cold and pain, but may be devoid of heat spots. If it is dipped into water at 40 C. the pain spots alone are stimulated and a disagreeable, painful sensation re- sults. If the temperature is raised to 45 C. the cold spots also are stimu- lated, the pain is displaced by a vivid sensation of cold. About the corona of the penis heat spots also occur. If this region is also immersed, the quality of the sensation changes to one of exquisitely pleasant warmth. If the water employed in the experiment is at a temperature higher than 45 C. the painful sensation persists and no sensation of warmth is felt. The sensations of pain and pleasant warmth are incompatible and cannot occur simultaneously. Which one will succeed in gaining control of consciousness and in suppressing the other depends on the relative strengths of their stimuli. An entirely analogous phenomenon occurs in the competition of incompatible reflexes for the control of a common motor path (page 947). Noxious stimuli give rise to an exceedingly impelling sensation and to reflexes which can dominate over any others which may be set up at the same time. The response to painful stimulation is of a protective character and it is imperative that such stimuli and their sensations should control the activity of the organism when they arise with in- tensity. On the other hand, if the noxious stimuli are near the thresh- old value of intensity, and little danger is threatened, it is an advan- tage that its effects be suppressed so that the organism may react with a discretion based on data derived from other sensations as well. The Distribution of Sensitivity in the Body Sensations of touch, heat, cold, and pain are felt generally throughout the surface of the body, with the exception of certain limited areas such as we have referred to, from which one or another quality of sensation 864 CENTRAL NERVOUS SYSTEM may be lacking. The presence of these cutaneous senses is obvious to any one, and the sensations which they arouse form the basis of many volitional acts. The presence of receptors in the deeper parts of the body, on the other hand, may be recognized only by careful introspec- tion, as when some pathological condition produces deep pain, or de- ranges the unconscious motor responses which depend on the receptors of deep sensibility. That afferent nerve impulses may arise from the muscles, connective tissue, tendons and joints is shown by a number of considerations. We are accurately aware of the position of our limbs and of any change in their position, whether made actively or passively. This knowledge does not depend on the cutaneous sensibility because the sense of position is not impaired when the cutaneous sensibility is destroyed by cutting the sensory nerves to the skin. When this is done sensations of pain and pressure may still be felt if the limb be pressed upon forcibly. Painful sensations which are referred directly to the muscles may arise during a muscular cramp or in the soreness which fol- lows the severe use of muscle groups which are unaccustomed to such activity. The fibers conducting afferent impulses lie in the trunks of the motor nerves, and when these are damaged muscular sensibility is lost. On cutting the dorsal root of a spinal nerve, it is found that a number of fibers in the motor nerve trunks to the muscles undergo degeneration. The receptors in the muscles, connective tissue, tendons, and joints may give rise to sensations of pain and touch and give in addition information which we shall see is invaluable in coordinating the movements of the body (page 914). The receptors of the internal organs of the trunk may give rise to sensations of pain, referred either to the inside of the body or to some region of the skin. That the sensibility of these organs is of a low or- der is attested by the fact that in healthy life we are rarely aware of their presence in spite of the almost continuous activity of many of these organs. The study of the distribution of the internal receptors for pain has been made by Lennander 14 during operations performed under local anesthesia. It has been found that the connective tissue about the ten- dons, the synovial membranes, periosteum, and perichondrium, are all very sensitive to pain. The parietal peritoneum is very sensitive to pain, especially from traction. On the anterior abdominal wall, at least, it is not endowed with end organs of pressure, heat, or cold. The mesen- teries are free from pain when cut, but are sensitive to traction. When free from connective tissue periosteum or perichondrium, the bone sub- stance and marrow, cartilage, and arteries and veins are insensitive to cutting, except those bony structures such as the maxilla and teeth which are traversed by sensory nerves. The muscles are insensitive to cutting, although excruciating pain may arise in them as the result of a cramp. THE KKCKPTORS 865 The brain substance and pia arachnoid are insensitive on the convexity of the brain and beneath the occipital bone, but pain may arise from it beneath the frontal bone and toward the zygomatic arch. In the thoracic cavity the visceral pleura, which is innervated by the vagus and sym- pathetic, is insensitive to the pressure of a stiff wire. The parietal pleura, innervated by the intercostal nerves, is the seat of pain which is accurately localized. The peripheral portion of the diaphragmatic pleura is innervated by the intercostal nerves over a band about two inches wide. Pain arising in this region is referred to the lower thorax, the abdomen and lumbar region. The central portion of the diaphragmatic pleura, innervated by the phrenic nerve, is the seat of pain referred to the neck. The lungs are insensitive, as is the heart, except under trac- tion. In the abdomen all organs receiving a nerve supply only from the sympathetic nerves and from the vagus below the branching of the re- current nerve have no sensation. The substance of the stomach, intes- tines, and liver may be cut into without causing discomfort. The fibrous capsule and parenchyma of the kidney do not give rise to sensation if the fatty capsule is removed. The bladder may be cut or pinched, but not pulled, without giving pain. It must be remembered that these observations have been made on individuals undergoing operation with local anesthetic. Sensitivity may have been modified by exposure of the organs to the air, or by spread of the anesthetic. They give valuable information from a surgical point of view, but do not tell us about the conditions which give rise to sen- sation during the normal or pathological activity of the viscera. It will be noted that the most adequate stimulus for visceral pain is trac- tion, which may produce discomfort when cutting is ineffective. It is probable that the pains of labor, menstruation, colic, gastric ulcer, etc., are set up by tension within the muscles of the uterus, or gastrointes- tinal tract, or to traction upon the mesenteries and their insertion into the parietal peritoneum by the arching of these organs when their walls attempt to contract while in a distended condition. The sensibility of the mucosa of the stomach has been made the ob- ject of extensive study, particularly in men possessing gastric fistulae. The mucosa is quite insensitive to pain, when pricked with a pin or pinched. Pain is felt as the result of such stimuli only when' they are sufficiently severe to spread to underlying structures. The mucosa is also insensitive to touch. A stiff test tube brush may be thrust into the stomach and moved about vigorously without producing any sen- sation. On the other hand, the temperature senses are represented in the wall of the stomach. The introduction of water below 10 C. pro- duces a sensation of cold, and above 50 one of heat. Intermediate tem- peratures are without effect. CHAPTER XCIII THE AFFERENT PATHS OF SENSORY IMPULSES - The insulation of conduction in the nerve fiber, and the fact that im- pulses can pass from neuron to neuron in one direction only, makes the arrangement of neurons in the nervous system a matter of great sig- nificance. We have already seen that the recognition of the quality and location of a stimulus depends upon the connection of each receptor in the skin with a definitely corresponding part of the brain. The ana- tomical arrangement of the paths conducting afferent impulses from receptors for each quality of sensation and from the different parts of the body is of importance in elucidating the sensory phenomena of disease. The Segmental Distribution of Afferent Nerves The vertebrate embryo develops as a segmented organism, at least so far as its nervous, muscular, and skeletal structures are concerned. The primitive segmentation of the vertebrate embryo is preserved in the arrangement of the spinal nerves of man. Each dorsal root of the spinal nerves contains afferent fibers coming from a definite segmental area of the skin. This may be shown by cutting all of the dorsal roots except one and then determining what parts of the skin retain their sensitivity. The areas innervated by adjoining roots overlap considerably, so that most parts of the skin receive a double innervation. Consequently damage to a single dorsal root does not produce a considerable loss of sensation. The areas supplied by each dorsal root agree closely with the segmental areas to which visceral pain is referred, with the exception that the latter do not overlap (Fig. 214). The areas of referred pain appear to represent the central portion of the segment innervated by a single dorsal root in which the overlapping is less considerable. The position of the skin areas do not correspond to the level of the dorsal roots which innervate them because of the downward slope of the spinal nerves. The skin areas, particularly of the lumbar and sacral roots, are somewhat below the corresponding segments of the spinal cord. In the limbs the segmental arrangement becomes obscure, until considered with respect to the segmental origin of the limb buds in the embryo. The areas innervated by the different roots contributing to the brachial and lumbar plexuses are nevertheless quite distinct. 866 THE AFFERENT PATHS OF SENSORY IMPULSES 867 The dorsal roots contain afferent fibers not only from the skin, but from the receptors of deep sensibility located in the muscles and viscera. We will see that the primitive segmentation of the muscles is consider- ably disturbed in the course of development (page 890). The afferent nerves from the muscles enter the cord, however, in the segment from which the muscles originally arose. The same is also true of the viscera. The heart, for example, which arises in the cervical part of the embryo has moved to a lower position, retaining, however, its innervation from Fig. 214. Diagram showing the segmental arrangement of the sensory nerves. (From Purves Stewart.) the cervical segments. Pain from the heart is referred consequently to the neck and arm. Because of this separation of the muscular and cutaneous distribution of the fibers composing a single dorsal root, injuries to the peripheral nerves may destroy cutaneous sensibility without affecting deep sensi- bility. This is particularly liable to occur because of the collection to- gether in nerve trunks, especially in the limbs, of fibers from adjacent dorsal roots which have a common (overlapping) distribution. When this occurs the cutaneous sensibility to light touch, two dimensional localization, heat and cold, and the prick of a pin, may be destroyed over 868 CENTRAL NERVOUS SYSTEM an area which is still sensitive to deep pressure and the resulting pain, and in which the sense of position of the part remains normal. Such a lesion is said to cause a dissociation of sensation. Dissociation between cutaneous and deep sensibility occurs only in the case of peripheral nerve lesions. Since the fibers of deep sensibility in muscles lie in the trunks of motor nerves, a lesion affecting it is usually accompanied by certain disturbances in motor function. Ascending Pathways in the Spinal Cord Our knowledge of the course of afferent impulses in the cord is gained by determining which tracts degenerate when the dorsal roots are cut, by destroying certain regions of the cord in animals and attempting to correlate the resulting degeneration with such disturbances in sensation as can be made out, and by studying the disturbances in sensation which result from injuries or disease of the cord in man. The latter method especially has been profitable because by it alone can accurate informa- tion concerning sensation be acquired (Head and Thompson, 15 Holmes 16 ). Unfortunately, however, an exact knowledge of the site of the lesion can- not usually be had in these cases, so that at present the course of the afferent impulses concerned with the various qualities of sensation from different parts cannot be stated with the precision which will ultimately be attained. On entering the spinal cord the course of the dorsal root fibers branch. A few fibers pass posteriorly in the fasciculus inter fascicularis of the dorsal column. The majority, however, extend upward in the ascending tracts of the dorsal funiculiis. As these proceed upward their number becomes less and less, because most of the fibers pass into the gray matter and terminate within a few segments of their point of entrance into the cord. As fibers enter the ascending tracts of the dorsal funiculus from higher spinal nerves, they lie lateral to those which have entered lower down, so that the funiculus takes on a laminated structure. Upon entering the gray matter of the cord the afferent fibers undergo synapse with association fibers which function as secondary afferent neurons conducting the impulses either to the motor neurons within the cord which are involved in spinal reflex acts, or leading to higher cen- ters in the brain, some of which give rise to sensation. The fibers of the secondary afferent neurons which are concerned with sensory im- pulses cross to the opposite side of the spinal cord and pass upward to the brain in the ascending tracts (dorsal and ventral spinothalmic) of the spinal lemniscus. Studies of the sensory disturbances due to lesions of the spinal cord in man indicate that on entering the cord the impulses which give rise to the various qualities of sensation undergo a charac- teristic grouping. All fibers conducting impulses which give rise to pain whether from the cutaneous or deep distribution of the sensory nerves THE AFFERENT PATHS OF SENSORY IMPULSES 869 SENSE or POSITION HOVEMENT [TOUCH PAIN HEAT COLD l r ig. 215. Diagram of the afferent paths followed by sensory impulses within the spinal cord and brain. 870 CENTRAL NERVOUS SYSTEM become grouped together in a single tract which is composed of second- ary afferent fibers situated on the side of the cord opposite to that by which they have entered. The impulses giving rise to the sensation of heat, and those giving rise to the sensation of cold are also each col- lected into separate tracts ascending through secondary afferent neurons on the opposite side of the cord. All impulses concerned with the tactile aspect of sensation, including the sensations involved in the recognition of position and passive move- ment are grouped together in the cord irrespective of their origin from cutaneous or deep lying receptors. These impulses, however, pass up the cord in two distinct groups. Almost all impulses concerned with the recog- nition of touch and pressure and the recognition of the location to which these stimuli are applied eventually pass on to secondary afferent neurons and cross to form a definite tract ascending the opposite side. But whereas the impulses from painful stimuli, heat and cold cross within five or six segments from their point of entrance into the cord, some of the tactile impulses may not have crossed until the upper cervical region is reached. Impulses upon which depend the recognition of two dimensional local- ization, the sense of position, and passive movement pass up the cord on the side on which they have entered it, in the fibers of the primary affer- ent neuron which lie in the dorsal f uniculas and terminate in the nucleus gracilis and cuneatus of the medulla. As a result of this arrangement of the afferent fibers in the cord, cer- tain characteristics are imposed upon the sensory disturbances which result from spinal cord lesions. A lesion which destroys the greater part of one half of the cord will obliterate the sensation of heat, cold, and pain on the opposite side of the body in those parts whose nerves arise below the level of the lesion. The ability to recognize touch and pres- sure may be unimpaired by such a lesion, because the impulses on which this depends cross over gradually as they ascend the cord, and conse- quently there exists at any one level two paths over which such impulses can travel. If the lesion is located high in the cervical region, a point at which the crossing over of these tactile impulses is complete, this qual- ity of sensation will also be lost from the opposite side of the body. Dis- turbances in the recognition of the position of the limbs and of passive movement and the distinctness of two points applied to the skin at once (two dimensional localization) occur, in the case of a considerable uni- lateral lesion, on the the same half of the body as the lesion, because the ascending tracts for the impulses involved in these sensations are uncrossed. On the contralateral half of the body dissociation occurs between heat, cold and pain, which are obliterated, and the tactile sen- sations which persist in part. On the homolateral half of the body the disso- ciation is between the sensations of two and three dimensional localization, THE AFFERENT PATHS OF SENSORY IMPULSES 871 which are obliterated, touch which is greatly impaired, and heat, cold and pain which remain normal. Lesions of more limited distribution may produce dissociations of a more simple character. Because the fibers conducting the impulses for pain, heat, and cold are grouped together into separate tracts for each quality of sensation a limited lesion may destroy one of these qualities without affecting the others. The close association of the three groups, however, makes it probable that a lesion affecting one will affect them all, so that in the majority of cases heat, cold and pain will all be affected together. When dissociation occurs as the result of spinal lesions, cu- taneous and deep sensibility of any given quality suffers alike, and as a result these dissociations have a distinctly different character from those due to peripheral nerve lesions. The loss of sensation which results from interruption of the afferent tracts in the cord affects parts which may be remote from the lesion. But if the destruction involves only the gray matter of the cord, it will not disturb those impulses which pass through either the crossed or uncrossed tracts of the cord at the level of the lesion, but only those whose paths entered the gray matter and cross the cord. As a result the sensation of heat, cold, and pain may be absent from a segmental area on the side of the body corresponding to the lesion, or if the destruction is more extensive, these sensations may be absent from a segmental band completely encircling both sides of the body, without any disturbance affecting the sensation of the lower segments, impulses from which have crossed the cord at a lower level and pass the lesion in the tracts of the lateral column. Segmental disturbances of this type are distinguished as the local effects of an intramedullary lesion. If the lesion is exten- sive so that both white and gray matter is destroyed on one side of the cord, a combination of the local and remote effects occur, with the re- sult that pain and the temperature senses are lost over the entire half of the body opposite to and below the lesion and also over a local segment at the level of, and on the same side as, the lesion. Afferent Paths in the Brain Stem On reaching the medulla all impulses producing sensations of pain, heat, cold, and touch (including one dimensional localization) have crossed the cord. They continue without interruption through the brain stem in the tractus spinothalamicus lateralis to terminate in the nuclei of the thalamus. In their course they are joined by impulses from ie cranial nerves, which have also crossed to the side of the brain stem opposite to that from which they have entered. Impulses which give rise to the recognition of posture, passive movement, and two dimensional localization reach the medulla on the side from which they have origi- 872 CENTRAL NERVOUS SYSTEM nated. They have been conducted along fibers of primary afferent neurons which terminate in the nucleus gracilis and nucleus cuneatus, from which arise the secondary afferent neurons which carry them to the thal- amus. These fibers cross the medulla immediately and take up a position in the medial lemniscus close to those which are conducting impulses for the sensations of pain, temperature, and touch. In their passage through the brain stem impulses for all qualities of sensation follow tracts which are grouped together closely in the opposite side from that in which they have originated. Lesions in the brain stem consequently tend to produce a complete, contralateral anesthesia. Impulses for any one quality of sensation are still grouped together in tracts which are distinct from those carrying impulses for other qualities of sensation. Consequently lesions of limited extent may abolish sensation of any one quality with- out disturbing the other qualities of sensation. Observations on cases of lesions of this sort indicate that impulses involved in the recognition of posture and movement, which have been associated with those for the two dimensional localization in their passage up the spinal cord, become separated in the brain stem, so that the power of recognizing posture and passive movement can be effected independently of the discrimination of two points applied simultaneously to the skin. Afferent Impulses Which Fail to Produce Sensation Those impulses which are destined to give rise to sensation will be traced in their course beyond the termination of the secondary afferent neurons in the nuclei of the thalamus, but we shall first consider the courses followed by those afferent impulses which do not reach the level of consciousness. These are of three types: (1) the afferent im- pulses of spinal reflexes, (2) the afferent impulses of visceral reflexes, (3) the afferent impulses of cerebellar reflexes. The Afferent Paths of Spinal Reflexes. The connections within the cord for spinal reflexes are undoubtedly very primitive. They do not, however, appear to have been worked out in many cases or in great de- tail. The simplest reflexes probably involve neurons lying in a single segment and on one side of the cord. The flexion reflexes which result from noxious stimuli applied to the limbs are of this type. In other cases the reflex pathway may extend through many segments, but lie totally within one half of the cord. An example is seen in the scratch reflex of the brainless dog. The method by which its course was worked out by Sherrington was described on page 849. It was found to consist of an afferent neuron connecting the skin of the shoulder with the grey matter of the cord in the upper thoracic region, a propriospinal neuron having its cell body in this part of the cord and its axon descending in the lateral column of the same side to connect with the motor neurons of the limb THE AFFERENT PATHS OF SENSORY IMPULSES 873 muscles which are involved in the scratching movements. Other reflexes are executed through paths which cross the cord. In addition to pro- ducing a flexion of the foot to which it is applied, a strong noxious stim- ulus causes an extension of the opposite limb. This is known as the crossed-extension reflex. Crossed reflexes also involve paths which trav- erse considerable lengths of the cord. Thus a flexion of the hind limb, produced by a noxious stimulus applied directly to it, is frequently ac- companied by a flexion of the fore limb on the opposite side of the body. The Afferent Paths of Visceral Reflexes, The visceral reflexes are con- cerned chiefly with the regulation of the circulation, respiration, and the motor activities of the internal organs. Such reflexes are set up by af- ferent neurons extending to the visceral organs themselves as well as to the skin and muscles. Visceral afferent fibers are found in the ninth and tenth cranial nerves and in the spinal nerves. Their cell bodies lie in the ganglia of the medulla and in the dorsal root ganglia of the spinal nerves. The fibers reach the viscera by following the course of the pre- and postganglionic fibers of the autonomic nervous system, passing through the ganglia and plexes without interruption. In spite of this close anatomical associa- tion with the autonomic nervous system, the visceral afferents are analo- gous in function and homologous in origin and structure with the affer- ent neurons from the skin and muscles. It is logical to classify the vis- ceral afferents with the latter, rather than with the autonomic system which is wholly motor in function. While certain impulses from the visceral afferents reach the sensory centers of the brain and give rise to visceral pain and the other sensory symptoms of visceral disease, the greater number never affect consciousness. They take part rather in the execution of visceral reflexes, which modify the activity of the muscles of the internal organs and regulate the blood flow through them in ac- cordance with the varying demands of their functional activity. The most important visceral afferents are those which modify the action of the cardiovascular and respiratory centers of the medulla. The depres- sor nerve and the afferent fibers of the vagus which extend to the lung are examples with which we are already familiar (pages 243 and 227). The centers regulating blood pressure and respiration are also influenced by impulses which have their normal origin in the receptors of the skin and which may be initiated by stimulating the nerve trunks in the limbs. Ranson and von Hess have studied the location of the afferent pathways in the cord over which such impulses travel. Two kinds of vascular reflexes were studied, pressor and depressor, the former being elicited by strong and the latter by very feeble stimu- lation of the central end of the sciatic and brachial nerves. They found that the pathways for the pressor and depressor afferent impulses were 874 CENTRAL NERVOUS SYSTEM quite different. Thus, after lateral hemisection of the cord, the depres- sor reflex obtained by weak stimulation of the sciatic on the same side as the lesion was normal, whereas it was greatly reduced when the sciatic nerve on the opposite side from the lesion was stimulated. On the other hand, the pressor reactions that were most conspicuously diminished were those from the sciatic on the same side as the lesion. The depressor fibers evidently cross in the cord, whereas the pressor do so only to a limited degree. Further it was found, after cutting across the dorsal part of the cord, that the pressor reflexes were interfered with but not the de- pressor, thus indicating that the former are transmitted either by the dorsal columns of white matter or by the gray matter of the posterior horns. To determine which, experiments were also performed in which the dorsal columns were alone destroyed and the results compared with others in which the tip of the dorsal horn was included. Since it was only in the latter experiment that any interference with pressor re- flexes was found to occur, it was concluded that the dorsal horn alone was concerned in the transmission of pressor impulses. Regarding conduction of the afferent impulses which in consciousness produce pain and of those concerned in the reflex changes in respiration, it was found that the dorsal horn of gray matter is not concerned, from which it is inferred that such impulses are conducted by the same afferent path that is involved in the depressor reflex; that is to say, as we have indicated above, the impulses cross in the cord to the opposite side and ascend in the lateral funiculus. The Afferent Paths of Cerebellar Reflexes. The cerebellum is con- cerned with the coordination of muscular movement, and must be in con- tinuous receipt of information concerning the changes in the position of the limbs which result from voluntary and reflex movement. The pri- mary afferent neurons of cerebellar reflexes are doubtless those which extend to the muscles, joints, and tendons along with the afferents of deep sensibility. Inasmuch as cerebellar injuries produce no loss of sensation, impulses extending into the cerebellum probably produce their effects without entering into consciousness. Consequently we can learn about the afferent paths of cerebellar reflexes only by inference from the anatomical arrangements and by observing the disturbances in co- ordinated movement which result from various lesions in the nervous system. The chief tracts in the cord which degenerate in an anterior direction and extend into the cerebrum are the tractus spino cerebellar is dor sails and the tractus spinocerebellaris ventralis. The fibers of both these tracts lie in the lateral column of the cord, and many of their fibers ex- tend directly into the cerebellum, the former by way of the inferior THE AFFERENT PATHS OF SENSORY IMPULSES 875 peduncle, the latter by way of the superior peduncle. Experiment shows that after cutting the tractus spinocerebellaris dorsalis a slight degree of ataxia and loss of tone in the muscles innervated from below the lesion may result, thus confirming the inference from the anatomical arrange- ment that this tract is one of the afferent paths of cerebellar reflexes. CHAPTER XCIV THE SENSORY CENTERS OF THE BRAIN Sensory experience and the recognition of the quality and location of stimuli acting upon the receptors of the body depends upon the arrival of impulses at certain stations in the brain which correspond to the re- ceptors in question. Knowledge concerning the location of the centers involved in the perception of sensations arising from each kind of recep- tor and from each part of the body has been gained by inference from the anatomical arrangements of the fibers connecting the sense organs with the various parts of the brain and from studies of the reactions of animals which have had certain parts of the brain removed. But this information is of slight value, since we can learn about a sensation di- rectly only through the verbal report of the person who is experiencing it. Consequently the important contributions to the sensory physiology of the brain have come from the clinical study of individuals who have suffered injury in some part of the cerebrum. Gushing 12 induced two patients in whom part of the brain was exposed to allow him to stimulate it while they were in a conscious state. As the result of the stimulation of the postcentral convolution definite sensory impressions were experi- enced, consisting of a . sensation of numbness, deadness, or tactual im- pressions. No muscular groups underwent movement unless the precen- tral convolution was stimulated, when no sensations w r ere experienced by the patient except those which accompanied the change in the posi- tion of the part that was moved. The sensations which were thus shown to be represented on the cortex are those of touch discrimination and those relating to the position and movements of the muscles. A com- prehensive analysis of sensory localization has been made by Head 9 from observations on soldiers suffering from the wounds of war. The Sensory Center of the Optic Thalamus It has long been recognized that the cerebral cortex is the site of cen- ters concerned in the perception of many qualities of sensation. Ex- periments by Goltz on a dog from which the cortex had been removed, suggested that certain subcortical centers might also give rise to sensa- tion, since this animal responded to various sensory stimuli, and when hungry gave evidence, so far as his actions were concerned, of experienc- ing sensations of hunger. Head's clinical observations have led him to 876 TlIK SKNSOKY CKNTKKS OF THE BRATN 877 assign a very important part in the origin of certain aspects of sensation to a center which he terms the essential organ of the thalamus. According to this view, afferent impulses may affect consciousness in two distinct ways on arriving in the thalamus. They may act upon this sensory center of the thalamus, or they may pass on to the sensory areas of the cerebral cortex. The presence of a sensory center in the thalamus, and the nature of the sensations aroused as the result of its activity, are indicated by the sensations which are experienced by in- dividuals in whom the sensory areas of the cortex have been destroyed. In their path through the cord and brain stem afferent impulses have been grouped on a strictly physiological basis, all impulses arising from a common type of receptor traveling together. On reaching the thalamus, a regrouping occurs on a psychological basis, the subsequent course of the impulse depending on the kind of appeal which it is to make to consciousness. The thalamic organ is the center for "aware- ness," responding to all stimuli capable of producing sensations of a change of state. The cerebral centers, on the other hand, are recipients of impulses which give rise to the discrimination of the detailed qualities of a sensation. A patient suffering from the destruction of the cerebral sensory area will exclaim, "Something is happening to me, I am being hurt," instead of "You are sticking a pin into me," because he fails to recognize the distinctive characters of the stimulus in question. The cerebral cen- ters, on the other hand, are concerned with the recognition of fine detail in sensation, enabling us to perceive not only the presence of a stimulus and its gross quality, but also to discriminate between stimuli of differ- ent intensities, to recognize the shape, size, weight, and texture of the stimulating object and to recognize the position of the hand as it explores its surface. Thus, to compare the sensations evoked by stimuli of each of the primary qualities in individuals who have lost the function of the cerebral centers with those of normal individuals, it is found that contact is recognized, but the distinction of differences in the intensity of the stimulus cannot be made by the unaided thalamus. The special aspects of tactile sensation, including the impulses involved in one, two, and three dimen- sional localization make no thalamic appeal, so that neither the location of the point stimulated, nor the position of the parts of the body are recognized. Painful stimuli* affect the thalamic center powerfully, giving rise to sensations of discomfort, deprived of the distinctive qualities which we recognize in painful sensations when the cortex is intact. The recognition of gradations of pain cannot be accomplished by the thala- mus alone. The thalamus can distinguish between heat and cold, as such, but makes no distinction between various degrees of warmth or coolness. Thus if a glass of hot water is placed in the hand of an in- 878 CENTRAL NERVOUS SYSTEM dividual whose cortical area corresponding to the hand is damaged, he will recognize the contact of the object, and will know that it is unpleas- antly hot. He will be unable, however, to perceive the roundness, size, or weight of the vessel, to know how hot it is, or to recognize the posi- tion of the hand which grasps it. The Sensory Centers of the Cerebral Cortex The Area of Cutaneous and Deep Sensibility. Impulses giving rise to cutaneous and deep sensibility which pass from the termination of the secondary afferent neurons in the thalamus to the cerebral cortex divide themselves into seven afferent streams, which may be affected by cortical lesions more or less independently. These comprise impulses concerned with the appreciation of (1) touch, (2) one dimensional local- ization, (3) two dimensional localization, (4) three dimensional local- ization, (5) pain, (6) heat, (7) cold. They pass to areas in the cortex which are more or less distinct for sensations arising from different parts of the body, and for sensations of different psychical quality. The sen- sory area for these sensations consists of the pre and post central convo- lutions, the anterior part of the superior parietal lobule and the angular gyri. Afferent impulses arising from one half of the body cross the cord or medulla in their ascent and affect this part of the cortex of the opposite side of the brain, so that the sensory disturbance which arises from a unilateral injury of the cortex expresses itself by a loss in sensory discrimination in contralateral parts of the body. Each of these parts is represented in a definite part of this sensory cortex. The lower ex- tremity is represented in the upper part of this area, the upper extrem- ity in the middle portion and the head in the lower portion. The extent of the cortical area corresponding to the different parts of the body is proportional to the functional complexity of their acts and sensations. Consequently the hands are represented by a very large region; the feet and face by extensive areas compared with which the representation of the proximal parts of the limbs and trunk is insignificant. The chances are consequently greatly in favor of an injury to the sensory cortex af- fecting one of these parts of the body, and it is very, rare that a prox- imal part of a limb is affected without the distal part sharing in the dis- turbance. In the sensory area for the hand, distinct regions exist for each finger and the corresponding proximal part of the hand. The area for the little finger adjoins the area for the lower extremity, with the result that loss of sensation involving the little finger also usually is accompanied by a disturbance of sensation in the foot. The ring, mid- dle, index fingers and thumb are represented one below the other in the sensory cortex, the thumb area adjoining that for the face, so that sen- sory disturbances in the thumb and face are apt to occur together. THE SENSORY CENTERS OF THE BRAIN 879 When sensory loss is occasioned on any part of the body as the result of a limited injury to the cortex, all aspects of sensation are not affected equally, with the result that the ability to respond with accuracy is lost more completely and over a larger area in the case of certain tests than in others. Such dissociations in the sensations which depend on cor- tical activity conform to two general principles. First: The more com- plex and difficult the psychical act required for an accurate answer to the test, and the more completely the test appeals to the cortical center in contrast to the thalamic center, the greater is the area of disturbed sensibility, and the more complete is the sensory loss in any one part of this area. Thus the extent and degree of disturbance is greater in the case of recognition of posture and movement than in the case of two dimensional localization, and is least for one dimensional localization, which obviously involves a simpler psychical judgment. Sensibility to touch is modified more than to temperature, and temperature more than to pain, because these sensations depend in this order on the cortex as contrasted to the optic thalamus. Second: The various aspects of cortical activity depend upon the in- tegrity of different parts of the sensory area. These aspects are (1) spa- cial recognition, typified by the sense of posture and movement, which is dependent chiefly on the part of the sensory cortex which occupies the precentral convolution; (2) the recognition of similarity and difference, on which depends the comparison of weights held in the hand, etc., which resides in the postcentral convolution; and (3) the recognition of the intensity of the sensation, whether it be of touch, temperature or pain, which is centered in the foot of the postcentral convolution, and in those parts of the sensory area lying behind this convolution. It appears that the sensory area of the cortex may be divided into certain horizontal zones which are each involved in sensation arising from stimuli acting on various parts of the body and into three nearly vertical zones involved each in the three fundamental aspects of discrimination. The cortical localization is based therefore, not on purely anatomical relationships by which each part of the body would be represented in all its sensory manifestations by a single part of the cortex, but on functional require- ments, according to which each psychical act of sensory discrimination is centered in a different group of cortical cells. Since these processes are distinct for the different parts of the body, these parts have a sepa- rate representation in the cortex, but the magnitude of the area devoted to each part of the body is dependent solely on the degree in which sen- sation arises from it. The Olfactory, Gustatory, and Auditory Areas. The cerebral cortex contains in addition to these centers for cutaneous and deep sensibility certain areas on which depend the perception of olfactory, gustatory, 880 CENTRAL NERVOUS SYSTKM auditory, and visual sensations. Very little of importance can be said of the physiology of the olfactory and gustatory centers, which are thought to lie in the hippocampal convolution of the median aspect of the temporal lobe, the former occupying the more distal position. The auditory center lies in the lateral side of the temporal lobe. Complete destruction of both temporal lobes causes deafness, but if one lobe only is destroyed, hearing is not impaired in either ear. It appears from this that the afferent paths from each ear lead to both temporal lobes, so that if the auditory center in one lobe only is injured, the center in the other lobe can carry on auditory perception for both ears. Consequently it is very unlikely that deafness will result from a cerebral injury. The Visual Areas. The visual centers are known with much greater precision. In order to explain the disturbances in vision which result from lesions in the visual centers, a word must be said about the formation of images upon the retina and the course of the afferent fibers which pass from the retina to these centers. The optical mechanism of the eye is such that the image of any object at which one looks is in- verted when it falls upon the retina. Consequently the upper part of the visual field falls on the lower part of the retina, the left half of the visual field falls on the right half of the retina, etc. The optic nerves from the two eyes meet at the optic chiasm, and there about half the fibers in each nerve cross to the opposite side of the brain and follow a course which leads to the visual center, which is contralateral to the eye in which they arose. The other half of the fibers in each nerve do not cross in the chiasm, but continue through the brain by a path which leads to the visual center on the same side of the body as the eye in which they arose. The remarkable thing about this arrangement is that the fibers which cross in the chiasm are those which arise from the median half of both retinae. As a result, the left visual center receives all im- pulses which arise from the left halves of the retinae of both eyes, and since these impulses are set up by objects whose images are inverted upon the retinae, this visual center is affected by the right half of the visual field. Conversely the right visual center is affected by the left half of the visual field. A consideration of Fig. 216 will make this rather complicated situation more clear. This arrangement is associated with binocular vision, that is, the simultaneous use of both eyes in viewing a single object. In the lower vertebrates, in which the eyes are on op- posite sides of the head and consequently have different visual fields the crossing in the chiasm is complete. The arrangement in man is of obvious importance in causing the images formed by the two eyes to effect simultaneously the same sensory centers in the brain. Because of this arrangement certain characteristics appear in injuries to the various parts of the optic tracts. A lesion located distal to the chiasm THE SENSORY CENTERS OF THE BRAIN Visual Field 881 Occipital Lobes Fig. 216. Afferent paths connecting the retina with the visual area of the cerebral cortex. will cause blindness in one eye alone. Lesions, such as those produced by the pressure of a pituitary tumor upon the chiasm, may destroy the function of those fibers which are crossing in the chiasm, and as a re- sult a type of blindness known as bitemporal hemianopsia results. This is a loss of sight in the temporal half of both visual fields, occasioned by destruction of the decussating fibers, which, as we have pointed 882 CENTRAL NERVOUS SYSTEM out, arise in the nasal half of each retina. Complete destruction of one of the visual tracts between the chiasm and the sensory area of the cortex produces a condition called homonymous hemianopsia, or blindness in the same half of the visual field of both eyes. Thus destruction of this part of the left optic tract which contains fibers from the left half of both eyes will produce blindness for the right half of their visual field. The visual area of the cortex is located about the calcarine fissure on the median and posterior surface of the parietal lobes. Its area is sufficiently great to make it probable that a lesion will involve only cer- tain parts of it, and as a result produce blindness in only part of the visual field. By correlating the position of lesions with the resulting loss of vision it has been possible for Holmes and Lister 10 to determine what part of the visual area corresponds with each part of the retina. They found that the center of each retina, or macula, is represented in the posterior part of the visual area. The superior quadrant of each eye is represented in the upper, and the inferior quadrant in the lower half of the visual area anterior to the center for macular vision. It has previously been held that the center of each retina was represented in the visual area of both sides of the brain. This was because in the lesions of civil life homonymous hemianopsia rarely affected the center of the visual field in the blind half of the eyes. Holmes and Lister found, however, in cases where the visual area of one occipital lobe was completely destroyed that vision was lost in the central part of the corresponding half of the retina quite as completely as in the more peripheral portions: They conclude consequently that the macula, like the rest of the retina, is not represented bilaterally in the cortex. The discrepancy in the condition, as they observed it, in wounded soldiers, and as it occurs in civil life, depends upon the fact that in the latter case the cause of the lesion is usually a disturbance of the circulation of the cortex as the result of thrombosis, hemorrhage, etc. The macular part of the visual area is supplied with capillaries from two sets of ar- teries, those of the median and of the lateral surface of the occipital lobe. An occlusion of the median supply would destroy the visual area for the peripheral half of the retina, but would leave intact the macular region which would be sufficiently nourished by the blood from the lateral arteries. Eiddoch 11 has noted that in case of injury to the occipital lobe, disso- ciations in visual sensations occur which are quite comparable to the dis- sociations which may occur in lesions of the sensory cortex for cutaneous and deep sensibility. These manifest themselves in the case of patients who are recovering from a functional disturbance of the visual areas. The first visual perception to appear is the recognition of the movement of an object in the visual field, which occurs long before the object as THE SENSORY CENTERS OF THE BRAIN 883 such can be recognized. A case is also described in which vision persisted in one half of the visual field on recovery from an occipital injury, and yet things which were seen quite well could not be oriented in space, and thickness and depth found no place in the visual perception. The sensory areas of the cortex and thalamus are the end points to which we trace the afferent impulses which give rise to sensation. We are not justified, however, in concluding from this that they are the regions in which the phenomena of sensation and consciousness occur. Bather should they be 'thought of as important junction points on the afferent side of the complex network of neurons which links up the various centers of the cerebrum and in which are carried out our mental processes, which give rise to consciousness. In a similar way the motor centers which we are to consider in the next chapter are the junction points from which start out the efferent impulses for voluntary move- ment. Sensory Hallucinations. It seems probable that under pathological conditions disturbances may be set up locally in the sensory centers which resemble closely those naturally occurring as the result of peri- pheral stimuli. Thus in Jacksonian epilepsy the irritation arising from a splinter of bone pressing upon one of the sensory centers may give rise to vague sensations or aura, such as flashes of light, loud noises, or tingling in the skin. If such disturbances resemble closely enough those occurring naturally, they may give rise to those conscious phenomena known as hallucinations and the sensory disturbance manifests itself as a definite vision, the sound of a bell or whistle, or a sensation of taste or smell. Use has been made of the fact that hallucinations may be set up by cortical stimulation, in tracing out the sensory areas in animals. If an irritant such as strychnine be applied locally to the cortex, the sensation is referred by the animal to the corresponding portion of the body and an effort made which is directed toward removing the irritant from this region. Thus irritation of certain regions in the cortex will cause the animal to shake one paw and attempt to brush away from it the supposed source of the sensation. CHAPTER XCV THE MOTOR AREAS OF THE CEREBRUM AND THE EFFERENT PATHWAY TO SKELETAL MUSCLE It is a debatable question whether motor acts are ever initiated by the nervous system except in response to some stimulus which sends afferent impulses into the brain or cord, or as the result of changes in the im- mediate environment of the nerve centers due to abnormal conditions in the circulation. There are, however, a large group of responses the nature of which is conditioned not only by the immediate stimulus which calls them forth, but by the previous experience of the organism. Situations which have existed in the past leave their mark upon the nervous system in the form of memories, associations and the like, and these determine how the animal or man will behave when new groups of stimuli, or sit- uations, arise. We shall see later, how previous experience may alter the nature of even involuntary responses to simple stimuli, when we take up the formation of conditioned reflexes (page 954). In the present place it will suffice to point out that unless we have considerable knowl- edge of the past experience of an animal it is impossible to predict how it will respond to certain situations. Responses of this type consequently are not obviously and invariably related to any particular stimulus, as are the simpler reflex responses, and consequently appear to arise spon- taneously in the nervous system. They are consequently called voluntary acts, which we shall take to imply that their nature and occurrence is related quite as closely to preexisting conditions in the nervous system as to the immediate situation which brings them forth. In animals from which the cerebrum has been removed, motor responses of a very perfect nature may still be carried out. A pigeon in this con- dition can walk, fly, coo, etc., quite normally, and if fed may live indefi- nitely. A dog from which the cerebral cortex is removed shows strik- ingly little difference in its behavior from a normal animal. Its equilib- rium is good, it moves easily, avoiding objects in its path, swims when thrown into water, feeds himself if food is brought into contact with his nose and rejects food of disagreeable taste. The reactions of such animals become almost strictly predictable, because they show no signs of being influenced by past experience. The pigeon is no longer fright- ened by a loud noise or sudden movement, nor does it suddenly become active for no obvious reason as a normal bird would. The dog shows no 884 MOTOR AREAS OF THE CEREBRUM 885 sign of affection or memory of its master, it does not recognize food as good to eat by its mere appearance, nor does it give signs of dreaming as normal dogs do. The mechanism by which the retention and association of the effects of past experience modifies behavior and produces acts of an apparently spontaneous and volitional nature evidently resides in the complicated meshwork of neurons which compose or connect the various parts of the cerebral cortex. The Motor Areas of the Cerebral Cortex Just as we saw in the last chapter that there were certain junction points, or sensory areas, by which afferent impulses are led into the Anus & Vagina Toes// Ankle ' Knee /5u/c(/5 centralis ,-Abdomen Chest Shoulder- Fingers sthum YES jaw Vocal cords Sulcus central is Mastication Fig. 217. Outer aspect of the brain of the chimpanzee, showing the position of the motor centers. Electric stimulation at the parts indicated causes coordinate movements of the corresponding mus- cle groups. (After Sherrington.) cerebral cortex so there are certain foci from which efferent impulses leave the cortex to initiate movement in the skeletal muscles. These are the motor areas of the cortex. Their situation and the groups of muscles related to each part of the motor area have been made out by two methods. In animals, and, under exceptional conditions, in man, the cortex may be excited locally, preferably by some method of unipolar electrical stimulation, and the groups of muscles brought into action may then be noted. By this method the most precise data has been obtained, especially in experiments upon the higher apes, the topography 886 CENTRAL NERVOUS SYSTEM of whose brains most closely resembles that of man. Conversely parts of the cortex may be removed and the distribution of the muscles which are then no longer under voluntary control may be determined. By these methods it is learned that the principal motor area lies in the cortex immediately in front of and extending into the fissure of Rolando. The Representation of Functional Activity in the Motor Area. When this area is explored with a localized electrical stimulus it is found that definite parts of the body are excited by the stimulation of definite parts of the motor area. There is then, just as in the case of sensory areas, a region corresponding to each anatomical part of the body. Movements of the muscles of the head are occasioned by excitation of the lower portion of the area; above it the neck, arms, trunk, and legs are represented in turn. Again, like the sensory areas, the representation is in respect to the functional use of the parts, so that each part of the cortex is the focus for impulses giving rise to an orderly act rather than to the contraction of a single muscle. This is shown by the fact that a weak, sharply localized stimulus gives rise to a coordinated movement such as the animal might make volitionally, in which certain groups of muscles contract while their opponents relax by virtue of a reciprocal inhibition. Thus stimulation of an appropriate spot in the motor area of the monkey will cause the fist to be clenched, an act which involves the setting of the extensors of the wrist, the relaxation of the extensors of the fingers, and the contraction of the flexors of the fingers. Because the thing represented in the motor area is a complete functional act, the areas related to each region of the body vary in size with the num- ber and complexity of the acts performed by these regions. Consequently the head and arm occupy a large part of the cortex because of the intricacy of the muscular acts which may be carried out by the face, tongue, and fingers. The leg likewise has a large representation when compared with the trunk, the functional reactions of which are obviously limited. In the cat the sensory and motor areas of the cortex for the head, limbs and trunk also coincide in their position, but in the higher apes and man they have become separated for the most part. Only those aspects of sensation which are concerned with the recognition of spacial relationships, particularly of the muscles and joints are repre- sented in the part of the cortex which lies in front of the fissure of Ro- lando, and in which the motor functions lie. This is an obvious relation- ship since a very close association is to be expected between the func- tions of coordinated movement and of recognition of the position and movement of the limbs, etc. The Visuo-Motor Areas. In addition to the large motor area there are two smaller areas, excitation of which gives rise to movements of the ocular muscles. One of them, the frontal visuo-motor area, is located MOTOR AREAS OF THE CEREBRUM 887 on the frontal lobe. Its excitation causes conjugate deviation of both eyes to the opposite side. The second area lies in the occipital lobe coinciding approximately with the center for visual sensation. Stimula- tion of it also causes eye movements, and these are not simply the result of referred sensations which result in movements initiated from the frontal visuo-motor center, since they persist after the latter has been destroyed. Consequently the occipital visual center is both sensory and motor in function. Physiological Bases of the Clinical Effects of Lesions in the Motor Area of the Cortex. Stimulation of the motor area of the cortex causes usually a response on the part of the muscles on the opposite side of the body. From this it appears that the connections between the two halves of the cortex and the muscles they control is a crossed one. In agreement with this is the fact which has been known since very ancient times that in- jury to one side of the brain frequently results in paralysis of certain voluntary movements on the opposite side of the body. But since cortical lesions are usually small in comparison to the area of the motor regions, the derangement more commonly affects muscular acts represented in a limited area of the cortex and as a result the move- ments of a single arm, of one side of the face, or of one leg are removed from voluntary control. Such a limited, unilateral paralysis is called monoplegia. Jacksonian Epilepsy, When the strength of the stimulus applied lo- cally to the cortex is great, its influence spreads so that adjoining motor cells are brought into activity and larger and larger groups of muscles take part in the response. This progressive " march" of the response to cortical stimulation is usually limited in its spread to the opposite half of the body, in this way differing markedly from the spread of spinal reflexes, which we have seen to occur when the stimulus is increased (page 842). The foregoing results obtained by experimental stimulation in animals, are very similar to the symptoms observed in man when the cerebral cortex is stimulated by the pressure on it of a meningeal tumor or a spicule of bone. Such stimulation causes contraction in the correspond- ing muscular area; the contraction then spreads to neighboring groups of muscles, and may ultimately involve the whole musculature of the body in a convulsive fit. This is known as Jacksonian epilepsy, and it is to be distinguished from ordinary epilepsy by its localized onset and its progressive march. Like ordinary epilepsy, however, the Jack- sonian type is usually preceded by a peculiar sensation of numbness or tingling in the area that is to show the first contraction. One of the great achievements of modern surgery is the cure of a Jacksonian epilepsy, by 888 CENTRAL NERVOUS SYSTEM opening the skull over the affected center and removing the menmgeal tumor or spicule of bone which is responsible for the stimulation. To enable the surgeon to locate exactly the position of the irritating body, it is necessary to examine the patient very closely as to the muscular group which is initially affected during the convulsions, and then to examine an outline map of the cerebral hemisphere indicating the position of the various motor and sensory areas as deduced mainly from experi- ments on the higher monkeys and verified by the experience gained by previous operations. Topographic maps indicating the surface mark- ings corresponding to the various convolutions of the cerebrum must also be used. In such operations the surgeon often has the opportunity of experimentally verifying the position of various centers. The entire cortical representation of motor acts does not follow this strictly crossed unilateral arrangement. Voluntary movements which involve muscle groups limited totally to one half of the body for their completion are generally represented in the cortex of the opposite side. Certain symmetrical muscle groups which commonly operate in syn- chrony such as those involved in mastication also are represented in and controlled by the contralateral cortex. On the other hand stimula- tion of the frontal visuo-motor area of one side causes deviation of both eyes towards the opposite side so that the movement of each eye must be represented on both halves of the brain. When the motor area of one side of the brain is destroyed, the movements of certain bilaterally acting muscles, such as those of inspiration, of movements in the diaphragm, intercostal and abdominal muscles and those of the larynx are not affected on either side. This may be due to a bilateral representation of these muscles in the cortex, as in the case of the ocular movements or to the fact that certain of the efferent fibers connecting the cortex with the lower motor neurons do not cross to the opposite side of the cord. The Efferent Pathway in the Brain and Cord The crossed connection between the cerebrum and the functional groups of muscles which it controls is due to the arrangement of the efferent neurons which link it with the motor neurons of the cord and brain stem. The most important tract involved in conducting efferent impulses from the motor areas arises from the large pyramidal cells (Betz cells) which are characteristic of the motor areas of the cortex and do not occur in its other parts. This is the pyramidal tract (tractus cortico-spinalis) . Its fibers converge from the various parts of the motor area as they approach the internal capsule of the cerebrum, and pass through the midbrain without crossing. At the level of the pons fibers MOTOR AREAS OF THE CEREBRUM 889 are given off which cross to the nucleus of the facial nerve on the op- posite side of the body. Similarly fibers leave the main tract in the upper part of the medulla to cross and connect with the hypoglossal nucleus. The main decussation of the pyramidal tract occurs in. the lower part of the medulla. A small bundle of fibers continue beyond this point uncrossed and descend the cord in the direct pyramidal tract, (tractus cortico-spinalis ventralis) in the ventral column of the cord. The fibers which enter the decussation in the medulla descend the cord in the crossed or lateral pyramidal tract (tractus cortico-spinalis lateralis) in the lateral column of the cord. In these tracts the pyramidal fibers descend to the level of the motor neurons of the peripheral nerves. The connection between cortex and muscle is consequently probably con- summated by no more than two nerve cells, the pyramidal and the lower motor neuron. It is quite inconceivable that other descending tracts in the cord are not involved in voluntary acts, but their relationships are not clearly enough understood to enable us to base our explanations of the motor symptoms of nervous disease upon their action. The Paralysis Resulting from Injuries to the Pyramidal Tract. The arrangement of the pyramidal fibers in their descent through the cord imposes certain characteristics on the distribution of the paralysis which results from lesions in different parts of their course. We have seen that lesions in the cortex rarely result in a complete destruction of the motor area, so that the resulting paralysis is usually limited to a few groups of muscles on the opposite side of the body. Such monoplegia would be accompanied by no loss of sensation, or by impairment in the special aspects of sensation in a limited region. In deeper parts of the brain the pyramidal tracts converge with the result that a lesion the size of a pea in the internal capsule will result in a complete destruction of the tract. Monoplegia is consequently rare at this level, the paralysis usually involving the entire opposite side of the body. Such a condi- tion is known as hemiplegia. If lesions in this region affect sensibility, the discriminative aspects of it will be destroyed, but those sensations which make a thalamic appeal will persist. Lesions in the brain stem as far back as the pons will similarly produce complete contralateral hemiplegia, accompanied usually, if they affect the afferent paths of sensation, by complete anesthesia on the paralyzed half of the body. At the pons the first group of pyramidal fibers cross to the opposite side to connect with the facial nerve. Lesions in the lower part of the pons consequently do not involve these fibers and the facial muscles of the opposite side do not share in the paralysis. This lesion, however, will interrupt the tracts from the opposite cerebral hemisphere which have crossed to join the facial nucleus on the side of the lesion. Consequently the face becomes paralyzed on the same side as the lesion. The decussa- 890 CENTRAL NERVOUS SYSTEM tion of fibers to the hypoglossal nerve produces a similar alternating paralysis of the muscles of the tongue and of the limbs in case of lesions in the medulla. The tongue is paralyzed on the same side as the lesion, the arms and legs on the opposite side, while the facial muscles of both sides escape. Such a condition is rare, however. Unilateral lesions below the decussation in the medulla which affect the pyramidal tracts cause paralysis which may affect those muscles whose motor neurons lie below the level of the lesion and on the same side of the body. If the af- ferent paths in the cord are affected also, sensation will be disturbed, and, as we have seen, heat, cold and pain may be lost over the opposite side of the body, the sense of position, passive movement, and two di- mensional localization will be impaired over the same side of the body, while touch may be unimpaired on both halves. The Peripheral Distribution of Efferent Nerves The motor neurons which conduct impulses for voluntary movement from the central nervous system to the muscles have their cell bodies in the ventral horn of the grey matter of the cord. Like the primary afferent neurons they have a segmental origin, and are distributed to those muscles which arose in the corresponding segments of the embryo. The segmental arrangement of the muscles has become greatly obscured dur- ing development, especially in the limbs, so that only in the case of the intercostal muscles does it remain perfectly evident. By the careful study of comparative anatomy and by correlating lesions, such as may occur in anterior poliomyelitis, which affect only a single segment of the grey matter, with the resulting muscular paralysis, it has been pos- sible to make out the segmental origin and innervation of the various muscles of the body. In man the distribution of the ventral root fibers according to seg- ments for the cervical and lumbosacral regions is as follows: 05 Deltoid, biceps, brachialis, supinators, rhomboids. Occasionally radial extensors. Barely pronator radii teres. 06 Pronators, radial extensors, pectoralis major (clavicular fibers), serratus anticus. 07 Triceps, extensor carpi ulnaris, extensors of fingers, pectoralis major. 08 Flexors of wrist and fingers. Tl Intrinsic muscles of hand. S3, 4 Levator ani, sphincter ani, perineal muscles. S2 Glutei, biceps, semitendinosus and semimembranosus. SI Intrinsic muscles of foot, tibialis posticus, and muscles of calf. L5 Muscles of ventrolateral leg (except tibialis anticus). L4 Extensors of leg and tibialis anticus. MOTOR AREAS OF THE CEREBRUM 891 The knowledge of the segmental innervation of the limb muscles, as furnished in the above table, is of value in the localization of spinal lesions. Paralysis of the extension movements of the wrist and fingers, along with the triceps, for example, usually indicates a lesion of the seventh cervical. It is more particularly in the trunk, however, that the segmental innervation of the muscles is evident. The innervation of the intercostal muscles being unisegmental, one may diagnose the level of a lesion of the upper thoracic region of the cord by observing their behavior during deep inspiration. If the fingers are placed in the in- tercostal spaces, the paralyzed muscles will feel limp and the fingers sink into the space during the act. Localization may also be shown by studying the paralyses of the abdominal muscles when the lesion involves one of the lower six thoracic segments. When the patient with a lesion of the eleventh thoracic raises his head from the bed or coughs, the rectus contracts, but the iliac re- gions bulge owing to paralysis of the lower portions of the obliques. Under the same conditions, when the ninth segment is involved the rectus contracts from about one inch above the umbilicus, whereas below this level it remains uncontracted, so that the umbilicus is pulled up. The motor fibers leave the ventral horn and pass through the ventral roots of the spinal nerves into the nerve trunks which supply the muscles. In the case of those roots which contribute to the cervical and lumbo- sacral plexus fibers from several segments may be combined into a sin- gle trunk. These trunks moreover break up into branches in which the fibers are sorted out so that motor fibers become separated from sensory fibers to the skin, and so that all fibers from several segments which are passing to neighboring groups of muscles may become combined. Consequently lesions to the nerve trunks of the limbs may show disso- ciation between the paralysis and the loss of cutaneous sensation, and at the same time certain muscles originating from several segments may be affected while other muscles derived trom the same segments remain under normal control. Spinal Reflexes In addition to voluntary movements, activated by impulses descend- ing through the pyramidal tracts from the brain, many reflex responses of the skeletal muscles may occur which owe their initiation to afferent impulses which do not affect consciousness. These impulses are conducted through the cord by propriospinal neurons, and reach the muscles by traveling over the same peripheral motor neurons which complete the path for voluntary acts. Consequently if paralysis is due to an injury to the motor neurons, either in the ventral horn of the gray matter, or along the peripheral course of their fibers, the muscles can be excited 892 CENTRAL NERVOUS SYSTEM neither reflexly nor by volition. Injuries to the pyramidal tracts, how- ever may cause paralysis without affecting the reflex response of the muscles. Consequently an examination of the reflex excitability of the paralyzed muscles will reveal whether the injury involves the motor neu- ron or not. Since, as we shall see (page 951) impulses from the cere- brum may have an inhibitant effect on certain reflexes, disease which cuts off this influence may cause an exaggeration of certain reflex re- sponses. Moreover since these reflexes are carried out over arcs which lie within definite segments of the cord the failure of a reflex may indicate in what part of the cord the lesion lies. The segments involved in the more important reflex tests are indicated in the following table : LOCALIZATION OF MUSCULAR REFLEX ACTS IN THE SPINAL CORD (After Starr) Pupillary reflex through the sympathetic: Dilatation of the pupil produced by irritation of the neck. Scapular reflex: Irritation of the skin over the scapula produces contraction of the scapular mucles. Biceps and supinator longus: Tapping their tendons produces flexion of the forearm. Triceps reflex: Tapping tendon produces extension of forearm. Scapulohumeral reflex: Tapping the inner lower edge of the scapula causes adduction of the arm. Tapping extensor tendons at the wrist causes extension of the hand. Tapping flexor tendons at the wrist causes flexion of the hand. Palmar reflex: Stroking palm causes closure of fingers; finger clonus. Abdominal reflex: Stroking side of abdomen causes retraction. Genital reflex: Squeezing the testicle causes contraction of the abdominal muscles. Patella tendon: Striking tendon at knee causes exten- sion of the leg; "knee-jerk." Achilles tendon reflex: Tapping the Achilles tendon causes flexion of ankle. Foot clonus: Extension of Achilles tendon causes flex- ion of the ankle. Plantar reflex: Tickling sole of foot causes flexion of toes, or extension of the great toe and flexion of the others. Fourth cervical to first dor- sal. Fifth cervical to first dorsal. Fifth and sixth cervical. Sixth cervical. Seventh cervical. Sixth to eighth cervical. Seventh to eighth cervical. Eighth cervical to first dor- sal. Ninth to twelfth dorsal. First to third lumbar. Second and third lumbar. First to third sacral. First to third sacral. First to third sacral. CHAPTER XCVI THE AUTONOMIC NERVOUS SYSTEM, OR THE EFFERENT PATH- WAY TO SMOOTH MUSCLES AND GLANDS The development of complex masses of association neurons in the cen- tral nervous system of the higher animals is associated with the acquisi- tion of more and more specialized motor mechanisms and of a diversity in the responses which these muscular structures can make. The organs con- cerned with nutrition have retained many of their primitive characters such as walls of smooth muscle and innervation by nonmedullated neu- rons, arranged in some cases, at least, as a nerve net. The nervous mechanism for the control of skeletal muscle has developed, consequently as an adjunct to the visceral system, and has reached such a dominating position that the latter has been rather neglected in the hands of neu- rologists. There can be little doubt, however, as the interesting book of Pottenger 18 suggests, that the study of visceral neurology will con- tribute greatly to elucidating the symptoms of disease. Because the neurons of the autonomic nervous system are organized somewhat differ- ently than are the efferent paths to skeletal muscle, and innervate organs of a different function, the mistake should not be made of thinking that it functions independently of the central nervous system. There is no evidence that afferent impulses play upon these neurons except after passage into the central nervous system. We have seen that afferent impulses from the viscera may give rise to sensory effects in the central nervous system (page 873). Similarly reflex change in the viscera may be set up through impulses having an efferent path in the autonomic nervous system, but originating from stimuli acting upon the surface of the body. The pupillary reflex elicited by pinching the neck is a case in point as is the psychic secretion of gastric juice, the voluntary emptying of the bladder, and the syndrome of gastrointestinal and circulatory changes which accompany great emotion. The autonomic nervous system comprises the group of neurons which carry impulses to the smooth muscles and glands of the body. It owes its interest and importance to the functions to which these tissues contribute (nutrition and reproduction), rather than to any supposed autonomy it may possess. The Organization of Efferent Nerves to the Viscera The reflexes in which the autonomic neurons take part are conducted over reflex arcs. While the afferent side of such arcs may arise from 893 894 CENTRAL NERVOUS SYSTEM somatic as well as visceral sources, it is interesting that the functional acts which these reflexes may perform find no representation in the motor areas of the cerebral cortex, and that in only exceptional cases can they be initiated voluntarily. The efferent side of the arc is interesting because of its anatomical organization. The motor path connecting the spinal cord with the smooth muscles and glands of the viscera consists of two neurons. The cell body of the first lies in the lateral horn of the gray matter of the cord. It is known as an internuncial neuron and its fiber, which is med- ullated, is called a connector fiber or preganglionic fiber. These fibers terminate in synapse with the cell body of the second neuron or effector Fig. 218. Diagram illustrating the different arrangements of the internuncial neurons of the voluntary and autonomic nervous systems. In both systems the afferent fiber terminates (by col- laterals) around a cell of the gray matter of the cord. In the voluntary system this cell is sit- uated in the posterior horn, and its axon travels to an anterior horn cell. In the autonomic system, on the other hand, it is located in the lateral horn, and its axon leaves the cord by the anterior root and travels by the white ramus into a sympathetic ganglion, where it connects with a nerve cell, whose axon forms the postganglionic fiber. (From Gaskell.) neuron, which lies in an outlying ganglion. These neurons give off fibers known as postganglionic fibers which extend to the effector which is innervated. The postganglionic fibers are not medullated (except in the case of the path to the sphincter pupillaB from the third nerve). Connector fibers are given off from three distinct regions of the cen- tral nervous system. The anatomical arrangements and physiological activities of these regions are distinctive. The bulbar outflow consists of connector fibers lying chiefly in the vagus, but also in the third, seventh, ninth, and eleventh cranial nerves. The sacral outflow consists of fibers, leaving the cord with the second to fourth sacral nerves, which join to form a common nerve trunk (the pelvic nerve or nervus erigens) Fig. 219. Diagram of the autonomic nervous system. The preganglionic fibers are in red, and the postganglionic in black. S.c., superior cervical ganglion; I.e., inferior cervical ganglion; T, stellate ganglion; S.p., great splanchnic nerve; (7, ganglia of solar plexus; m, inferior mesenteric ganglia; h, hypogastric nerves; N.B., nervus erigens. The arrows indicate the direction of nerve conduction. The numerals indicate the spinal nerves. (From Howell.) THE AUTONOMIC NERVOUS SYSTEM 895 on each side. Because they have certain characteristics in common these two outflows are classed together as the bubo-sacral (sometimes called the parasympathetic) division of the autonomic nervous system. The thoracico -lumbar outflow consists of connector fibers leaving the cord between the first thoracic and second or third lumbar segment. This out- flow is sometimes called the sympathetic division of the autonomic nervous system. The terminology applied to these systems is confusing because of the different usage of the same name by different authors. The following table indicates the classification adopted in this book, together with the synonymous terms in current use. THIS BOOK SYNONYMS (Langley) (Meyer) (Gaskell) Autonomic Nervous System Autonomic Nervous System Vegetative Nervous System Involuntary Nervous System 1. 2. Thoracico-lumbar Autonomic Bulbo-sacral Autonomie 1. Sympathetic 2. Parasympathetic 1. Sympathetic 2. Autonomic 1. Sympathetic 2. Enteral Oculomotor While the details of the anatomical courses of the fibers to the great variety of structures innervated by the autonomic system is beyond the scope of a textbook of physiology, it is appropriate to outline certain generalities concerning the arrangement of the connector and effector neurons which supply different organs. In addition to the obvious meth- ods for tracing the paths of nerve fibers by degeneration and by stimu- lating the roots of the motor nerves knowledge of the course of the connector neurons has been gained by the use of a special method dis- covered by Langley. This depends upon the fact that nicotine in cer- tain concentrations specifically blocks the passage of nerve impulses across the synapse between the connector fiber and the effector neuron without disturbing conduction in the course of nerve fibers unin- terrupted by a synapse. Consequently when nicotine is painted upon an outlying ganglion it is possible to determine, by stimulating the con- nector fibers, whether they pass through the ganglion without inter- ruption or not. In this way it has been learned that the motor paths in the autonomic system fall into three groups with regard to the position of the synapse between the connector and the outlying neuron. Position of the Effector Neuron. The outlying neuron lies wholly in the walls of the organs innervated by the vagus nerve, the sacral outflow, and that part of the thoracico-lumbar outflow which supplies the organs which have developed from the Wolfian and Mullerian ducts, i. e., the ureters, uterus, and vas deferens. The cells of Auerbach's plexus in the gastrointestinal tract represent outlying neurons from 896 CENTRAL NERVOUS SYSTEM the vagus, while the sacral plexus is composed of the postganglionie fibers from the sacral outflow. The remaining parts of the thoracico- lumbar outflow undergo synapse in one of two sets of ganglia. The seg- mental chain of sympathetic ganglia contain the cells of the outlying neu- rons of those thoraeico-lumbar paths which follow the course of the spinal nerves to smooth muscles and glands located in the skin and muscles, and of those which innervate the organs of the thoracic cavity. The typical arrangement of this group is shown in Figs. 218 and 221. The connector fiber reaches the ganglion through the white ramus and the postganglionic fiber passes back to the trunk of the spinal nerve in the gray ramus and follows this trunk to the peripheral structure which it innervates. Many connectors do not terminate directly in the ganglion of their own segment, but send collaterals forward or backward through the sympathetic chain to the other ganglia, where they connect with ef- fector neurons. The third group of thoracico-lumbar paths leads to the organs of the abdominal viscera, including their blood vessels. The connector fibers of these paths pass out over the white rami and through the segmental sympathetic ganglia without interruption to terminate in one of the mesenteric ganglia in connection with an effector neuron. The Double Innervation of the Visceral Organs, The innervation of the smooth muscles and glands is peculiar in that each effector may be acted upon by two neurons which effect its activity in opposite ways. Impulses from one neuron tend to increase the secretion of these glands, or augment the tone or degree of contraction of the smooth muscles, while impulses from the other neuron set up changes in the other direc- tion which inhibit or depress these activities. Because of this double innervation of structures supplied by the autonomic system, the activ- ity of any of them will depend on the balance which is struck between the effects of these antagonistic neurons. The details of this arrangement may be learned in the case of any particular organ from Fig. 221. In general it may be stated that the bulbar and sacral outflows do not overlap. In the gastrointestinal tract all parts cephalad of the ileocolic sphincter are innervated by the vagus and other cranial nerves, and are replaced below this point by the sacral outflow. At the same time, the thoracico-lumbar outflow supplies this entire tract below the cardiac part of the stomach with fibers which act upon it in the opposite sense. The action of the bulbo-sacral outflow on the gastrointestinal tract is excitatory, except for the ileocolic, and internal anal sphincters which are inhibited. It consequently favors the movement of food along the digestive tract and the secretion of the digestive fluids of the pancreas and salivary glands, and the emptying of the gall bladder. The thora- cico-lumbar outflow, on the other hand, tends to diminish the activities Heads Neck (Posting.; Medulla =Key PreGangiionic Sympathetic Post Ganglionic Sympathetic Pre GanglionicBulbo-Sacral (Para5ympathetic) Post Gang!ionicBulbo-5acral Splanchnic (Posrgang.) Coeliac Plexus & 5wp.Mes.Gang. minal Viscera (Preqang.) 'Pelvic visceral nerve Fig. 220. Diagram showing the main parts of the autonomic nervous system. For the sake of clarity several of the preganglionic fibers of the sympathetic autonomies are omitted, but the posi- tion of their egress from the cord is indicated in the side notes. The diagram shows clearly the distribution of the bulbosacral autonomic system by way of the vagus and the first, second and third sacral nerves. THE AUTONOMIC NERVOUS SYSTEM 897 of the gastrointestinal muscles and of the salivary glands and to close the sphincters of the lower tract. These systems of fibers also act in an antagonistic way on the heart, bladder, and interocular muscles. The bulbar outflow through the vagus inhibits the action of the heart, through the third nerve constricts the sphincter pupillae and probably inhibits the radial fibers of the iris. The sacral outflow through the nervous eri- gens causes contraction of the bladder musculature and inhibition of its sphincters. In each of these cases the thoracico-lumbar outflow acts on these muscles in the opposite sense. A large group of smooth muscles are innervated only through the thoracico-lumbar outflow. Some of these, i. e., the pilo-motor muscles and the muscles of the sweat glands, receive so far as we know only ex- citor fibers from this system. Others receive both excitor and inhibitor fibers from this system, so that an antagonism exists in the action of different neurons from the same outflow. This fact has been discovered by the use of a drug, ergotoxine, which prevents the excitor fibers from acting upon the muscles but does not impair the action of the inhibitor fibers. After the application of ergotoxine to these organs the stimula- tion of their nerves results in an inhibition of the muscles rather than the normal excitation. Inhibitory fibers are found in the thoracico-lum- bar supply to the cutaneous blood vessels of the bucco-facial region and of the kidney and their activity results in a vasodilatation in these re- gions. The smooth muscles of the uterus also receive inhibitory fibers from the thoracico-lumbar system, and in the virgin uterus these may dominate the activity of the organ, so that its muscles relax upon stimu- lation of their nerves. In the pregnant uterus, on the other hand, the constrictor fibers in these nerves predominate and a contraction follows their excitation. Further details of the organization of the Autonomic system may be obtained from the Monograph by Gaskell. 17 The Function of the Autonomic Nervous System When we examine the contribution which the autonomic nervous system makes to the organization of bodily activity, we are struck by .the fact that these nerves regulate the activity of a group of organs and tissues which possess a high degree of autonomy, so that their various functions can be carried out quite successfully when they are completely isolated from the central nervous system. The heart is perhaps the most complex organ under autonomic control, yet not only will isolated strips of this organ contract in the rhythmic fashion characteristic of the heart's activity but the entire organ will beat in a perfectly coordinated way when freed from nervous control. The tonic contraction of the arter- ioles, essential to the maintenance of blood pressure, is only temporarily deranged by the destruction of their nerves. The musculature of the 898 CENTRAL NERVOUS SYSTEM gastrointestinal tract carries out its rhythmic movements and maintains polarity in its action after it is separated from its extrinsic nerve supply. Even the mechanism for operating the pyloric sphincter does not de- pend on nervous connections extending beyond the gut wall. The secre- tion of the gastric, pancreatic and intestinal juices is also brought about by hormones of the secretin type so that nerves are unnecessary for this activity. Goltz and Ewald have succeeded in keeping dogs alive and in good condition for long periods after removal of the spinal cord below the cervical region. After such animals have recovered from the immediate effects of the operation, the skeletal musculature of the posterior part of the body is paralyzed and soon atrophies. The organs under auto- nomic control recover their normal function to a surprising extent. The tone of the blood vessels recovers, and they react to temperature changes much as a normal vessel would. The digestion becomes normal, defeca- tion takes place regularly, and the bladder is emptied periodically and spontaneously. A pregnant bitch gave birth to puppies a few hours after 9.4 cm. of cord had been removed, and suckled one of them suc- cessfully. In these animals it appeared that the organs innervated by the autonomic nervous system could function successfully after the cord, and with it the connector neurons, had been destroyed. The experiments suggest that the autonomic system may contain reflex arcs within itself, which do not pass into the central nervous system. A difficulty arises here because it has not been possible to demonstrate any connection in the outlying ganglia between the afferent fibers passing through the autonomic nervous system and the effector neurons. A type of reflex which may operate here is that known as the axon reflex. The only axon reflex which is known to function, except under laboratory conditions of stimulation, affects the cutaneous blood vessels, and causes a local inflammation to be set up in the skin when mustard is applied to it. This reaction was shown by Bruce to persist after cutting the dorsal roots of the spinal nerves and consequently could not be attributed to a spinal reflex. After the sensory fibers had degenerated, however, the effect could no longer be elicited. It appears from this that the mechanism depends on the integrity of the peripheral end of the sensory fibers. It is believed that these send off collaterals which connect with the cutaneous blood vessels. Impulses set up by the mustard in the sensory termination pass up the fiber to these collaterals and down them to the blood vessels where dilation is produced. This mechanism also explains the observation of Bayliss that stimulation of the peripheral stump of the dorsal root of a spinal nerve causes a local vasodilatation in the skin. A similar axon reflex has been found to occur in certain connector fibers of the thoracico-lumbar outflow which supply the smooth XP//O motor muscle Lachrymalrtand Submaxillar Submaxilldryublingual) ganglion ll/ocecal sphincte B/a Ves/ca/ sphincter-^ Urethra I sphincter *' r f \ mi. aiitji Pelvic(Hypoqastricjnteriliac) < sphincter Cranial and Sacral nerves motor = red inhibitory = blue Thoraclco-lumbar nerves mrPostganglionic fibers aredotted.thus c. N.XI Sup. cervical ganglion Thyroid gland Inf. cervical ganglion Z-Ansa subclavia -Stellate (/$? Thoracic) ganglion Sweat gland Vaso-motor fibers Pilo motor muscle Celiac (Semilunar) ganglion(5olarplex) Splanchnic nerves Sympathetic chain ll rtumbar splanchnics -Sup. Mesenteric ganglion s. Inf. Mesenteric ganglion Hypogastric nerves *. r i v, ^ i f ^ v^ w w _/ i | r v-,ii i i N. i ifw vy . . y p/exu5 . ( Vestcal $ reefs/ portions) Pelvic nerves (Nervus erigens) Fig. 221. Schematic representation of the autonomic nervous system. (From Jackson.) THE AUTONOMIC NERVOUS SYSTEM 899 muscles of the bladder, the blood vessels of the rectum, and the internal anal sphincter. These fibers send collaterals to the bladder through the hypogastric nerves. When one of these is cut and stimulated cen- trally, the muscles innervated by the collaterals in the other hypogastric nerve are seen to respond. There is no evidence, however, that this mechanism is brought into play in normal life. The organs supplied by the autonomic nervous system appear to be able to carry out their functions in an orderly way by virtue of their inherent properties, of the presence of the primitive nerve network which makes up their intrinsic nervous supply, and possibly by the assist- ance of simple axon reflexes through the postganglionic fibers of the thoracico-lumbar outflow. Since their reactions are simple and either Post, root gang.- Fig. 222. Diagram of an axon reflex in a sensory nerve fiber of the skin. A stimulus applied to the skin is transmitted by the sensory fiber (N), part of it going to the spinal cord (SO, and part of it passing by the collateral (C) to the arteriole (A), which it causes to dilate. strictly local or generally diffuse, a simple nervous mechanism suffices, just as it does for the simple activities in the coelenterates (page 829). Function of the Bulbo-sacral Division. The function of the connector fibers in the autonomic system would appear to consist largely in adjust- ing the activities of these structures as a whole to the conditions of activity brought about in the somatic musculature under the influence of the central nervous system. Correlation takes place between visceral activity, and somatic activity, so that the closest cooperation can ob- tain between the organs of the body. This is brought out by considering the way the three divisions of the autonomic system manifest themselves. The bulbar outflow is concerned with conserving the resources of the organism. Its action on the heart is to reduce its activity so that there 900 CENTRAL NERVOUS SYSTEM may be a reserve power for times of need. The gastrointestinal tract is brought into activity by the vagus which thus contributes to the nu- tritional processes of the body. When an animal has acquired food, cer- tain rather specific reflexes take place through the bulbar outflow which result in the psychic secretion of salivary and gastric juice and the re- flexes of swallowing, and thus initiate the process of digestion. The succeeding stages in the process can no doubt be executed by the intrinsic mechanisms of the gastrointestinal tract, but activity of the vagus fibers should be expected to counterbalance the inhibitory influence of the thorac- ico-lumbar outflow and thus to reinforce the action of the gastrointestinal muscles. The bulbosacral outflow brings about those reactions in the sphincters and muscular walls of the bladder and cloaca which result in the dis- charge of waste materials from these receptacles. Although these re- sponses may occur automatically when the spinal connections are de- stroyed, under normal conditions these acts are made volitionally at the convenience of the individual. In them a close cooperation occurs between the action of voluntary and involuntary muscles in which affer- ent impulses from these viscera play an important part. The mechanism for emptying the bladder illustrates this correlation between voluntary and involuntary action. Reflex micturition is set up when, through the accumulation of urine, the intravesicular pressure reaches 15 to 18 cm. of water. As the tension increases, rhythmic con- tractions of the bladder musculature occur which increase in strength and result in afferent impulses being set up in the pelvic nerves which pass to the sacral cord and to the higher parts of the central nervous system. Reflexes are thus set up through centers in the lower cord, which result in excitation of the sacral autonomic supply to the bladder, causing a contraction of the bladder walls and inhibition of the sphincter. The pressure within the bladder rises to 20 or 30 cm. and forces the urine through the neck of the bladder and through the urethra. Normally the emptying of the bladder is accompanied by a voluntary effort ini- tiated by the afferent impulses which ascend to the cerebrum, and there give rise to the sensations aroused by the distended vesicle as well as to efferent impulses which contract the respiratory and abdominal muscles, so as to increase the intraabdominal pressure, and help squeeze the urine out of the bladder. Reflex emptying of the bladder depends then on the development of a certain tension in the vesicular muscle. It may be in- hibited to a certain extent by voluntarily contracting the striated peri- neal muscles which help in the closure of the urethra, either before or during the reflex act. Micturition may be initiated before the bladder is full enough to start the reflex, either volitionally or by virtue of im- pulses arising through sensory nerves from other parts of the body. THE AUTONOMIC NERVOUS SYSTEM 901 These types of micturition must be set up by impulses which affect the spinal centers in much the same way as impulses arising from the blad- der itself. The bladder may consequently be emptied by three mecha- nisms, (1) by its intrinsic activity as in Goltz and Ewald's dogs; (2) by a purely spinal reflex through visceral afferent and efferent impulses, and (3) through voluntary effort acting on the spinal centers and re- inforced by the contraction of skeletal muscles. The former mechanism is relatively inefficient, as is seen in the behavior of the bladder during the failure of the reflex mechanisms which results from the shock of a spinal cord injury. The urine is retained until the intravesicular pres- sure becomes great enough to force the sphincter, and then it dribbles out feebly. The bladder is not completely emptied and the residual urine is apt to putrefy, giving rise to cystitis and other infections which constitute the chief danger in such injuries. If the local condition of the bladder remains good, the automatic emptying of the bladder may become periodic and complete whenever about 225 c.c. of fluid accumu- lates, even though the injury has completey destroyed the reflex con- nection between badder and cord. If the injury to the cord does not interfere with the reflex centers, recovery makes automatic reflex mic- turition possible and the bladder is emptied completely at periodic in- tervals as it becomes full, but is no longer under voluntary control (Fearnside, 19 Head and Riddoch 20 ). The Function of the Thoracico-Lumbar Division. The thoracico-lum- bar outflow, in contrast to the bulbo-sacral, inhibits the activity of the digestive tract and brings about changes in the organs of circulation which are appropriate to increased activity of the skeletal musculature and of the nervous system which controls them. Whereas the different parts of the cranial and sacral outflow are brought into activity sep- arately, so that the digestive secretions may be stimulated without the heart or iris being affected at the same time, and micturition may occur independent of defecation, discharge over the thoracico-lumbar system frequently has a diffuse nature, affecting all of the organs controlled by it simultaneously. A definite syndrome consequently results which is brought about under conditions of great emotion, as in fear, pain, and rage. It consist of acceleration of the heart rate, constriction of the arterioles in the skin and splanchnic viscera, inhibition of the mus- cular activity of the gastrointestinal tract, inhibition of the salivary se- cretion, erection of the hair and secretion of the sweat glands. These changes occur under conditions when severe muscular effort is apt to be exerted by the animal, and are appropriate because they tend to shift the circulating blood to the muscles and nerves of the body at the ex- pense of the viscera so that the maximum energy may be devoted to 902 CENTRAL NERVOUS SYSTEM muscular acts. For this purpose the diffuse activity of this system is an obvious advantage. In this connection the relation of the adrenal medulla to the thoracico- lumbar outflow is of interest. Its cells are derived in the embryo from the same neuroblasts which give rise to the effector neurons of the sym- pathetic ganglia. They are innervated directly by the connector neurons of the thoracico-lumbar outflow so that they are strictly homologous with the effector neurons of this system. It has been shown that without exception the effects produced on any organ by epinephrine, the secre- tion of these glands, is the same as that produced by the excitation of its thoracico-lumbar innervation. These gland cells may be considered as nerve cells which have changed their mode of acting on their effectors, but still produce the same effect through their secretion. If these glands are brought into action, as Cannon 21 has shown them to be during emotional conditions, they will reinforce the action of the thoracico-lumbar nerves and because their hormone is distributed through the blood, ensure the general, diffuse response characteristic of the action of the thoracico- lumbar system. It must not be supposed that the reactions of the thoracico-lumbar system always has this diffuse character. The regulation of vasomotor reactions is largely carried out through its neurons, and it is possible for vasoconstriction to occur in one part of the vascular bed without affecting other parts. The shifting of the mass of the circulating blood from one part of the body to another in response to the varying needs occasioned by the varying conditions of each organ must be controlled by these nerves. The local reflex response of the cutaneous blood vessels to temperature changes is a case in point (page 744). The Effects of Impulses from the Viscera upon Central Nervous Ac- tivity. Just as the visceral organs may be inhibited through the thora- cico-lumbar outflow in adaptation to the activity of the skeletal muscles, so visceral conditions may influence the state of the central nervous system and the muscles which it controls. We have considered the effects of afferent impulses from the viscera in producing pain (page 864). In the presence of visceral disease afferent impulses tend to produce "de- pression" in the central nervous system, so that muscular activity is avoided and the sufferer seeks retirement in which to recover. The dis- agreeable mental conditions produced by constipation are probably also nervous in origin, as is indicated by their prompt relief following a satis- factory movement of the bowels. The vigorous contractions of the stom- ach musculature in hunger not only give rise to this sensation, but make the subject restless and irritable. Certain reflex acts are reinforced by impulses set up during hunger, as the knee jerk. The cough attending diseases of the pulmonary region is believed by Pottenger 18 to be due to THE AUTONOMIC NERVOUS SYSTEM 903 irritability set up reflexly in the pharynx in much the same way as is the hypersensitivity of the skin in regions of referred pain. Spasms in skele- tal muscles are also produced in tuberculosis by reflexes arising from the lung. These are reactions depending on visceral afferent fibers. Reflex conditions arise from the viscera which affect other viscera through the autonomic efferents. Dmitrenko accelerated the respira- tion and pulse in dogs and raised their blood pressures by stimulating the stomach in various ways, as by distention with balloons. The vom- iting of pregnancy, menstruation, and the menopause, and of whooping cough, the gastrointestinal disturbances which may follow injury to the testis, etc., are attributed by certain clinicians to reflex effects carried out through the autonomic nervous system. CHAPTER XCVII MUSCULAR CONTRACTION Voluntary and reflex nervous activity expresses itself in the reactions of the muscles of the body. In order to interpret the normal working of the motor mechanism, and the abnormal manifestations which dis- ease of the nervous system imposes on the muscles of the body, the na- ture of the contractile processes in skeletal and smooth muscle must be understood. A muscle is an elastic body. That is to say, for every length to which it is stretched it will exert a definite tension on its origin and insertion, tending to pull them together until it has returned to its unstretched length. When a muscle "contracts," it does not assume a smaller vol- ume; rather it tends to change its shape so as to become shorter and thicker. The contracted muscle has become a body with new elastic properties, i. e., for each length to which it is stretched, it now exerts a greater tension on its origin and insertion than it did in the "uncon- tracted" condition. Consequently if opposition is presented to the short- ening of the stimulated muscle it will exert a tension on the opposing object equivalent to the tension necessary to stretch the contracted mus- cle to its resting length. The muscle's length does not change, and hence it is said to contract isometrically. If, however, the opposition is not strong, i. e., is due to a light weight, the tension developed on contraction will overcome the opposition, and the muscle will shorten, thus lifting the weight. Since the tension exerted by the weight is the same at all times during the contraction of the muscle, such a contraction is called isotonic. When it shortens against a weight the muscle assumes a new length at which it exerts a tension equal to the weight which is lifted. A muscle is consequently a machine for developing tension, and this tension may or may not do work in lifting a weight, or moving a joint, depending on whether or not the tension developed is great enough to overcome the opposition. The elastic properties of skeletal muscle may be modified in two dis- tinct ways which differ in respect to the energy required for the processes which cause the muscle to tend to shorten. A tonic contraction is one in which the processes which cause the muscle to change its elastic con- dition so as to take on a shorter length when at a given tension (or to exert a greater tension at a given length) are maintained with great 904 MUSCULAR CONTRACTION 905 economy. A tetanic contraction, or tetanus, is one in which the new elas- tic condition is maintained by processes which require a considerable expenditure of energy. The properties of these two forms of contraction will now be considered in some detail, together with the uses to which they are put by the organism. The Tonic Contraction of Skeletal Muscle The skeletal muscles of the body are normally maintained in a state of slight tension even when at rest. This condition, to which the word tone or tonus is applied, is due to the action upon the muscle of nerve impulses which come to it over reflex arcs which we will describe in the next chapter. When these reflex arcs are interrupted the muscles loose their tone, that is, their elastic properties change and they can now extend from origin to insertion without developing any tension. Limbs in which the muscles have lost their tone become " loose jointed" and readily assume under the influence of gravity, a variety of postures which a normal limb would not exhibit. Consequently it is easy to dis- tinguish between death and sleep by the postures of the limbs, since the tone of the sleeper's muscles is retained. Tonic contraction is thus seen to be connected with the maintenance of posture in the limbs. The posi- tion of the body, and particularly of the limbs is constantly changing, and with each change the muscles assume new lengths, maintaining meanwhile the same tension which they exerted before. To do this a new elastic state must be set up in the muscle so that the muscle can exist at its new length without exerting a greater tension on its inser- tion. The tone of the muscles consequently may be changed to fit the new position of the joint and to maintain this new posture. It is conse- quently called plastic tonus. (Sherrington 27 ). Since the greatest ten- sion must be exerted by those muscles which support the weight of our limbs, or the weight of the body which falls on the limb, these muscles have developed the greatest powers of tonic contraction. Consequently they become affected most in conditions which tend to increase the tone of the muscles. Tonic contraction, as contrasted with tetanus, is characterized by the economy with which the elastic state of the muscle is maintained. Roaf was unable to detect any difference in the respiratory exchange of cats during the highly developed tone of decerebrate rigidity when com- pared with the same animals in which all muscular contraction was abol- ished with curare. Evans has found that the metabolism was less after curare, but the gaseous exchange was clearly much less in the state of tone than if the muscles had been thrown into tetanic activity. Bayliss also found a slight heat production in muscles in decerebrate rigidity, which varied with the degree of tonic contraction, but very much less than 906 CENTRAL NERVOUS SYSTEM would have been produced by a tetanic contraction of the same height. Since the metabolic changes underlying tonic contraction are not great, it is not surprising that tension can be maintained by this form of con- traction for long periods without fatigue. It is a peculiar fact that although tonus is maintained in skeletal muscle only by means of a reflex arc, it is impossible to produce it by any known form of artificial stimulation applied to either the efferent or afferent nerve trunks. Under the normal reflex control the tonic re- action may involve either a shortening or a lengthening to a new state of tone, in which the tension remains the same. In the tonic contraction of skeletal muscle only a comparatively low degree of tension can be maintained. Consequently it does not require much force to move a joint out of the posture in which it is held by the tone of its muscles. In certain muscles the tension exerted by the tonic contraction is not inconsiderable, however. In the tonic rigidity which certain muscles assume after the cerebrum is removed the ex- tensors of the limbs may exert a tension well in excess of that required to support the weight of the animal. In order to exert the maximum tension, however, muscles must resort to the tetanic mode of contraction. Tetanic Contraction of Skeletal Muscle When a skeletal muscle is stimulated directly, or through its nerve with a single shock from an induction coil, it gives a momentary con- traction or twitch. On page 178 it was explained that if a series of stimuli are applied to a muscle the resulting twitches may follow one another so rapidly that the muscle does not relax between them, and a maintained or continuous contraction results to which the name teta- nus is applied. That tetanus is really a series of discrete contractions is shown by the nature of the electrical changes, or action currents set up by a muscle contracting in this way, for it is found that each part of the muscle becomes alternately positive and negative as each twitch is set up in it (see page 188). It is extremely unlikely that the volun- tary and reflex contractions of our muscles ever consist of single twitches, since a nervous discharge must usually consist of a series of impulses, in order that summation may occur and the resistance of the synapses be overcome. Whenever our movements are not due merely to changes in muscular tone, they are tetanic in nature. The action currents produced by the voluntary contraction of skel- etal muscle enable us to discover the rate at which component twitches of the tetanic contraction occur, which is about 50 per second (Fig. 223). This rate is determined by the fact that immediately after one contractile process has occurred a second cannot take place until a brief time, known as the refractory period, has elapsed. In the case of skeletal muscle one fif- MUSCULAR CONTRACTION 907 tieth of a second is required after one twitch has been initiated before the tissue has recovered sufficiently to respond to a second nerve impulse. Forbes and Rappleye 22 have shown that the rhythm is not due to the rate of discharge of nerve impulses from the higher centers, by demonstrat- ing that the rate of the oscillations of the electromyo graph is slowed by chilling the muscles of the arm in ice water. Since this procedure does f I f If Fig. 223. Electromyogram of the voluntary contraction of the flexor muscles of the forearm. (From Forbes and Rappleye.) not alter the body temperature as a whole, it cannot be supposed that the temperature of the centers in the nervous system are changed, or that the diminished rate of the rhythm is due to any change .in the rate of discharge of nerve impulses from these centers. They give evidence to show, moreover, that the rate at which nerve impulses follow one an- Fig. 224. The contraction of a single fiber of the sartorius muscle of the frog. The appearance before stimulation is shown at the left; during stimulation at the right. Note the tense appearance of the muscle fiber (X) , the slight pull on the deep blood vessel (I 7 ), and the change in the relative position of the surface capillaries which are indicated by the heavy, wavy lines. (From Fyisenberger.) other down the motor nerves is much faster than 50 per second. Many nerve impulses must reach the muscle while it is still refractory and con- sequently do not take effect upon it. Since tetanic contractions are composed of a series of twitches it is instructive to study the conditions which produce and modify this form of contraction in skeletal muscle. These muscles are composed of large numbers of fibers, each one of which may contract quite independently 908 CENTRAL NERVOUS SYSTEM of any of the others. This may be shown by applying an electric stimu- lus to a single fiber, by means of the very delicate technic devised by Pratt, 23 when it will be observed that this fiber alone responds to the excitation (Fig. 224). The fibers of the muscle are insulated from one another in such a way that the disturbance set up by the stimulus in one of them does not spread to any other. In this respect skeletal mus- cle differs markedly from cardiac muscle (see page 177). The unit of function in skeletal muscle is consequently the twitch of the individual fiber. The All-or-none Law. The question obviously arises how the strength of contraction of the fiber is affected by the strength of the stimulus ap- plied to it. As soon as it was realized that a single nerve impulse did not vary in strength, except under conditions which affected the con- ducting power of the nerve fiber, it was difficult to imagine how the nerve impulse could be made to alter the degree of contraction of the muscle fiber which it excited. It was consequently suspected that the all-or-none law applied to the activity of the individual fiber of skeletal muscle just as it does to heart muscle as a whole. The final direct proof of this view is supplied by the experiments of Pratt and Eisenberger who showed that when a single muscle fiber is excited its response is maximal if it responds at all. This fact is shown in Fig. 225, in which the movement of a droplet of mercury placed on the contracting fiber has been photographed. On increasing the strength of the stimulus no change occurs in the amount of contraction until the current strength becomes strong enough to affect an adjoining fiber. At this point the amount of movement increases by a definite step, and then continues at the new level until a third fiber is brought into action and another step-like rise in the record occurs. As the strength of stimulus is de- creased again the contractions fall off through the same series of steps. It is consequently believed that if a skeletal muscle fiber contracts at all, it does so to the full extent to which it is capable. Graded series of muscular contractions in response to graded strengths of stimuli, such as are shown in Fig. 45, are due to the fact that as the stimulus is in- creased in strength more and more fibers, each contracting maximally, are brought into play until finally all are excited and the contraction of the muscle as a whole becomes maximal and cannot be further in- creased. The adjustment of the strength and degree of muscular move- ment consequently depends on bringing into action the proper num- ber of muscle fibers. If the fibers were not insulated from one another, so that one could contract without the others joining in, graded muscular movements would be impossible and our skeletal muscles as a whole would act in an all or none way just as the heart does. Although the skeletal muscle fiber contracts to the utmost if it con- MUSCULAR CONTRACTION 909 tracts at all it must not be supposed that under all circumstances the maximal contraction is of the same magnitude. The ability of the fiber to develop tension varies from time to time and may be shown to depend on a variety of factors. The energy set free in the contractile process is greater, the longer the muscle at the time when it begins to contract. This is shown by the fact that a muscle which is stretched at the moment when it begins to contract is able to develop a greater final tension than an unstretched muscle. The heat liberated in the act of contraction, which measures the total energy set free, increases in a similar manner with the initial length of the muscle. This observation is of importance Fig. 225. The all or none nature of the contraction of a single fiber of skeletal muscle. The lower line represents the strength of the stimulus applied to the muscle, which rises to a maximum, and then is reduced to its initial level. The upper record is of the movement of a drop of mercury resting on the contracting fiber. The amount of contraction does not change as the stimulus is in- creased until the point A is reached, when a second fiber became excited causing a pronounced, step-like increase in the record. Later a fiber responded and produced another step. The same steps are observed as the stimulus strength is gradually decreased. (From Pratt and Kisenberger.) because it shows that the strength of the contraction is dependent upon the surface area of the contractile elements in the muscle fiber, which naturally becomes increased as the muscle is elongated, and not on the volume of muscle substance, which is unchanged by stretching. We have seen on page 217 the importance of this principle in enabling the heart to compensate for an increased load by dilating. The previous history of the muscle fibers also has a great influence upon the magnitude of the contractions of which each fiber is capable. We have seen on page 178 that if a muscle is excited to a maximal 910 CENTRAL NERVOUS SYSTEM contraction several times in rapid succession each twitch is somewhat higher than its predecessor. This phenomenon is known as treppe and is explained by the fact that chemical changes arising from one con- traction make the muscle better able to contract the next time. If the successive stimuli are continued for some time the height of the contractions soon reaches a maximum and then begins to fall off as the muscle becomes fatigued. Fatigue is due, in part at least, to the fact that after prolonged activity each fiber in the muscle is able to develop less tension when it contracts. The Chemistry of Tetanic Contraction. In order to understand the nature of fatigue, the chemical changes which occur during the activity of muscle must be considered. When a muscle is excited, energy is lib- erated which sets up a state of tension. The tension may result in doing external work, as in lifting a weight, or it may dissipate its energy as heat if the muscle is not allowed to shorten. In either case a supply of potential energy stored in the muscle has been drawn upon. It has been shown by Roaf that the hydrogen-ion concentration of muscle substance becomes increased at the moment of contraction, and chemical analysis shows that lactic acid appears in muscle as the result of pro- longed excitation. It is consequently believed that the liberation of lactic acid in the muscle substance is connected with setting free the energy for contraction. In the process of recovery which follows the activity of muscle when it is allowed to rest, it is found that the lactic acid disappears provided a supply of oxygen is available. Since at the same time carbon dioxide is set free from the muscle, one might suspect that the disappearance of the lactic acid is due to its being oxidized to carbon dioxide and water. This, however, cannot be the case, for it is known that after the repeated fatigue and recovery of an isolated muscle the total quantity of lactic acid which may be extracted from it is undiminished. In other words lactic acid does not disappear from the muscle during rest, but is re- stored to the condition in which it occurred before contraction took place. Further evidence that lactic acid is not oxidized is afforded by the fact that the disappearance of 1 gram of lactic acid from fatigued mus- cle is accompanied by the production of 450 calories of heat, whereas the oxidation of 1 gram of lactic acid would set free 3700 calories. Apparently the oxidation of some other substance is necessary in order to restore lactic acid to the percursor condition, and to replace the po- tential energy lost in the contractile process, and in the course of the oxi- dation of this substance carbon dioxide is liberated and heat is given off. The nature of the substance oxidized is not definitely known, but it is presumed from the high respiratory quotient of muscular work that it is chiefly carbohydrate. (Bayliss, 24 Fletcher and Hopkins. 25 ) MUSCULAR CONTRACTION 911 Treppe and Fatigue. Applying these facts to the phenomenon of treppe and fatigue, we see that the continued use of a muscle will be attended with an increase in the hydrogen-ion concentration as the re- sult of the liberation of lactic acid in the muscle substance. For all tis- sues there is an optimal concentration of hydrogen-ions at which their activities are carried out to the best advantage. The initial change in the hydrogen-ion concentration which accompanies the first few contractions brings the muscle into a more favorable condition and treppe results. On further production of lactic acid the optimal condition is exceeded and the muscle fibers become successively less and less able to perform their work. If the muscle is supplied with an adequate circulation a point is soon reached at which the excess of lactic acid is restored to the precursor condition as fast as it is formed, by virtue of the oxygen supplied by the blood, and beyond this point fatigue does not proceed further, successive contractions following one another for a long time with undiminished force. The muscle is then said to have reached a fatigue level in which the con- structive processes during the rest between contractions just balance the destructive processes during activity. If the circulation is inadequate, or lacking, as it is in an isolated nerve muscle preparation, the increase in the hydrogen-ion concentration goes on as the result of the accumula- tion of lactic acid until the contraction of the muscle becomes impossible. The fatigue of muscle is consequently due to the accumulation of lac- tic acid and probably other waste products in the muscle substance, and in protracted exertion to the using up of the materials which upon oxidation restore the energy lost in the act of contraction and replace the lactic acid in the condition in which it existed in the rested muscle. Under normal circumstances skeletal muscle is protected from the development of any harmful degree of fatigue. This is because its ac- tivity is initiated through the nervous system, parts of which become fatigued long before this condition comes on in the muscles. We have already seen that the nerve fiber is unfatigable. The synapses, on the other hand, and the myoneural junction are easily fatigued and cease to conduct excitations to the muscles long before the muscle it- self becomes too fatigued to respond to direct stimulation. A further protection against fatigue in protracted muscular activity is afforded by the fact that the threshold of excitation of the individual muscle fibers is raised by repeated stimulation. It is quite possible that as the result of this tendency each fiber may fail to respond to the nerve impulses reaching it as soon as it becomes fatigued, and consequently it has an opportunity to rest, while other fibers in the muscle carry on the work in hand. The falling off in the height of the successive con- tractions of a muscle which is being fatigued is probably due not only to the reduction of the degree of contraction of which each fiber is capable, 912 CENTRAL NERVOUS SYSTEM but also to the cessation of activity on the part of many fibers as their thresholds rise above the level of the exciting stimulus. Smooth Muscle The smooth muscles of the mammalian body differ markedly in their histological appearance from the skeletal muscles. Their origin in de- velopment is also different as they arise from the mesenchyme, and not from the mesodermic somites as the skeletal muscles do. It is not sur- prising consequently to find that the properties of smooth muscle differ markedly from those of the skeletal muscles. The contraction of smooth muscle is sluggish and never develops very great tension. In this respect it resembles closely the tonic contraction of skeletal muscle, as contrasted with the tetanic contraction. The most outstanding feature is the ability of this tissue to alter its tonic condi- tion so that it may exist now at one length, now at another, under equal degrees of tension. This is seen to be a most important property when it is considered that smooth muscle forms the walls of the various hollow viscera and that these organs must constantly alter their capacity to fit the varying volume of their contents. In the stomach and urinary bladder, especially, the muscular walls must be capable of relaxing as the organ becomes filled, so that the tension exerted on the contents may not increase unduly. The pressure in the urinary bladder is much the same whether its contents be 50 or 150 c.c. of urine. Similarly after injecting 400 c.c. of water into the stomach of a dog the pressure within it returns almost immediately to its original level through a change in the condition of the gastric musculature. The conditions in the blood vessels differ only in degree, for here the smooth muscles adjust their tone to the requirements of maintaining a uniform pressure of the blood, and differ from the smooth muscles of the abdominal viscera only in the relatively high degree of tension which they can maintain. Like the tonic contraction of the skeletal muscles, the contraction of smooth muscles is maintained with very little expenditure of energy and consequently without fatigue. The sphincters of the gastrointestinal tract and bladder remain closed almost continuously and the muscles of the arterioles support the relatively great pressure of the blood unremit- tingly and without becoming exhausted in the least. The smooth mus- cles which hold the shells of the mollusca closed can support great weights. The muscle of the fresh water clam, Anadonta, can support a weight of 3 kilos for three hours without consuming any oxygen in excess of its re- quirements when at rest. The economy of this is seen when it is considered that the gastrocnemius of the cat consumes when supporting a similar load by tetanic contraction 4500 times as much oxygen as does the Anadonta muscle. MUSCULAR CONTRACTION 913 Like the heart, smooth muscle is capable of setting up automatic con- tractions of a rhythmic nature even when removed from the body. This automatic rhythmicity is probably a fundamental property of the smooth muscle cells, just as it is of heart muscle, for it has been shown by Gunn and Underbill 26 in the case of intestinal muscle that it continues after the nerve plexus which lies between the layers of muscle has been removed. It is also to be seen in isolated smooth muscle cells grown in tissue cultures in which nervous elements can be observed to be absent. CHAPTER XCVIII POSTURAL COORDINATION The maintenance of posture is accomplished by the tonic contraction of the skeletal muscles. Not only must the muscles assume a new tonic state in each new position of the body, but the tone of the muscles must alter rapidly and in harmony with voluntary and reflex contractions of a tetanic nature. Each voluntary act must also be made with reference to the preexisting posture of the parts involved. The smooth continuity of the normal movements of the body is dependent on the mechanism which correlates postural with voluntary and reflex tetanic contraction, and disturbances in this mechanism give rise to incoordinated movement, ataxia, and the abnormal states of contraction seen in the muscles of sufferers from nervous disease. The Reflex Adjustment of Tone. The tonic contraction of skeletal muscle is maintained only in the presence of a reflex arc. The afferent neuron of this arc arises from the muscle itself. This is shown by the fact that after cutting all the nerve trunks to neighboring muscles and the skin the tone of the muscle persists, but it disappears at once on sever- ing the dorsal root through which the afferent fibers from the muscle pass. The receptors of the afferent neuron which lie in the muscle substance are called proprioceptors, and the reflexes which they ini- tiate proprioceptive reflexes. The efferent path of the arc is completed by the motor neurons of the muscle. In the presence of this arc tone is not only maintained, but plastic changes in tone may be induced by impulses traveling over it. Two types of reaction are recognized which bring about changes in the postural tone of skeletal muscle. If the knee of a spinal or decere- brate mammal is flexed by pushing the foot with the hand, the resistance which the tone of the extensors opposes to the movement may be felt to give way, almost suddenly, as the joint moves into its new position. On releasing the limb, it will now remain in the flexed condition as the result of a reflex change in the tone of its muscles. The explanation of this is that stretching the extensor muscle has stimulated its proprio- ceptors and set up a reflex which causes a lengthening reaction of the mus- cle. If the knee is now forced into an extended position a similar phenome- non occurs and the tone of the muscles adjusts itself to the extended posi- tion. The proprioceptors of the extensors have in this case been excited by a decrease in the tension of these muscles and have given rise to a short- 914 POSTURAL COORDINATION 915 ening reaction. By this mechanism the tension which muscles exert on their insertions is kept approximately constant, in spite of the changes in their length which are occasioned by alterations in the position of the body (Sherrington 27 ). The plastic tone of a skeletal muscle is effected not only by afferent impulses arising in its own proprioceptors, but by impulses arising from other muscles as well. When the lengthening reaction of the extensors of one limb is produced, a shortening reaction occurs in the extensors of the opposite limb. Conversely the stimulus which sets up a shortening re- action of one limb causes a lengthening reaction of the opposite exten- sors. The crossed lengthening and shortening reactions may be seen to be appropriate to the normal mode of using the two legs, for under usual circumstances when one limb is flexed the opposite leg becomes extended to support the weight of the body. The arrangement is conse- quently one which ensures a certain degree of harmony in the tonic ad- justments of muscles of parts which are used synchronously. A similar adjustment of the tonic condition of the muscles must also be made when the position of the body is changed through voluntary or reflex movement. When such movements are produced tone changes of two types occur: (1) The tone of the muscles which oppose the movement is inhibited; (2) the muscles which produce the movement enter into a tonic contraction which maintains the limb in the new position after the ex- citation has come to an end. The first phenomenon is seen when the leg of a spinal or decere- brate mammal is thrown into flexion by stimulating a sensory nerve, or the pain receptors of the foot. The tone of the extensors, which in the decerebrate cat is strong enough to support the animals weight, might be expected to offer some resistance to the bending of the joints. It may be shown, however, that at the moment the flexors contract, the tone of the extensors vanishes, so that they do not oppose the flexion. This is demonstrated by separating the flexor muscles from their insertion at the knee and attaching them to a weighted lever, by which changes in their length may be recorded. Since they are freed from their insertion, any increase in their length cannot be due to stretching by the flexors, but must be due to a loss in tone. When flexion is pro- duced, it is found that the extensor muscle lengthens and the lever falls, in synchrony with contraction of the flexor (Fig. 226). This is an exam- ple of the phenomenon of reciprocal inhibition of antagonistic muscles, which ensures their cooperative action. The maintenance of the new position of a limb after the stimulus which caused the change has subsided is called the after-discharge of the re- flex. It is due in certain cases at least to a tonic shortening reaction which is set up during the response to a stimulus applied to receptors 916 CENTRAL NERVOUS SYSTEM Fig. 226. Reciprocal inhibition. Tracings made by myographs connected with E, and ex- tensor muscle (vastus crureus), and F, a flexor muscle (semitendinosus), of a decerebrate cat. At signal / the homolateral peroneal nerve was excited, causing contraction of the flexors and in- hibition of the tone of the extensors. At signal // the flexors were again thrown into contraction by exciting the homolateral peroneal nerve, and (without removing this stimulus) the contralateral peroneal nerve was excited (as shown in the lower signal), with the result that the contraction of the flexors was inhibited at the same time that the extensors contracted. On removal of the latter stimulus, the former one reasserted its influence. This experiment demonstrates very clearly the accurate coincidence of the reciprocal action. (From Sherrington.) POSTURAL COORDINATION 917 other than the proprioceptors of the muscles (called exteroceptors). Fig. 227 shows how greatly the reflex contraction of the extensors of the knee of the cat may outlast the stimulus. If the afferent fibers from this muscle are destroyed by cutting the dorsal roots the reflex contrac- tion scarcely outlasts the stimulus. It appears that the reflex contrac- tion is accompanied by a tonic shortening reaction which is maintained after the exciting stimulus has come to an end. Its occurrence is de- pendent on the proprioceptive reflex arc of the muscle, so that if this is interrupted the contraction of the muscle cannot be maintained. The reflex response fuses with the proprioceptive reaction so that it cannot be said where one ends and the other begins. It is obvious that this is a mechanism which greatly facilitates the "smoothness" of muscular Fig. 227. Records of the contraction of the isolated extensor muscle (vasto crureus) of the knee of the cat produced by stimulating the popliteal nerve of the opposite leg. Periods of stimulation indicated by the signal below the record. A, illustrates the after-discharge in the normal muscle; B the absence of after-discharge following the cutting of the afferent nerves from the muscle. (From bhemngton.) acts, enabling the limbs to remain in the position assumed under the effects of certain stimuli, until other stimuli cause new postures to ap- pear. The Posture of the Body as a Whole, When the position of the body changes as the result of voluntary and reflex activity the tone of its entire musculature must be modified so that each part may make a har- monious contribution to the act as a whole. Part of the function of the central nervous system is consequently to correlate the simple proprio- ceptive reflexes such as we have described, not only with one another, but with the voluntary and reflex acts which are initiated through the ex- teroceptors. The afferent impulses which give rise to these harmonious changes in the posture of the body as a whole arise in part from the pro- 918 CENTRAL NERVOUS SYSTEM prioceptors of the muscles and tendons, and in part from the labyrinth. The former are influenced by the position of the parts of the body, the latter by the position of the body as a whole in space. The attitude con- sequently depends not only on the harmonious adjustment of the posture of its parts, but all these are brought into relation with the position of the body in space, whether right side up, inverted, etc. The in- teraction of these two sets of proprioceptors is illustrated by ex- periments on the decerebrate cat. The tone of the extensors of such animals is sufficient to support the body's weight. If the head of the standing animal is forcibly flexed, the postural contraction of the ex- tensor muscles of the fore limbs is inhibited, and the forequarters sink, while at the same time the postural contraction of the extensor muscles of the hind limbs increases, raising the hind quarters. The animal as- sumes the appropriate attitude for looking under a shelf. On the other hand, if the head is passively tilted up and back, the postural contrac- tion of the extensor muscles of the fore limbs increases, raising the fore quarters, and at the same time the postural contraction of the extensors of the hind limbs is diminished so that the hind limbs sink. The at- titude is now that of a cat looking up at a shelf. These reactions per- sist after the labyrinths have been destroyed and consequently must be due to proprioceptors in the muscles of the neck. The influence Oi the labyrinth was studied by rendering the neck immobile with a plas- ter cast, or by cutting the afferent roots of the upper cervicle nerv.es. On changing the position of the head the same adjustments occurred in the position of the limbs, so that the labyrinth must reinforce the proprioceptors of the neck in their action. In addition the labyrinth affected the posture of the neck in such a way that it would, had its afferents been intact, have set up the appropriate change in the limb posture. It has long been known that destruction of the labyrinth causes very abnormal postures to be assumed and these can be attributed to unusual positions set up in the neck muscles. In case of destruction of both labyrinths a great loss in tone results. If the organ on one side only is destroyed, tone is diminished on the same side, with the re- sult that the trunk is curved and the animal tends to roll over and over. Compensatory Movements of the Eyes. The position of the eyes is very markedly influenced by stimulation set up in the labyrinth. This is of obvious importance, since as our bodies move, compensation must be made by the eye muscles in order that the gaze may remain fixed on any object. This relationship is nicely demonstrated in the dogfish where the labyrinth is large and easily experimented upon. As the head of the fish is turned from side to side, as it is in swimming, the eyes move so as to compensate for the change in position. When the head turns to the left the left eye is turned forward, the right eye backward so as to re- POSTURAL COORDINATION 919 tain the original point of fixation. On rotating the head to one side, the eye of that side is turned- upward, that of the other side downward in compensation. Similar movements can be induced by stimulating the sense organs in the semicircular canals directly. Stimulation of the hori- Fig. 228. Compensatory movements of the eyes and fins of the dogfish. A and C illustrate the position of the fish when at rest. B shows the compensatory movements of the eye. 5 which occur during swimming, or on bending the tail into the position indicated. D illustrates the compensatory movements which occur on rotating the fish about its horizontal axis. Fig. 229. The semicircular canals of the ear, showing their arrangement in the three planes of space. (From Howell's Physiology.) zontal canal, which is normally excited by a movement in its plane, i. e., by turning the head to one side, causes the eye of the same side to turn forward; similarly the anterior vertical canal causes a rotation of the eye upward and forward, the posterior vertical canal a rotation upward and backward (Lee 28 ). Appropriate movements of the fins accompany 920 CENTRAL NERVOUS SYSTEM these reactions. In the dogfish the positions of the eyes is also con- trolled by the proprioceptors of the tail muscles, for if the tail is bent from side to side compensating movements of the eye are produced of the same nature as those which accompany the movements of the tail in swimming (Lyon 29 ). Eye movements of a compensatory character are also seen in man during rotation of the body. As the body turns the eyes swing slowly in the opposite direction so as to maintain their fixa- tion. Having turned as far as possible, they swing quickly back in the opposite direction to fix a new object which in turn they follow by a slow deviation. This slow deviation alternating with a rapid movement in the opposite direction is called nystagmus. It also occurs during the sen- sation of turning which persists for some seconds after the rotation of the body has come to an end, and in certain pathological conditions. Clinical Tests of the Labyrinthine Mechanism. The influence of the labyrinth on the postural coordination of the eye and limb muscles, forms the basis for certain useful clinical tests by which the condition of the labyrinth and of its central connections can be determined. These have been developed chiefly by Barany. In normal individuals when the semi- circular canals are stimulated in addition to the turning sensation and nystagmus, a phenomenon known as past pointing occurs. If the sub- ject is directed to close his eyes, extend his arm, raise and then lower it in a vertical plane he will have no difficulty in bringing it back to its original point. If he attempts to do this while the labyrinth is being stimulated he will, if normal, return the arm to a position which de- viates from the original point in the direction from which he thinks he is turning, i. e., in the rotation test in the direction toward which he has been spun. The methods used to excite the canals are called the rotation test and the caloric test. The rotation test is performed by spinning the subject about two or three times in a pivoted chair, with the head held so that the pair of canals to be tested lie in the plane of rotation. The caloric test depends on the fact that if warm (112 F.) or cold (68 F.) water is poured into the external auditory canal it will set up convection cur- rents in that semicircular canal which lies in a vertical position, and there give rise to excitation. By holding the head in various positions any one of the three canals can be stimulated. The caloric test has the obvious advantage that it excites the canals of one labyrinth only. If labyrinthine stimulation by these methods does not produce after turning sensations, nystagmus, or past-pointing it is concluded that a lesion occurs in the sense organ or some part of the nervous system in- volved in the reaction. The past-pointing test is particularly useful, since it can be applied to any joint and in any plane, and consequently it has been possible to study the localization of the centers in the cere- POSTURAI; COORDINATION 921 bellum which are concerned with the postural tone of each of these parts. (Black. 30 ) Other Clinical Tests of the Proprioceptive Reflex Mechanism. Certain phenomena may be considered at this point which form the basis for practical tests of the integrity of the mechanism by which posture and tone is maintained. The posture of the body is adapted to the position which it occupies in space, i. e., equilibrium is maintained, not only by virtue of afferent impulses arising from the semicircular canals, but as the result of visual sensations and of information supplied to the central nervous system by the receptors of deep sensibility in the limbs. Loss of function in any of these three groups of sense organs may be com- pensated by the other two. As a result sufferers from a destruction of the deep sensibility of the legs have little difficulty in maintaining, an upright position, so long as they are aided by the use of their eyes. In the dark, however, their balance is kept with great difficulty. It is consequently possible to test the integrity of the afferent paths from the limbs involved in the maintenance of equilibrium by noting the ability of the subject to stand with the feet close together and the eyes shut. Under these circumstances a normal person will stand quite steadily, but a sufferer from locomotor ataxia, in whom the afferent neurons from the limbs are diseased, will sway violently and tend to fall. This test is known as Romberg's sign. The tendon jerks are a group of reactions which result from tapping the tendons of the muscles of the knee, ankle, elbow and wrist. The contraction of the muscle which results is not solely one of postural tone, but it depends for its elicitation on the integrity of the reflex arc between the proprioceptors of the tendon and the muscle. It may consequently be used as a test for the condition of the afferent neurons from the deep structures in the limbs and for the condition of the lower motor neurons to the muscles, i. e., for the integrity of reflex arcs quite similar to those involved in the maintenance and adjustment of tone. When a lesion affects either the afferent neuron, as in locomotor ataxia, or the lower motor neuron, as in anterior poliomyelitis, the tendon jerks and tone alike are wanting. The character of the tendon jerks is also pro- foundly modified by conditions in the central nervous system which affect the tone of the muscles. In the normal individual when the patellar ten- don is tapped, the response of the extensor muscles is prolonged by a tonic contraction which gives way slowly as the flexors draw the leg back into its original position. The tone of the antagonistic muscles checks the limb on its return to this position, with the result that there is little tendency for the leg to bounce up and down after the response is over. When, however, the tone of the muscles is reduced as the result of in- jury to remote parts of the central nervous system, as in cerebellar le- 922 CENTRAL NERVOUS SYSTEM sions, or the later stages of spinal shock, the knee jerk is much more brisk in character because the contraction of the muscles is unimpeded by the tone of their antagonists. The return of the leg to its original posi- tion is very rapid since there is no tonic prolongation of the contraction of the extensors, and the shank tends to bounce up and down in the absence of any constraining tonic action on the part of the muscles Fig. 230. A, tracing of the knee-jerks of a normal man. B, tracings of the knee-jerks of the hypotonic leg of a man with a cerebellar injury of eight years' duration. The records should be read from right to left. (From Holmes.) (Fig. 230). Under these conditions the return is a passive act produced by the weight of the leg, and not by a compensating flexor contraction, for if the limb is supported on a bed when the knee jerk is elicited, it shows no tendency to flex again after the extension has been produced. Impulses from other parts of the nervous system may prevent the occurrence of the tendon jerks by preventing the afferent impulses set POSTURAL COORDINATION 923 up by tapping the tendons from controlling the activity of the motor part of the reflex arc (see the Final Common Path, page 945). If the leg of an animal be thrown into flexion, the extensor muscles are in- hibited from contraction and the knee jerk can no longer be elicited, even though the extensor muscles are separated from their insertion and consequently do not have to pull against the contraction. In a similar way the knee jerk may be inhibited by influences arising in the cerebrum, and consequently it is frequently necessary to distract the subject's attention before the response can be brought out. CHAPTER XCIX THE CENTRAL CONTROL OF POSTURAL REACTIONS; THE CEREBELLUM The prime essential for muscular tone and its plastic reactions is the integrity of its own proprioceptive reflex arc. Certain centers in the brain exert a modifying influence upon the degree of tone which this arc maintains, but if the afferent fibers from the muscles are damaged, these centers cannot replace them in their effect on the motor neuron of the proprioceptive reflex arc. Thus the exaggerated tone of the ex- tensor muscles which is produced by the action of the centers in the brain of animals from which the cerebrum is removed disappears at once if the afferent roots are cut. Since the proprioceptive reflex arc is necessary for the production of tone in muscles, conditions of diminished tone or flaccidity appear in disease affecting either the afferent fibers from the muscle or its motor neurons. If the former alone is damaged tone will be dimin- ished or lost without paralysis of the muscle. This is an unusual con- dition since the afferent and efferent paths are only separated during their passage into the cord through the spinal nerve roots. It occurs, however, in locomotor ataxia, in which the primary lesion lies in the dorsal spinal roots, and in the ganglia of the cranial nerves. If the motor neuron alone is affected the loss of tone will be accompanied by paralysis of the muscles, that is a flaccid paralysis will result. This is an important principle in determining where the lesion which gives rise to paralysis is situated, since paralysis produced by lesions of the higher motor centers and tracts is not accompanied by permanent flaccidity. A typical flaccid paralysis occurs in anterior poliomyelitis in which the lesion is situated in the ventral horn of the gray matter of the cord, involving the cell bodies of the motor neurons. The Influence of the Brain on the Local Tonic Reflex When the proprioceptive reflex arc is isolated from the brain by com- plete section of the spinal cord, a condition called spinal shock inter- venes for a period during which reflexes may not be elicitable and the muscles become atonic. After a period of time, which is longer the higher the animal in the evolutionary scale, the reflexes return and tone is regained, but not quite in normal degree. This condition holds true 924 CENTRAL CONTROL OF POSTURAL REACTIONS 925 for complete transections of the cord at levels up to the medulla. It appears that the higher centers exert a reinforcing effect on the local tonic reflex, which may be compensated for when these influences are removed, but never with complete success. The reinforcement of the tone of skeletal muscles by the higher centers of the nervous system represents the resultant of two opposing tendencies, one augmenting the tonic contraction, the other inhibiting it. The augmentation of tone is seen in the condition known as decere- brate rigidity, to which we have already referred, in which the tone of the extensor muscles is greatly exaggerated. Experiments on animals show that the rigidity is not the result of separating the cerebrum from the central nervous system, as the name would imply, for if successive slices of the brain be removed from above downward the condition does not develop until the section separates the optic thalamus from the mid- brain. The researches of Sherrington, 31 Thiele, 33 and particularly of Weed, 32 indicate that the mechanism supporting the rigidity is probably somewhat as follows. The afferent impulses on which the " reflex stand- ing" depends arise in the muscles themselves, since cutting the posterior roots of the spinal nerves supplying the muscles in question abolishes the rigidity. They pass up the cord in the ventrolateral column of the same side and probably enter the cerebellum through the superior cerebellar peduncles and pass to the cerebellar cortex. From thence the path leads back through the same peduncles to the midbrain in which lies the main center for maintaining this condition, probably the nucleus ruber. From there the paths descend through the cord in extrapyramidal tracts, prob- ably the rubrospinal. Afferent paths also probably lead directly to the midbrain center without passing through the cerebellum, for in some animals removal of the cerebellum is not followed by loss of rigidity or its loss is not immediate. The Inhibition of Tone. The rigidity may also be inhibited, once it has developed, by stimuli applied to various parts of the nervous system. If the "decerebration" is limited to one side of the body rigidity may develop on that side only and it becomes possible to study the effect of stimulating the cortex of the other half of the cerebrum. Stimulation of the sensory-motor area inhibits the existing tone in the extensor mus- cles. Excitation of the cerebral peduncles also inhibits the rigidity. Weed 32 considers that the cerebellum forms an essential link in the path over which these inhibitory impulses pass from the cerebrum to the centers which maintain the rigidity, because severence of the middle cerebellar peduncles eliminates the inhibition which is produced by stimulation of the cerebral peduncles. In support of this observation is the fact that excitation of the anterior part of the superior vermis or of 926 CENTRAL NERVOUS SYSTEM the stump of the superior or middle cerebellar peduncle inhibits the rigidity of the limbs. The maintenance of the excessive extensor tonus known as decere- brate rigidity consequently depends upon the reflex connection of a cen- ter in the midbrain with the muscles. Normally the activity of this reflex mechanism is held in abeyance by the inhibitory influence of the fore brain. The fact that both the afferent impulses from the muscles and the inhibitory impulses from the cerebrum pass through the cere- bellum suggests strongly that this organ must have a very important relation to the regulation of postural tone, and that this is the case is indicated conclusively by what is known of the physiology of this structure. The Function of the Cerebellum, The function of the cerebellum has been the subject of exhaustive study, particularly on the part of Luci- ani. 3G Stimulation of the cortex of the cerebellum with an electrical current does not give rise to any detectable reactions unless the current is so strong as to spread to the deeper ganglia of the brain. Conse- quently it has been necessary to study the effects of removing all or part of the cerebellum and to attempt to deduct from the resulting dis- turbances the function which the ablated parts perform. When this is done it is found that three stages can be distinguished in the condition of the animal, following the operation. In the initial stage a definite group of symptoms are presented which are presumably due to certain immediate effects of the operation. These effects change quite com- pletely after several days and a set of conditions present themselves which appear to be the permanent result of the loss of the cerebellar tissue. Finally, however, a certain improvement in the behavior of the animal occurs which may be attributed to the compensatory action of other parts of the nervous system which modify their activities so as to correct for the loss of cerebellar influences. The second stage is ob- viously of chief interest in the interpretation of the normal function of the cerebellum. The conditions existing in this stage in man, following gunshot injuries, have been exhaustively described by Holmes, 35 and as they agree closely with the results obtained on animals and are of greater interest to human physiology, we may draw our conclusions from them. The destruction of tissue in one half of the cerebellum manifests it- self in the condition and behavior of the muscles on the same side of the body. When compared to the muscles of the uninjured side, these ex- hibit several forms of abnormality which may be designated as atonia, asthenia, astasia, and ataxia. To atonia are attributed those symptoms which manifest themselves as the result of a diminution in the tone of the muscles. As the result of this condition the muscles feel soft and flabby and the limbs tend to assume unnatural positions. When the arm CENTRAL CONTROL OF POSTURAL REACTIONS 927 is held upright, for example, the wrist is flexed under the weight of the hand. If the joints are passively bent, their movement is not checked by the action of the muscles, but continues until the articulations cause them to lock. The characters which the disturbance in tone imposes on the knee jerk have been described on page 921. Asthenia expresses the fact that the muscles of the injured side are weaker than on the normal side. Not only are the patients conscious that these muscles feel weaker, but measurement with a dynamometer shows that they can exert in some cases only 50 per cent of the force of which the normal muscles are capable. The limbs in which the mus- cles are asthenic are also unusually subject to fatigue. It should be emphasized that the muscles which have been deprived of a cerebellar influence are never paralysed: the asthenia is not a failure of function, but rather an expression of inefficiency in the act of voluntary contraction. Closely associated with the asthenia is the discontinuity or irregu- larity of a maintained muscular contraction known as astasia. The af- fected muscles frequently tend to give way under the load they bear, so that objects held in the hand are liable to be dropped, or the leg on the injured side may suddenly collapse under the body's weight and thus cause the patient to fall as he attempts to walk. A tremor may occur in maintaining an attitude, if it requires the exertion of some force, par- ticularly as the muscles begin to tire. It is a less prominent symptom in man, however, than in animals. The destruction of the cerebellum seems to disturb the mechanism by which the individual twitches of the con- tracting muscle become fused so as to maintain a constant tension. We have pointed out on page 917 that the continuity of muscular movement is facilitated by the reinforcement of the primary response by an adjust- ment in the postural tone of the muscle, and it would seem that this is the mechanism whose disturbance gives rise to the astasia of cerebellar injuries. Under asynergia may be grouped those symptoms which are due to ir- regularities in the voluntary movements of the body. The limbs of the affected side are ill directed when an attempt is made to perform some precise movement. The finger may strike the eye, when its true objec- tive is the mouth and consequently the patient fears to hold his cigarette in the affected hand while smoking. It is impossible to stop the move- ment at the right point, with the result that in trying to reach some ob- ject, the hand strikes it forcibly or else falls short of its objective. When voluntary movements are made they are interrupted in their progress by a tremor, particularly as they approach their objective, and greater need for accuracy becomes necessary. This jerky character of the mo- tion is obviously closely associated with the static tremor described as astasia. Certain abnormalities in movement indicate that difficulty ex- 928 CENTRAL NERVOUS SYSTEM ists in the synchronous adjustment of the activities of various muscle groups which should cooperate for a common end. When the fingers are flexed the extensors of the wrist normally contract synergically in order to prevent simultaneous flexion of the wrist. If a patient with a cere- bellar lesion grasps a small object quickly, the wrist may be extended excessively so that the hand is bent backwards before the fingers are half flexed. Movements which involve the activities of several joints are frequently decomposed into their component parts which are exe- cuted one at a time instead of all at once. In bringing the finger to the nose the patient will first depress the arm by a movement of the shoulder, and then flex the elbow after the first act is complete. Difficulty is also experienced in making rapid alternate movements such as flexion and extension of the fingers. The actual movement may be made nearly as rapidly as with the normal hand, but a considerable delay intervenes be- tween the successive acts, indicating a difficulty in adjusting the neuro- muscular mechanism for the new act. In making such rapid alternating movements groups of muscles not concerned in the desired action may come into play. When, for example, the ankle is voluntarily flexed and extended in rapid succession the knee and hip may flex and extend also. The asynergia produced by the destruction of cerebellar tissue consists then in a disturbance of the normal harmony and correct cooperation in time and degree of the various muscular contractions concerned in movements and in the maintenance of posture. We may conclude that the cerebellum is actively concerned with the maintenance of tone and in the adjustment of voluntary contraction and plastic tone to the posture of the body, not only as it is maintained in rest, but as it changes when in action. Injuries to it result in a mis- coordination, rather than an incoordination of muscular activity. Sher- rington 37 has called the cerebellum the main ganglion of the proprioceptive system. The passage of impulses from the proprioceptors and from the cerebrum through the cerebellum in their course to the centers of the midbrain which are involved in the maintenance of decerebrate rigidity consequently assumes an important functional significance. No Sensory Disturbances Follow Injury to the Cerebellar Cortex. In considering the function of the cerebellum it should be pointed out that although it receives afferent impulses from the proprioceptors of the body there is no evidence that it is concerned in any way with sen- sation. The only sensory tests to which those afflicted with cerebellar injuries fail to respond with normal accuracy are those involving the comparison of weights placed in the normal and the affected hand. In this test the weight in the latter is judged heavier than it should be be- cause of the greater effort required to support it with the asthenic mus- cles. Although the ability to maintain the equilibrium of the body may CENTRAL CONTROL OF POSTURAL REACTIONS 929 be disturbed as the result of cerebellar injury, this is because the mus- cles do not respond properly in the attempt to prevent falling, and not because the sense of equilibrium is in any way impaired. Localization of Function in the Cerebellum. The observations on cere- bellar injuries in man which we have described indicate that the two halves of the cerebellar cortex are each concerned with the regulation of tone and movement in the corresponding half of the body. Beyond that they do not afford any evidence of localization of function in the cere- bellum. By studying the correlation between the functional importance of different muscle groups and the development of the different parts of the cerebellum in different animals Bolk has assigned the control of each muscle group to a definite part of the cerebellar cortex, as is indicated in Figure 231. Basing his work on these anatomic conclusions, Van Rijnberk has studied the effect of circumscribed extirpation of certain lobules of the cerebellum Fig. 231. Schema of the parts of the mammalian cerebellum spread out in one plane. (After Bolk by Van Rijnberk from Luiciani. Op. cit.) On the right side of the figure the relation . of tne different lobules to the functional development of the musculature is indicated according to the theory of Bolk noted in the text. (From Davidson Black.) on the muscular control of the different parts of the body, with the following results. Total or partial extirpation of the lobulus simplex produces side to side oscillations of the head, indicating the removal of the influences of the cerebellum that control the movements of the muscles of the neck. Complete extirpation of the crus primum of the lobuli ansiformes causes as an imme- diate irritative effect dynamic disturbances of the fore limb of the same side, replaced later by a condition of atonia, which makes the limb hang limp, and of asthenia, which makes it feeble in its movement when it is excited to contract. Extirpation of the crus secundum has a similar influence on the muscles of the hind limb of the corresponding side. Extir- pation of both crura of the lobulus ansiformis causes marked asthenia and atonia in both fore and hind limb on the same side as the lesion. A char- 930 CENTRAL NERVOUS SYSTEM acteristic disturbance in walking develops as a late effect of this extirpation. It has been termed the "hen's gait." Extirpation of the lobulus para- medianus causes rotation on the longitudinal axis of the body, with pleuro- thotonus to the operated side. (Fig. 232.) Just as in the case of cerebral localization, so in cerebellar we find that within each of the largest centers a more particular localization can be made out; thus, in each of the centers for the upper and lower extremities, there is a definite arrangement of subsidiary centers for the direction of the activities of antagonistic muscle groups concerned in the movements of particular joints. Localization of function in the cerebellum of man has been worked out by Barany by correlating the position of cerebellar lesions with disturb- Fig. 232. Diagrams to represent respectively a ventral view of the left half and a dorsal view of the right half of the human cerebellum illustrating the scheme of subdivision according to Bolk. (From photographs of specimens from the Anatomical Museum, Western Reserve Medical School.) (From Davidson Black.) ance in the past-pointing tests (described on page 920) which appear when the action of each joint is examined in turn. Barany 's conclusions so far may be summarized as follows: (1) The centers for the extremities are located on the cortex of the hemispheres in the semilunar (superior and inferior) and digastric lobules (see Fig. 233). The representation is uncrossed or homolateral, thus con- trasting with cerebral localization, in which it is crossed or heterolateral. (2) Within each of these chief centers there is a further localization, which however does not refer to anatomical groups of muscles but rather to the functional performances of the different segments of the limb. Thus, within the arm centers there are subsidiary centers concerned in the movements of the limb in the various planes in rotation, in pronation CENTRAL CONTROL OF POSTURAL REACTIONS 931 and in supination. It is a functional rather than an anatomical localization. (3) When a center concerned in the movements of the limb in a certain direction, e. g., to the right, is suddenly destroyed, a spontaneous devia- tion is produced in the opposite direction (to the left). For further details see the paper by Black. 30 Compensation for Cerebellar Injuries. The final stage following Fig. 233. Fig. 234. Figs. 233 and 234 represent respectively the inferolateral and the posterior aspect of the human cerebellum indicating certain cerebellar localizations according to Barany. (After Barany, from Andre-Thomas et Durupt. Op. cit.) N. VII, Nervus facialis; N. IX, Nervus Glossopharyngeus; N. XII, Nervus hypoglossus. The signs in the above diagram indicate the exact localization of the centers for the tonus of the musculature concerned in some of the movements of the right arm and leg, marks the center for downward movements of the arm; X, for abduction of the arm; O, adduction of the hand; + adduction of the arm; , adduction of the hip. N. V. indicates Nervus trigeminus; N. VI, Nervus abducens; N. VII, Nervus facialis; N. IX, Nervus glossopharyngeus; N. XII, Nervus hypoglossus. (From Davidson Black.) the removal of cerebellar tissue is one in which compensation is made for this loss by other parts of the nervous system, so that in time the symptoms gradually tend to disappear. The initial disturbances in lo- comotion and the improvement which comes with time are illustrated in Fig 235, which represents the footprints of a dog from which the cere- bellum has been removed. 932 CENTRAL NERVOUS SYSTEM It will be of interest to consider for a moment the possible causes for the ultimate disappearance of the symptoms of cerebellar extirpation. These are either: (1) an organic compensation by the uninjured parts of the cere- bellum, or (2) a functional compensation by the voluntary centers of the cerebrum. Although the former of these methods of compensation may sometimes develop after partial destruction of the cerebellar cortex, it can not of course explain the recovery which we have seen to occur after the e d c b a Fig. 235. Footprints after destruction of the cerebellum in a dog: a, before the operation; b, four days after; c, five days after; d, a month after; e, two months after. (From Luciani.) entire cerebellum has been removed. The most important compensation no doubt is effected by the cerebrum, as the following observation clearly in- dicates. If half of the cerebellum of a dog is destroyed, and the animal kept alive until the symptoms of cerebellar extirpation have entirely dis- appeared, it will then be found, if the cerebral center on the opposite side is removed, that the symptoms return in their original severity. After this second operation the powers of standing in the erect position and of walking are permanently lost. CHAPTER C THE INTEGRATION OF ACTION WITHIN THE REFLEX ARC In considering the anatomical arrangements which give rise to sensa- tion and motor activity, we have taken the risk of creating a false im- pression that the action of the nervous system is carried out in a rigid and immutable manner, and that nerve impulses travel over the conduct- ing paths with the invariability with which an electric current flows over a fixed system of wires. While the constancy of the anatomical paths by means of which certain nervous functions are carried out is of ines- timable value in clinical neurology it must be emphasized that in the nervous system continuous adjustment is made to the end that the activ- ity of one part may adapt itself to the activities of other parts, and con- sequently the results of a given stimulation are not always strictly pre- dictable. In other words the activity of the various reflex paths is closely coordinated. Before we can consider how this coordination between reflexes is accomplished, we must first examine into how the activity of the various parts of a reflex arc are related so that the reflex may bring about an act of functional significance; i. e., a response in which a local- ized group of muscles act in a coordinated way so as to accomplish some purpose which is related to the exciting stimulus. The Receptors. Reflex acts are initiated by the stimulation of recep- tors. Because each receptor is specialized to respond more readily to one quality of stimulus than to any other (page 856), each reflex arc is brought into action only by stimuli of an appropriate sort. The selective excitability of the receptors of the different reflex arcs consequently enables the organism to respond in different ways to stimuli of different kinds when they are applied to the same receptive skin area. As an example we may consider the reactions which may result from stimulat- ing the foot of a spinal or decerebrate cat. By pressing against the under surface of the paw a reflex may be elicited known as the extensor thrust, consisting of a vigorous extension of hip, knee, and ankle of the corre- sponding leg. This response cannot be called out by any other form of stimulus. In life it would be expected to occur when in running the cat's foot comes in contact with the ground, thus throwing the animal's weight on the leg. If a harmful stimulus is applied to the same part of the foot the response is of a totally different character, consisting of a flexion of the joints of the limb. The foot is pulled away from the stim- 933 934 CENTRAL NERVOUS SYSTEM ulating object. This would serve to relieve the cat of pain, such as might be occasioned if it stepped on a thorn. The receptors for these two reflexes have their thresholds lowered each to a particular form of stimu- lus, and the nature of the response is such that the leg moves in a way which is appropriate to the conditions under which stimulation occurs in nature. When nerve impulses are set up in the afferent neuron of a reflex arc, the path over which they may travel is limited by the insulation of the fibers of the nerve trunk to those neurons with which the afferent fiber makes connection through synapses located in the grey matter of the spinal cord. The characteristics of the synapse, which we have de- scribed in Chapter XC determine the destiny of the nerve impulse and the characteristics of the reflex response which results. Summation. It was pointed out that a single nerve impulse frequently fails to pass across a synapse, over which a series of impulses may travel if they follow one another in rapid succession, so that their effects are summated. This characteristic of synaptic conduction is of importance in regulating reflex activity, as may be realized when it is considered that the organism is constantly in receipt of many unimportant stimuli. Be- cause of the necessity for summation only those stimuli call forth a re- sponse which are of some intensity and duration. Consequently mo- mentary and hence insignificant stimuli do not affect its behavior. 6. The Refractory Period. This has been well defined by Sherrington as being "a state during which apart from fatigue the mechanism shows less than its full excitability." We are already familiar with the re- fractory period in the cases of the heart muscle and the musculature of the esophagus and intestine. For example, the application of a stimu- lus to the quiescent frog heart while it is contracting in response to an im- mediately preceding stimulus fails to produce any further effect. The re- fractory period is extremely brief (one thousandth of a second) in a nerve trunk, but is much longer in a reflex arc, being probably longest in the case of the scratch reflex, in which it is demonstrated by the fact that, however frequently we apply suitable stimuli to the sensory surface, the rhythm of response of the contracting limb is always the same. After each stimulus, therefore, a refractory period must become developed during which a repetition of the stimulus has no effect. It is evident that the existence of the refractory period is the factor responsible for the rhythm of the movements. It is interesting to consider what part of the reflex arc is responsible for the existence of the refractory phase. It obviously can not be a function of the motor neuron, for through the same motor neuron may be discharged, at one time, impulses which bring about the scratching movement and, at another, those causing a tonic flexion of the INTEGRATION OF ACTION WITHIN THE REFLEX ARC 935 same muscles. Nor can the seat of the refractory period be in the sensory area of the skin or the afferent neuron, for if a scratch move- ment is elicited by stimulation at a point A in the proper skin area, the rhythm of response which it calls forth will not in any way be altered by the application of a second stimulus applied at B at some distance from A and having a different frequency (Fig. 236). There is evidently, therefore, some part of the reflex arc that is common to Fig. 236. Tracing from the hind limb of a spinal dog during the scratching movements pro- duced by applying stimuli at two skin points (A and B), the application of the stimuli being in- dicated by the signals. Not only were the stimuli applied at different points, but at B they were of much greater frequency than at A. Although there is a slight change in "local sign," it will be observed that there is no alteration in rhythm, indicating that this property can not be a function of the final common path. (From Sherrington.) impulses starting both at A and at B, for if in each of these spots a refrac- tory phase occurred, then there would be interference before the two im- pulses had reached the centers of the spinal cord. By exclusion, there- fore, "the seat of the refractory phase seems to lie somewhere central to the receptive neuron in the afferent arc" (Sherrington 18 ). Many other types of reflex activity illustrate rhythm due to the re- fractory phase. Two laboratory examples may be given: (1) When 936 CENTRAL NERVOUS SYSTEM the central end of an afferent root is stimulated in the lumbar region of the spinal cord, the movement produced is distinctly rhythmic in character. (2) Upon stimulating the central end of the sciatic nerve in a frog whose spinal cord has been cut some days previously, a clonic action of the contralateral foot occurs, and the rate of the rhythm is not affected by variation in the frequency of the stimulus. Fig. 237. Diagram showing the reflex arcs involved in the scratch reflex. Ra and R0 represent ihe afferent neurons connected with hairs on the skin of the back and flank. The afferent im- pulses are transmitted by these fibers, and on entering the corresponding segments of the spinal cord terminate by synapses on cells of the internuncial neurons, whose arrows Pa and PQ travel down in the lateral columns to terminate similarly around the cells of the motor neurons that innervate the muscles of the hind limb. Since afferent impulses coming from elsewhere, par- ticularly from the skin of the leg (R and L), also terminate on these neurons and may excite them to a different type of action, the motor neuron is called the final common path (F.C.). (From Sherrington.) Fig. 238. The region of body of dog from which the scratch reflex can be elicited. (From Sherrington.) In all the above cases the refractory period may be held responsible for the rhythmic nature of the contraction. In other reflexes it exists for another purpose. In the case of the extensor thrust, which it will be remembered is elicited by pressure applied to the pads of the plantar aspect of the foot, the momentary extension of the leg lasts only for a little less than two-tenths of a second, but is followed by a refractory INTEGRATION OF ACTION WITHIN THE REFLEX ARC 937 period lasting nearly a whole second, during which a second stimulus elicits no response. The object of this long refractory period is no doubt that opportunity may be given for the flexor muscles to perform the contraction that would naturally ensue during the normal occurrence of the extensor thrust, as in the act of walking. When the animal places his foot on the ground, the sudden pressure exerted on the pad of the foot immediately calls forth the extensor thrust, by means of which the weight of the body is temporarily removed from the ground, and the muscles perform the contractions necessary to produce flexion of the limb. Although the refractory period is unaffected by the strength of the stimulus it is very dependent upon the internal condition of the reflex arc, such as that caused by changes in blood supply or by narcosis. Reciprocal Inhibition It might appear that to bend a joint or to move the eyeball the only muscular action required would be contraction of the muscles which flex the joint or rotate the eyeball, and that the antagonistic muscles would merely become passively elongated. When we remember, how- ever, that all the muscles of the body are ordinarily in a condition of slight tonic contraction, and that this tends to become increased when the muscles are passively stretched, then we see that for effi- cient movement there must be inhibition of the tone of the muscles which oppose those that are contracting. This reciprocal inhibition, as it is called, is a very widespread function throughout the animal body. Sometimes it is purely peripheral in origin, as in the claw of the crayfish, where stimulation of the nerve causes an opening of the claw due to the contraction of one set of muscles and the simultaneous inhibition of their antagonists. Instances of peripheral reciprocal in- hibition in the higher animals are not so common, but are illustrated in the case of the myenteric reflex, where it will be remembered a contraction of the intestine over a bolus of food is accompanied by inhibition in front of the bolus. The reciprocal action in this case is probably dependent on the myenteric plexus. On the other hand, reciprocal inhibition of central origin is very com- mon in the higher mammalia. Thus, in the case of the lateral movement of the eyes, if we cut the third and fourth nerves to one eye, say, the left, the external rectus of that eye will alone be under the control of the nervous system, through the sixth nerve; nevertheless, if we after- ward cause the animal to look toward the right, as by holding some ob- ject in that direction, it will be found that the left eye as well as the right follows the object. Obviously there must be an inhibition of the 938 CENTRAL NERVOUS SYSTEM external rectus muscle of the left eye, an inhibition which is pronounced enough to bring about a movement of the eyeball, and which exactly cor- responds in point of time with the contraction of the external rectus of the right eye. This movement, due to the atonicity of the external rec- tus, does not however succeed in causing the eye to rotate beyond the midline of the field of vision. This is an instance of a willed reciprocal inhibition ; i. e., a reciprocal inhibition brought about by stimuli coming from the motor centers in the cerebrum. The same result may be obtained by electric stimulation of the center for eye movements on the cerebral cortex. The most important details concerning the mechanism of reciprocal inhibition have been obtained by studying the flexion reflex in a spinal animal which has completely recovered from shock. In such an animal the tonus of the extensor muscles of the knees is well marked. If we pre- vent the flexors from acting on the knee joint and the leg is held in an ex- tended position, irritation of the skin of the leg will cause the flexion of the disconnected hamstring muscles simultaneously with a visible relaxation of the extensors. If the leg is held properly, this relaxation may be marked enough to cause a slight flexion at the joint under the influence of gravity. This experiment is very striking when performed on a de- cerebrate animal, in which, as we have seen, the extensor muscles of the limb are in a permanent state of hypertonicity. Reciprocal inhibition can also be demonstrated by stimulating the central end of suitable afferent nerves that is, certain afferent nerves acting on the same groups of neurons will produce a flexion reflex, others an extension reflex; thus, stimulation of the homolateral peroneal nerve produces a flexion reflex of the hind limb (excitatory for flexors, in- hibitory for extensors), whereas stimulation of the contralateral peroneal nerve produces an extension (inhibitory for flexors, excitatory for ex- tensors). (Fig. 239.) Before we can conclude that the two elements in reciprocal inhibition are part and parcel of the same reflex it must be shown that under what- ever conditions the contraction of one group of muscles is brought about, inhibition of their antagonists must also occur and that the responses take place at exactly the same time. If records are made of the move- ments of the flexors and extensors of the knee in an experiment such as we have just described, it is found that the latent period for the re- sponse of agonist and antagonist, whether it be contraction or inhibition exactly coincides (Fig. 226). If the strength of the stimulus is gradually increased the latent period becomes shorter for the inhibitory and active reaction alike and the synchrony of the reciprocal responses is still pre- served. Moreover the receptive field on the skin from which contraction INTEGRATION OF ACTION WITHIN THE REFLEX ARC 939 may be elicited coincides exactly with that for the inhibition of the antag- onistic muscles, as do also the kinds and strengths of stimuli which are effec- tive in each case. The inhibition of antagonists may consequently be considered a part of the same reflex which excites the agonists to contrac- tion. It is impossible to demonstrate any trace of inhibition of the skeletal muscles by stimulation of their motor nerves, thus indicating that in- hibition is dependent upon the nerve center. Furthermore, since inhibition occurs along with contraction of the antagonistic muscle, we must assume Fig. 239. Record from myograph connected with the extensor muscle of the knee. During the time marked by the lower signal, the skin of the opposite foot was stimulated, thus causing the crossed extension reflex. While still maintaining this stimulation, faradic shocks were ap- plied to the skin of the foot of the same side (as indicated by the upper signal), with the result that immediate inhibition of the contracted extensor occurred. (From Sherrington.) that the afferent impulse on entering the spinal cord divides into two branches, one going to one motor neuron so as to excite it, the other to another neuron so as to inhibit the tonic stimuli which it is con- stantly sending to the muscles (Fig. 240). According to this assumption the effect of impulses carried over each branch depends on the nature of the synapse which these make with the motor neurons of the antagonistic groups of muscles. Since the seat of the inhibition is in the nerve center, it is to be ex- pected that impulses transmitted from other parts of the nervous system 940 CENTRAL NERVOUS SYSTEM than the particular level of that reflex, will also be able to induce the inhibition. In the case of the decerebrate cat this can be demonstrated by stimulation of the lateral columns of the spinal cord; inhibition of the extensor muscles of the elbow joint occurs, which is all the more marked because in such a preparation these muscles are in a state of hypertonicity. Through the pyramidal tract impulses may descend from the cerebrum which exercise a marked inhibitory influence over the reflex activities of the cord. Finally, it must be pointed out that this mechanism of reciprocal in- hibition is by no means confined to the voluntary muscles. We have Fig. 240. Sherrington's diagram illustrating the mechanism of reciprocal inhibition. The afferent fibers (d) from the skin of the leg and (d) from the flexor muscles of the knee (in hamstring nerve) pass to the spinal cord, where each gives off a branch which divides into two others, of which one in each case goes to a motor neuron of the extensor muscles (H) and the other to a motor neuron (5) of the flexor muscles (F). Branches also pass across the median line to similar motor neurons on the opposite side of the cord. As indicated by the plus and minus signs, the afferent stimuli either stimulate or inhibit the activities cf the motor neurons, the determination of the exact effect being a function of the synapsis. (From Sherrington.) already seen that it occurs in the case of the myenteric reflex. It is also a most important function in the innervation of the blood vessels, dilata- tion in one vascular area being accompanied by constriction in another. These facts have been already sufficiently dwelt upon elsewhere (page 247). Sometimes also we may have reciprocal action between differently acting nervous mechanisms, as for example in the case of the submaxil- lary glands, which respond to stimulation of the chorda tympani nerve by dilatation of the blood vessels, an inhibition of their tone occurring along with stimulation of the activity of the gland cells. INTEGRATION OF ACTION WITHIN THE REFLEX ARC 941 The Action of Strychnine and Tetanus Toxin on Reciprocal Inhibition Under certain conditions reciprocal action may fail to occur, as, for example, at certain stages of strychnine poisoning and during the action of tetanus toxin. In order to demonstrate this failure of reciprocal ac- tion, it is necessary to examine muscles which act on one joint only, and to observe their behavior when an afferent nerve is stimulated which un- der ordinary conditions would throw them into inhibition. Such a preparation can be obtained in the hind limb of a dog by cutting all the muscles that act on the knee joint except the vasto-crureus, which in a normal animal invariably undergoes inhibition when the central end of the internal saphenous nerve is stimulated. If a suitable dose of strych- nine is injected, it will be found that stimulation of the internal saphenous nerve, in place of inhibition, causes contraction of the vasto-crureus muscle. The same result is obtained by injection of tetanus toxin. The failure of the reflex inhibition explains the symptoms produced by these substances. It explains, for example, the well-known rigidly extended condition of the limbs in strychnine poisoning, and the dis- tressing symptom of lockjaw in tetanus infection. In this latter con- dition the sufferer is subjected to extreme torture with every endeavor that he makes to open the jaw for the purpose of taking food or drink. Firmer closure is the result because the normal inhibition of the temporal and masseter muscles does not occur, but instead they become excited and the jaw all the more firmly closed. Not only does, the inhibition fail to occur, but the above muscles are usually in a state of constant hy- perexcitability, which it is impossible for the patient to restrain ; indeed, whenever he attempts to do so the opposite occurs and the excitation becomes heightened. Chloroform acts on reciprocal innervation in an opposite way from strychnine and tetanus; namely, it paralyzes the ex- citation of the contracting muscles. The Reflex Figure We have seen in the preceding paragraphs that the afferent fibers of a single reflex arc make connection with the motor neurons of a con- siderable group of muscles, exciting some and having an inhibitory in- fluence on others to the end that all may cooperate in producing an or- derly movement of some functional significance. The effects of stimulating a single afferent path may not be limited to the antagonistic muscles ar- ranged about a single joint, but may extend to the flexors and extensors of all the joints in a single limb and, further, to each of the other limbs and to muscles of the head, trunk, and tail as well. The response which follows the application of a strong stimulus to the receptors of a reflex arc may consequently involve nearly the whole musculature of the animal. The par- 942 CENTRAL NERVOUS SYSTEM ticular attitude which is assumed is called a reflex figure, and is the expres- sion of the complete central connections of the reflex in question. Fig. 241 illustrates three characteristic reflex figures obtained by applying a harm- ful stimulus to various parts of the skin of a decerebrate cat. The reflex figure which results from such a stimulus when applied to the foot of the hind leg consists in a flexion of this leg, an extension of crossed hind leg (the crossed extension reflex), extension of the homolateral fore leg, and flexion of the crossed fore leg. This group of responses may be considered to be a compensatory reaction, inas- much as the movement of certain parts is adapted to restore a balance which is disturbed by the movement of other parts, and the result is an orderly change in the position of the body as a whole which is Fig. 241. Reflex figures. A, the position of the cat in decerebra :ctiyely, are the reflex figures resulting from stimulating the left pinna, : hind foot. (After Sherrington.) . spectively left hind 1) decerebrate rigidity. B, C, D, re- the left forefoot, and the significant in meeting the exigency which has given rise to the reflex response. The reflex figure which we have just described might be brought into play, for example, when a cat steps on some object which hurts its hind foot. The hind foot is lifted from the ground by virtue of the contraction of the flexors and the compensatory inhibi- tion of their antagonists. The weight of the hind quarters is thus thrown on the contralateral hind leg, which extends to support this weight, preventing the animal from sitting on the source of discom- fort. At the same time the cat must prepare to move away so that the stimulus may not be encountered again, and for this act the exten- sion of the crossed hind leg and of the homolateral fore-foot, with their backward thrust, tend to throw the body forward and support it while the flexion of the stimulated hind leg and the crossed fore leg move INTEGRATION OF ACTION WITHIN THE REFLEX ARC 943 these limbs forward preparatory to supporting the body at the next step. The reflex figure is seen from this to be an integral mechanism out of which is built the functional act of stepping away from a stim- ulus which endangers the hind foot. The entire reflex figure may be considered to be an unified reflex act depending on the central connections of a single afferent path, for much the same reasons which lead us to conclude that excitation and inhibition of the antagonistic muscles at a single joint are parts of a single reflex. In all parts of the reflex figure reciprocal inhibition may be seen to occur, as may be proved by studying that component known as the crossed-extension reflex. As we have pointed out above, stimulation of an afferent nerve from one foot produces a contraction of the contralateral extensors and an inhibition of any contraction which may exist in the flexor muscles (Pig. 226). Certain collaterals of the afferent paths of the reflex must be assumed to cross the cord and exert an inhibitory effect upon motor neurons of the flexors of the crossed hind limb somewhat in the manner which Fig. 240 indicates. In a similar way the paths which lead to the fore limbs must give off collaterals which inhibit the contraction of those muscles which would oppose the assumption of the reflex figure. A single afferent path may in this way, not only produce contraction in a large group of muscles, but by the inhibition of the activity of their antagonists it can preoccupy a large part ' of the reflex mechanism of the spinal cord to the exclusion of other reflexes. Because of the compensatory nature of the component parts of the reflex figure, (including the occur- rence of reciprocal inhibition in each part), which is brought about by the connections of the various collaterals of the afferent path, the spinal cord is enabled, quite independently of the higher centers in the brain, to effect a high degree of coordination in reflex response. The various parts of the reflex figure do not respond to the same threshold value of stimulation. Weak stimulation brings into activity only those muscles which affect the part to which excitation is applied. If the strength of stimulation is gradually increased, more and more parts of the total reflex figure appear. In stimulating the hind foot with increasing intensity, first the ankle alone is flexed, then the knee, and finally the hip. The response then spreads to include the extension of the crossed hind leg and finally involves the fore legs also. The mechanism which determined the course of this march of the reflex figure is the graded resistance of the synapses described on page 842. It may be reemphasized, however, that as the reflex spreads to each new joint the inhibition of the antagonists occurs coincidently with the 944 CENTRAL NERVOUS SYSTEM contraction of the active muscles, and the resistance of the synapses lying in the path to the antagonistic muscle groups must be equal. Rules for the Spread of Spinal Reflexes Sherrington 37 has laid down the following rules for the spread of short spinal reflexes, as the components of a reflex figure which in- volve only a few spinal segments may be designated: 1. The degree of reflex spinal intimacy between afferent and efferent spinal roots varies directly as their segmental proximity. 2. For each afferent root there exists in immediate proximity to its own place of entrance in the cord, e. g., in its own segment a reflex motor path of as low a threshold and of as high potency as any open to it anywhere. 3. Motor mechanisms lying in the same region of the cord are un- equally accessible to the local afferent channels, if judged by pressor ef- fects, i. e., the production of contraction. This rule breaks down, how- ever, when it is considered that an afferent path may produce inhibition in many of the motor mechanisms to which it has access. 4. The groups of motor nerve cells contemporaneously discharged by spinal reflex action innervate synergic and not anergic muscles. As a consequence the muscular contractions are harmonious with one another (Reciprocal Inhibition). 5. The spinal reflex movement elicitable in and from any one spinal region will exhibit much uniformity despite considerable variety of the locus of incidence of the exciting stimulus. The spread of long spinal reflexes, which are parts of a reflex figure involving many segments of the cord, is too inconstant in a single re- flex figure and varies too greatly in different figures to permit any defi- nite rules to be laid down. CHAPTER CI THE INTEGRATION OF SIMULTANEOUS AND SUCCESSIVE RE- FLEXES The Principle of the Final Common Path A single reflex acting independently of the rest of the central nervous system does not really occur. An afferent impulse on entering the cord spreads so as to involve a large variety of motor neurons, each of which may, however, be excited through other afferent fibers arriving either from other receptors or from higher nerve centers. The motor neuron itself may therefore be a pathway occupied at different times by very different types of nerve impulse. Hence it is appropriately called the final common path, and its activity at any moment must depend on the nature of the various afferent impulses that are transmitted to it through the synapses. That each motor neuron must be at the service of several afferent neurons becomes evident when it is remembered that in the spinal nerve roots there are three times as many afferent fibers as there are efferent fibers, and if the afferent fibers of the cranial nerves are taken into account the proportion of afferent to efferent fibers becomes five to one. Reflex connections involve usually one or more internun- cial neurons, and these may act as a common path connecting several receptors with a common motor mechanism. The propriospinal neurons of the reflex arc for the scratch reflex described on page 935 act as a common path for impulses arising from distinctly different parts of the skin. The various reflexes which share the final common path leading to a single group of muscles may cause these muscles to respond (1) in some definite way as in the maintained contraction of the flexion reflex; (2) in some different way, as in the rhythmic response of the scratch reflex; or (3) inhibition may be produced so that no contraction can be brought about. Reflexes belonging to any one of these groups are allied reflexes, because they have a common effect on the motor mechanism. Reflexes belonging to different groups, which consequently affect the final common .path to different purposes are antagonistic reflexes. Inte- gration in reflex activity depends on the consequences which follow when allied or antagonistic reflexes act upon the final common path, either simultaneously or in rapid succession. 945 946 CENTRAL NERVOUS SYSTEM The Integration of Allied Reflexes Simultaneous Combination. The scratch reflex is well adapted for the study of this subject since the skin area from which this reflex can be elicited is very widespread (see Fig. 238). The type of reflex produced from any given area is in general the same, although "the local sign" that is, the point at which the animal scratches will vary according to the point stimulated. If we take point A in the reflex scratch area and apply to it a stimulus which is just inadequate to produce any reflex at all, and then, while this stimulus is still in progress, apply a similar subliminal stimulus to point B a little removed from it, the two sub- liminal stimuli will become effective and produce a typical scratching movement. In other words, the subliminal stimulus of point A be- comes added on the final common path with the subliminal stimulus of point B; the one has reinforced the other. In a similar way two stimuli each of which are adequate to excite a weak reflex response from a common motor mechanism, will reinforce one another and produce a strong response if they are applied at the same time. The receptors from which these mutually reinforcing impulses are re- ceived need not, as in the above example, be of the same kind, similar results being obtained by stimulation of receptors of widely different kinds, such as exteroceptors and proprioceptors. For example, if a stimu- lus inadequate to elicit a flexion reflex is applied to the skin of the leg, and another stimulus, itself also inadequate, is applied to the central end of some deep afferent nerve in the same leg, then the two subliminal stimuli will become effective in producing a flexion movement. We have seen that in the development and maintenance of posture the closest alliance takes place between the exteroceptive reflexes which initiate movement and the proprioceptive reflexes which adapt the tone of the muscles to the new positions of the limbs (page 917). Allied re- flexes may reinforce one another even though the exciting stimuli are applied far apart. The flexion of the fore limb which results from stimulation of the fore paw is reinforced, for example, by a simultaneous excitation of the contralateral hind foot a stimulation that gives rise to a reflex figure involving flexion of the fore limb as we have seen in the last chapter. Reflexes which give rise to inhibition may also reinforce one another in their action on the final common path. Successive Combination. The reinforcement which one reflex lends to a second allied reflex lasts for a short time after the stimulus for the first response has been removed. Consequently successive allied reflexes may reinforce one another. This reinforcement also can be illustrated in the case of the scratch reflex. If a stimulus, inadequate to excite when INTEGRATION OF SIMULTANEOUS AND SUCCESSIVE REFLEXES 947 acting alone, is applied to a point A on the skin, and immediately after a second stimulus also inadequate to excite, is applied to a point B, the combined effect of the two successive subliminal stimuli may give rise to a reflex response. The threshold of some common part of the reflex arc has been lowered by the effect of the preceding inadequate stimulus, even though it was applied to a somewhat remote part of the skin. For this reason a moving stimulus applied to the scratch area is far more effective than a stationary stimulus applied over the same extent of area. This phenomenon is called immediate induction, and it is by no means confined to the spinal cord. It is well illustrated, for example, in the case of vision. If a thin line drawn on a white card be looked at so that it falls on the edge of the receptive field of the retina, it will not be seen so well as a dot of similar width which is moved through the same distance as the line. The Integration of Antagonistic Reflexes Simultaneous Combination. Two antagonistic reflexes, which use the final common path to cross purposes, naturally cannot both succeed in occupying it at the same time. We must examine what results when two such reflexes come into competition for the control of a common motor mechanism. The crossed extension reflex and the flexion reflex are two responses utilizing a common group of muscles in opposite ways. If we apply a stimulus to the skin of the leg of a decerebrate animal while the limb is extended as the result of a stimulus applied to the contralateral leg, the extension gives way completely to the flexion reflex (Fig. 239). By choosing the relative strengths of the stimuli properly a preexisting flexion reflex may also be interrupted by a crossed extension reflex (Fig. 226). In a similar way the scratch reflex gives way before a flexion reflex. The significant fact is that when antagonistic reflexes are in simultaneous competition for the final common path, one of them occupies the path to the exclusion of all others. Accordingly there is no tendency for a fusion of their effects, and this is most fortunate, for fusion would only result in confusion, since the response would be ap- propriate for neither of the simultaneous stimuli. This is perhaps the most important principle in the integration of spinal reflexes by the final common path. In the competition of antagonistic reflexes for the control of the final common path several principles determine the outcome. These depend on (1) the nature of the reflexes, (2) the relative intensity of the stimulus, and (3) fatigue. The nature of the reflex is dependent on the quality of the stim- ulus and the purpose to which it is employed. The most prepotent re- flexes are the flexions which result from the application of harmful 948 CENTRAL NERVOUS SYSTEM stimuli. Their purpose is to protect the body from immediate danger and consequently they tend to displace other kinds of reflexes from occu- pancy of the final common path. The reflex responses to all stimuli which are capable in the conscious animal of giving rise to strongly affective sensations tend to prevail over all others and consequently the sexual reflexes share with the responses to painful stimuli a position of domi- nance in the competition of reflex activity. At the opposite extreme are the postural reflexes which arise from proprioceptive stimuli and con- cern chiefly the extensor muscles which must support the weight of the body. These give way before competing reflexes of other types with facility. It is important that they should do so, since postural tone must always adapt itself to the position into which the body has been forced in response to the stimuli of the environment. In the case of antagonistic reflexes of equal potency the result of competition for the common path depends on the relative strength of the exciting stimuli. Thus a flexion reflex of the hind leg will usually displace a simultaneous scratch reflex, but if the stimulus eliciting the scratch is strong, and that tending to produce flexion is weak, the scratch reflex may persist and the flexion fail to assert itself. When a reflex has occupied the final common path for some time it will become fatigued, and may then be displaced by an antagonistic reflex which could not previously compete against it successfully. Thus, ordinarily the scratch reflex is much less readily elicited than the flexion reflex, and if both are excited at the same time the latter will prevail; but if the flexion reflex is kept up until it shows signs of fatigue, then by simultaneous excitation of both reflexes the scratch reflex will obtain the mastery. The development of successive induction, which is de- scribed below, also assists the competing reflex in gaining control as its antagonist becomes fatigued. The susceptibility of reflex arcs to fatigue is probably of importance in assuring the development of variety in reflex responses, since it prevents prepotent responses from occupying the final common path for too long. Many characteristics differentiate reflex fatigue from the fatigue of an isolated nerve-muscle preparation. The most impor- tant of these distinguishing features are as follows: (1) The fatigue comes on intermittently; thus, when the flexion reflex is persistently elicited, the first sign of fatigue is an irregular decline in the flexion movement followed by its entire disappearance for a short time. These lapses become more and more frequent, until at last complete fatigue sets in and no flexion occurs. (2) Reflex fatigue soon passes off. (3) It appears earlier for weak than for strong stimuli. (4) The movement produced by the reflex action may also change in character during re- flex fatigue ; thus, the beat of the scratch reflex may become slower INTEGRATION OF SIMULTANEOUS AND SUCCESSIVE REFLEXES 949 and less steady and the foot be less accurately directed to the spot stimulated. The locus of the fatigue in the reflex arc can not be the motor neuron itself, for, after this has been completely fatigued by stimulation of the scratch area, the same muscles may quite readily execute the flexion reflex if a painful stimulus is applied to the skin of the hind leg. It must consequently lie in some part of the afferent side of the reflex arc. Since we know that the nerve trunk is infatigable, the natural assumption is that reflex fatigue is due to a change in con- duction across the synapses of the neurons. Successive Combination. If antagonistic reflexes occupy a final com- mon path in succession it is found that the use of it by one reflex Fig. 242. Successive induction illustrated by the crossed-extension reflex. The reflex, elicited periodically with a stimulus of low intensity, is recorded in A and B. Between B and C a strong flexion reflex was provoked and maintained for 45 seconds. C illustrates the immediately succeeding extension reflex, which is greatly augmented by successive induction. The next extension (Z?) is also slightly augmented, but in the third extension (E) the augmentation has passed off. The signal recording the stimulation is above; time is indicated in seconds below. (From Sherrington.) facilitates its subsequent use in a movement antagonistic to that which first occupied the motor mechanism. This phenomenon is known as successive induction. In the spinal animal successive induction is demonstrated by us- ing two reflexes that are of a more or less antagonistic character for example, the flexion reflex and the knee-jerk, or better still the crossed extension reflex and the flexion reflex. If we elicit the knee- jerk in a spinal dog at regular intervals, with stimuli of equal intensity, the extension movements (the kicks) will be approximately equal. If now we apply a nocuous stimulus to the skin of the foot and so throw the leg into flexion, it will be found, after the flexion movement has dis- 950 CENTRAL NERVOUS SYSTEM appeared, that the knee-jerk is much more pronounced than previously. Similarly, if we elicit the crossed extension reflex by nocuous stimuli of equal intensity applied to the opposite limb, the extension movements will be approximately equal. By now throwing the limb exhibiting them into the flexion reflex, the extensor movements will of course disappear, but after the flexion has been discontinued, they will reappear with increased intensity (Fig. 242). These facts show us, then, that after the final common path has been occupied by a reflex of one type, it becomes more available to a reflex of an opposite type. Successive induction gives rise to a series of re- sponses which afford a further example of the compensatory movements produced by the integrative action of the spinal cord. By facilitating the occupancy of the final common path by reflexes opposite in nature to those which have just occupied it, movements are brought about which restore the position which was disturbed by the primary response. In other words, it is evident that if the two opposite reflexes are constantly competing with each other for possession of the final com- mon path, they will tend alternately to occupy it, thus bringing about a rhythmic movement. Such is the mechanism involved in walking; the leg is lifted from the ground (flexion reflex) ; it is then brought on the ground, and the mechanical push given to the plantar surface of the foot brings out the extensor thrust, the appearance of which is greatly facilitated by the fact that immediately before the flexion reflex occupied the final common path. CHAPTER Oil THE INTEGRATIVE ACTION OF THE CEREBRUM The motor areas of the cerebral cortex are connected through the pyramidal tracts with the various lower motor neurons of the body. We have seen that the excitation of a localized area in the motor cor- tex may bring about a response of some localized group of muscles which is coordinated, involves recriprocal inhibition, and develops through an orderly march in much the same way as does the reflex figure which arises from a cutaneous stimulation. These responses utilize the same motor mechanisms as the spinal reflexes do, and con- sequently come in competition with them for use of the final common paths. Consequently cerebral influences may modify profoundly re- flex responses, reinforcing them when both affect the motor mechanism in the same way, inhibiting them when their actions are antagonistic. The inhibitory aspects of the cerebral influence are particularly prom- inent, and consequently many reflexes are elicited with greater cer- tainty in animals from which the cerebrum has been removed. In this competition probably much the same factors determine which influence shall control the common path as govern which of two antagonistic spinal reflexes shall prevail. Cerebral influences are apparently prepo- tent over all but the most intense reflex responses to harmful stim- uli and those which result from strongly affective sensations. We can, for example, inhibit the reflex withdrawal of the hand from hot water unless the pain is particularly intense. On the other hand it is diffi- cult to refrain from winking when the cornea becomes irritated. Within limits the respiratory reflex may be controlled by the will, but when the stimulus to the respiratory center becomes intense, the breath can no longer be held. A large group of reflex arcs concerned with the regulation of the visceral organs are not connected with the paths from the cerebrum and consequently cannot be brought under vol- untary control. In describing the motor areas in the cortex it was pointed out that these were points at which many neurons from widely separated parts of the brain converge upon paths which lead to the lower motor neu- rons and their muscles. The pyramidal fibers consequently are com- mon paths used in many diverse volitional responses. For their con- trol many different influences come into competition and the unpre- dictable nature of volitional response is no doubt due to the impos- 951 952 CENTRAL NERVOUS SYSTEM sibility of determining which of the antagonistic elements in this com- petition will prevail. Deviation from the " straight and narrow path" may well be due to the failure of the proper influences to gain control of the internuncial mechanism of voluntary action to the exclusion of all others. The paralyses of hysteria are perhaps also due to the con- trol of the common paths from the brain by an inhibitory influence which cannot be displaced by ordinary volitional impulses. The Relation of the Cerebrum to the Distance Receptors. The de- velopment of the brain in the leading segments of the body is asso- ciated in all animals with the acquirement of the distance receptors of the head, i. e., the eye, ear, and olfactory organ. These sense organs serve to acquaint the organism with parts of its environment with which it has not yet come into immediate contact. They are suited to bring about responses which are anticipatory of the consequences of more direct contact with objects in the environment, that is, of the seizure and consumption of food, the appropriation of a mate, or the avoidance of objects w r hich might prove harmful on closer contact. In order that responses toward distant objects may be made with dis- crimination, a mechanism has been evolved which apprehends the dis- tant object not merely as a stimulus possessing a single quality, but as a " thing" built up of a number of properties, and the response is determined by these properties as a group. Since the properties of such environmental objects appeal to a variety of receptors, the sensa- tions aroused by each must be combined in the nervous system and built up into a definite concept by a process of association. Moreover, since the response to the distant object is of an anticipatory nature, it must be made with reference to the past experience which the ani- mal has had with objects presenting a similar group of properties. Conse- quently the results of contact with the object must be associated as part of the concept with the various properties which have appealed to the distance receptors, in order that when such an object enters the environment at a later time the results of the previous encounter may be recalled and behavior modified accordingly. In other words distant objects appeal to the nervous system by virtue of the meaning which is placed upon the particular combination of receptors which they excite, and the sensations which arise as a result. The nature of the response which is thus called forth depends on the memory of past experience with similar objects. For this reason the development of distance receptors is associated with the development of the cerebrum in which association and memory mani- fest themselves. The modification of behavior through the correlation of present and past experience is the process of learning, which is one of the chief characteristics by which responses influenced by the cere- INTEGRATTVTE ACTION OP THE CEREBRUM 953 brum are differentiated from those of the loAver levels of the nervous system. The relation which the distance receptors bears to the cerebral proc- esses is well illustrated by a comparison of the responses of animals of different kinds toward light. Many of the lower organisms possess eyes incapable of forming an image such as is produced in the eye of the vertebrate. Their eyes are so arranged as to be stimulated by light coming only from a certain direction. Consequently the eye may form a guide which determines the direction of progression, which will be either toward or away from the light according to the kind of animal which is studied. Such an animal obviously cannot act with much discretion in regard to light from different sources, since the shape of the source of light and perhaps the color of the light make no appeal to it. The insects and Crustacea possess eyes which are capable of forming an image and consequently might be capable of distinguishing between the light of a candle and that from an open window. The development of the nervous system of these animals has not kept pace, however, with Fig. 243. Postures assumed by the robber fly when the eyes are unequally illuminated: illustrat- ing the influence of light on the tone of the muscles. A, the lower half of each eye is blackened; B, the upper half of each eye is blackened; C, the right eye is blackened. (After Garry.) the optical perfection of the ocular mechanism. Each part of the retina is connected in a rather inflexible way with certain parts of the motor mechanism. Unequal stimulation of the different parts of the retina causes certain parts of the musculature to become more active than other parts in other words sets up a characteristic reflex figure (Fig. 243). In the robber fly, for example, light falling on the upper half of the retina, when the lower half is covered by opaque cement, causes the trunk to be curved upward, the forelegs extended and the hind legs flexed. On taking flight the insect tends to swerve upward and backward and consequently loops the loop. Blinding the upper half of the retina has the reverse effect. If one eye only is covered, the legs of that side are extended, those of the opposite side flexed, and in walking the fly tends to circle toward the side on which the eye is exposed to light. By virtue of the physiological influence of the eyes on the muscles the direction of locomotion of these insects is guided with mechanical pre- 954 CENTRAL NERVOUS SYSTEM cision toward the light. The flight of the moth into a candle is deter- mined in a similar way. The nervous system of the insects is singularly incapable of modifying its responses as a result of past experience; it is weak in the display of association and memory. Consequently the moth does not learn that contact with a candle flame is attended by dis- aster and repeats its flights into the flame until it has achieved its self- destruction. It is because of the development of the associative powers of the cerebrum parallel with the perfection of the ocular mechanism that the behavior of the mammals and man is adjusted to past experience with greater success than is that of the insects. The human infant devel- ops in the fifth month a reaction which leads it to reach for any ob- ject brought sufficiently close within its field of vision. The response is elicited by a lighted candle as well as by a harmless object such as a piece of candy. When reaching for the candle is first developed, the movement is not checked until the heat of the candle actually reaches the fingers and sets up a protective flexion deflex. By repeated trials the baby develops within two months a modified response to the candle, the reaching reaction being completely inhibited. Reaching for candy still persists without diminution. The infant has developed an asso- ciation between the particular configuration of stimuli which the candle produces upon the retina and the harmful consequences of reaching for this object, and responds to the stimulus by a response suitable to the painful effects which have previously attended its experiences with the candle. Conditioned Reflexes The nature of the responses of animals with the cerebrum intact is less predictable than that of the spinal animal in which the cord is removed from cerebral control, because these responses are conditioned by the previous experience of the animal and the associations which it has formed between various stimulating objects and the consequences which result from its hereditary types of response. The altered responses which develop by virtue of the modifying influence which the cerebrum exerts over reflex action are consequently called conditioned reflexes, in contrast to the unconditioned reflexes of the spinal cord. It must be considered as one of the greatest advances of modern physiology that Pavlov and others should have succeeded in evolving methods by which we may arrive at conclusions regarding the nature of certain of the integrations which occur when such conditioned reflexes are formed, since they show us the elementary nature of the processes by which the association of the results of sensory stimuli leads to modi- fied forms of behavior. 1NTEGRATIVE ACTION OF THE CEREBRUM 955 The methods employed for the study of these higher integrations of the central nervous system all depend on the reactions of the animal that are associated with the taking of food. When the food is actu- ally placed in the mouth, it excites a secretion of saliva, whatever the circumstances may be. This is an unconditioned reflex. Suppose, how- ever, that every time food is given a particular sound is made; after some time it will be found that the occurrence of the sound alone is sufficient to cause a secretion of saliva. In other words, a conditioned reflex has been formed. Similarly, sight or smell or any other type of sensation may be made the excitant for the conditioned reflex. The secretion now becomes psychic instead of merely physiological. To quote Bayliss: "Any phenomenon of the outer world for which the animal in question possesses appropriate receptors can be drawn into temporary association with salivary secretion, so that it becomes an exciter of se- cretion if only it has been frequently presented at the same time with the unconditioned reflex stimulus, food in the mouth. " Work along lines similar to that devised by Pavlov has more recently been undertaken by students of animal behavior, who have utilized the acquired habits of an animal in searching for its food in order to study the influence of conditioning circumstances on its procedure. The advantage of this method depends mainly on the fact that it can be applied to all groups of animals. In carrying out such an observation, the animal is placed in one compartment of a cage, from which it is then released to a second compartment, the end of which is divided into two passage- ways, one leading to food, the other leading to some compartment in which the animal is punished for its mistake as by receiving an electric shock. Objects such as colored lights are placed in the different pas- sageways, and the animal by repeated trial comes ultimately to learn which particular colored light signifies the passage along which he will receive food. A reflex has therefore become established conditioned on the particular colored light. On account of the unavailability of his publications, it is impossible at present to give any complete account of Pavlov's discoveries. A few facts, however, are of such importance that it is necessary for us to state them here as far as we know them. (See Bayliss, Physiology.) Two mechanisms seem to be concerned in the conditioned reflexes: (1) that of temporary association, and (2) that of analysis. Temporary associa- tion is well illustrated in the above experiment in which the secretion of saliva is induced by a sound. Temporary association of the sound with the secretion of the saliva may readily be inhibited by all kinds of ex- ternal phenomena; thus, if the dog's attention becomes diverted while the conditioned reflex is being stimulated, the response does not occur. 956 CENTRAL NERVOUS SYSTEM In a dog that had been trained to secrete saliva at the sound of a par- ticular metronome beat, inhibition occurred one day because, just as the dog was being presented with the food, the laboratory servant made a noise outside of the building which diverted the animal's attention. The conditioned reflex may also be interfered with by internal inhibi- tion, which is illustrated by experiments in which, after a dog has been trained to respond to a given conditional reflex, several occasions follow when food is not given to the animal after the particular sensation to which it has been trained to respond. The condition for example, a sound loses its effect. This is internal inhibition, but it is a temporary condition since the reflex returns of itself after a period of rest. These experiments illustrate what is meant by the formation of tem- porary associations occurring in conditioned reflexes, but in order that there may be a fine discrimination between those stimuli which shall and those which shall not serve to call forth the conditioned reflex, an- other mechanism becomes involved that of analysis. This is performed by a sense organ the function of which is to separate and distinguish the complicated phenomena of the outer world. For example, it has been proved that small differences in the pitch of a musical note may determine whether or not a conditioned reflex will be excited or in- hibited, as in the case of one animal that was trained to respond by the secretion of saliva to a tuning fork vibrating at 100 per second. It was found that no secretion was produced by a tuning fork vibrating at 104 or at 96. Much work has also been done with the skin receptors. Thus, when a given spot of skin is stimulated every time that food is presented, this becomes an active spot for the conditioned reflex. At the same time another spot may be stimulated so as to be associated by the animal with the nonpresentatioii of food; it is a conditioned reflex for no food, and is associated with the absence of salivary secretion. By comparing the responses from active and inactive spots when both are stimulated either simultaneously or at close intervals, much can be learned concerning the delicacy of appreciation for external stimuli and the influence of the inhibitory on the excitatory process. Bayliss cites the following experiment. Along a series of spots on the skin of the leg five devices are arranged for producing equal mechanical stimulations of the skin. The four uppermost of these .are made active spots for the salivary reflex, and the lowest one inactive that is, when- ever it is stimulated no food is presented. Let us suppose that upon administering mechanical stimuli of equal intensity to each of the four active spots, a certain amount of saliva is produced in a certain time; if now the inactive spot is stimulated and then thirty seconds later one of the uppermost spots, there will be no secretion. The previous stimu- INTEGRATIVE ACTION OF THE CEREBRUM 957 lation of the inactive spot must have caused an inhibition to be set up in the nerve centers concerned in the reflex. This inhibition only gradually passes away, disappearing first in the spot farthest removed from that made inactive, but it may take several minutes before all the active spots have reacquired their original sensitivity. The persistence of the inhibition produced by stimulating the inac- tive spot in the above experiment indicates an important factor in con- nection with the production of conditioned reflexes. For example, an animal can be trained to know that in a certain number of minutes after the sound of a given bell food will be presented to him; the condi- tioned reflex will become established so that he salivates at exactly the same time after the bell is sounded. Something must be going on in the centers during this time something inhibiting the reflex. If during this interval of inhibition some other sensory stimulus is applied, it will be likely to cut short the inhibition; in other words, it produces an inhibition of inhibition, so that the secretion of saliva occurs. Another most curious combination of conditioned stimuli is illustrated in the following experiment. Suppose, for example, that a given light and sound are each separately made a stimulus for a conditioned reflex, but that when they occur together there is no reflex. Suppose now that while one of these active stimuli is being presented, the other stimulus is also presented; the result will be that the secretion produced by the one stimulus will stop. Evidently, although each is in itself a stimulus, acting together they cause inhibition. By studying the conditioned reflexes after a certain part of the cere- bral cortex has been removed, it has been found that the power of estab- lishing certain kinds of conditioned reflexes becomes abolished, while that for others is retained. The writing of Sherrington, 37 Loeb, 38 Watson, 39 and Margulis 41 should be consulted for further details concerning the material in this chapter. CHAPTER CHI THE HIGHER FUNCTIONS OF THE CEREBRUM IN MAN; APHASIA The study of the higher functions of the cerebrum leads us to the border- land between physiology and psychology, but into this vast and relatively unexplored field we can not venture here, unless just far enough to gain a suitable vantage point from which to understand the pathology of the condition known as aphasia* As we have seen from our studies on cerebral localization, the cerebrum must be regarded as a great sensorimotor gan- glion, whose functional activities are indicated by various movements. These movements may, in general, be classified as objective indications either of feeling and emotion or of intelligence. Although both classes are evident in all animals, it is particularly in the case of man that the evi- dences of intelligent activity are especially prominent, since they include gesticulation and the muscular activities required in spoken and written language. The movements that express emotional conditions are evolved earlier and from lower planes than those of intellectual activity. Thus, very young infants "make faces" when there is reason to believe they feel pain, and, as they develop, their power of expressing emotion is evolved long before they present evidence of intelligent motor activity, and still longer before they can articulate words. The phenomenon of human psychic activity which is of greatest im- portance is that of language, and to understand the nature of the cerebral integration required to produce it, we must briefly consider the cerebral processes involved in the intellectual development of the infant. The first step in this development is the storing away in projection centers of memories of the sensations which these centers have received. For ex- ample, when the child looks at a bell, there is stored in the visual center a memory of the shape of the bell, and when the bell moves so as to produce sound, this also is stored as a sound impression in the auditory center. Likewise, when he touches the bell impressions of its hardness and smooth- ness and temperature are stored in the centers for cutaneous sensations. At first each of these memory impressions occupies an isolated position; but later, association tracts open up between them, so that the calling forth of one memory impression is associated with others, and the child *Free use of the article by Bolton 40 is made in this chapter. 958 HIGHER FUNCTIONS OF THE CEREBRUM IN MAN; APHASIA 959 comes to be able to associate the appearance or image of the bell with a certain sound and with certain sensations of hardness, rotundity, etc. This preliminary use of observation is known as perception. It involves the fusion of direct sensations as well as their correlation with memory im- pressions, of former sensations. The number and variety of the latter called into activity by a particular sensation will obviously vary at dif- ferent times. On seeing a bell, for example, a child may associate it with sound on one occasion, and on the next with the feeling of the bell. On account of this difference in the detail of the method of association, it is evident that perception must be a product of cerebral integration rather than one depending on memory impressions stored in the isolated centers. It is a complicated process with an infinite variety of possibilites as to the exact way in which it is integrated on each occasion. The act of perception, however, becomes considerably simplified in the higher animals by the laying down of short-cut paths of association. These are formed first of all with the auditory center, in which the memory impression of an articulated sound representing the object for example, the word ''bell" is stored away. The child comes to learn that this par- ticular word is to be associated with the memory impressions it has stored awajr of the sound, the sight, and the feeling of the bell. Similar short-cut paths later become developed in connection with the visual centers, where a certain symbol, like the word "bell," is presented to the child as signi- fying all the other attributes of bell. In its most highly developed form, therefore, perception may be described as the act of calling up one or more sensorimemorial images when a name is seen or heard. Having acquired the ability to integrate sensorimemorial impressions in the above described manner, the child next learns to integrate the motor centers concerned in the control of the articulatory apparatus so as to produce a sound. This sound is the word indicating the object involved in the integrating process. It is the integration necessary to produce the sound which symbolizes the particular object. When the power of understanding and producing language has been acquired, the crowning process of intellectual development the forma- tion of a concept, or general notion becomes evolved. Thus, the evolu- tion of a general name will include a number of particular objects or acts. "This process of conception involves the revivification of numerous sen- sorimemorial images which present common points of similarity" (Bol- ton). It is relatively a simple process for such general objects as animal, man, building, but becomes very complex for such abstract concepts as heaviness, beauty, etc. It is obviously a process to which no one cerebral center can be assigned. The outward manifestation of the conception is spoken or written language. 960 CENTRAL NERVOUS SYSTEM Language consists, therefore, in an extremely complex symbolic system, involving various centers and association tracts in the cerebrum, and capable of an almost infinite degree of development by the laying down of new symbolic systems. Language, indeed, becomes the instrument of thought, practically all of the higher intellectual processes being dependent on its evolution. In this connection it is interesting to note that a great number of individuals, especially those who do not read, depend on the sense of hearing for the acquisition of the impressions required for their psychic development, while others depend on the sense of sight for the same purpose. At least four different types of center are involved in the integration of language; namely, auditory, visual, chirographic, and articulatory. We may call these "word centers," and we must assume that they lie near to the auditory, visual and general sensory projection areas of the cortex. To understand and to be able to produce spoken and written language, it is necessary that all these four word centers participate through associa- tion tracts, although the meaning of a word may be perceived without all of them being involved. PSYCHOPATHOLOGICAL APPLICATIONS In the study of mental diseases the most important conclusion which we can draw from the above facts is that language is essentially a sym- bolic mechanism for the integration of sensorimemorial images. It is therefore the symbolic system of the integrated processes of the brain; it is the servant of thought. When, as is often the case, language is used without the proper exercise of thought, it becomes merely an automatic affair. A practical deduction from these facts is that any considerable derangement of the language mechanism must necessarily involve some interference with the complicated processes of association that go to make up the psychic function. These considerations naturally lead us to the subject of aphasia. It has been usual to distinguish three varieties of this; namely, motor aphasia, sensory aphasia, and anarthria. In motor aphasia the patient, although he understands what is said to him, is unable to speak, and the intellectual powers are little, if at all, impaired. In sensory aphasia speech is possible in a more or less intelligible manner, but there is a distinct impairment of intelligence. In anarthria, or subcortical aphasia, the only disability is the loss or impairment of the power of articulate speech because of some lesion existing in the center coordinating the lower neurons concerned in the movements of the laryngeal and tongue muscles. Pierre Marie, as a result of very extensive experience in Paris, has shown that this classifi- cation is unjustified. He maintains that there is only one true form of HIGHER FUNCTIONS OF THE CEREBRUM IN MAN; APHASIA 961 aphasia, and that such a thing as pure motor aphasia as above defined does not exist, the condition being invariably accompanied by intellectual impairment. Marie points out that the various claims that aphasia may exist without intellectual impairment have been made without sufficient investigation of the intellectual status of the patient. He shows that many patients suf- fering from aphasia if asked to do ordinary things, such as cough or spit or raise the hand, can do them as well as a normal individual, but that these after all are very crude acts in the ordinary performances of a normal individual. To test the intellectual powers it is necessary to require the patient to perform acts which entail a considerable amount of cerebral integration. We must ask him to perform some sequence of events such as walking several times in one direction, then in another, touching cer- tain objects, etc., or better still we should observe the patient closely in his business transactions and everyday routine of life to see whether he does things exactly as he did them before. It is always possible by such tests to show that in aphasia the mental powers have become distinctly de- preciated. The portion of the cerebral cortex affected in aphasia is always in the neighborhood of the so-called area of Wernicke, which is closely related to the visual and auditory centers. In making this sweeping conclusion, Marie admits that cases of pure word-blindness but not of word-deafness may exist ; that is, a patient still retaining his intellectual powers may lose his ability to interpret correctly what he sees, although he can still interpret accurately what he hears. This conclusion conforms exactly with those of the psychophysiologists regarding the difference in the language mechanisms of educated and un- educated persons. Language is learned through the sense of hearing, and it is only by later education that more is learned by the sense of sight; that is to say, a person learns to read only after he has learned to under- stand spoken language. Word-blindness may therefore occur as a pure symptom, and is less likely than word-deafness to be associated with ab- normal integrative functions of the cerebrum. Word-deafness however de- pends upon a lesion involving the auditory center; it necessarily means disturbance in the association functions of the cerebrum, and is always accompanied by a certain amount of mental derangement. In corroboration of these facts may be cited the well-known fact that a deaf-mute is mentally far inferior to one that is congenitally blind. Loss of hearing leads to more serious cerebral disability than loss of sight. To quote Bolton again, "In such cases deafness is therefore a more serious deprivation than blindness, as, for the evolution of the functional activity of the cerebrum, an entirely new development of associational spheres to 962 CENTRAL NERVOUS SYSTEM replace those normally employed for auditory and spoken language has to be acquired. In the case of congenital or early-acquired blindness, on the other hand, the complex sphere of language, with all its psychic com- ponents, can be employed in a perfectly normal manner and almost ex- actly as it is brought into use in the case of persons who neither read nor write." It would be beyond the scope of this work to go into the clinical and pathological evidence upon which Marie bases his far-reaching conclusions. Suffice it to say that it is definitely shown that the old contention of Broca, that a special speech center exists, is entirely unjustified by the facts of clinical and pathological experience. Broca, it will be remembered, con- tended that motor aphasia is always due to destructive processes occurring in the lower portion of the ascending frontal convolution on the left side, and he concluded that this portion of the cerebrum represents the speech center. Marie has shown, however, that a patient may show distinct evi- dence of aphasia without any lesion involving this so-called Broca area, and, on the other hand, that cases not infrequently occur in which this is completely destroyed without any evidence of aphasia. Important though this discovery of the inaccuracy of Broca 's conclusion is, by far the most important conclusion which we may draw from Marie's work is that, since language is a product of an extended integration of impressions and memories stored in different parts of the cerebrum, it is not so likely to be interfered with by destruction of any one of the centers as it is by destruc- tion of the paths which connect the centers with one another. As a matter of fact, Marie has shown that in cases of aphasia the lesion is nearly always located in the course of the pathway connecting the visual and auditory centers with the other centers of the cerebrum ; it lies around the upper end of the fissure of Sylvius in the region which in previous years had been considered particularly associated with the condition known as sensory aphasia. Those interested in this subject should consult Bolton's article. CHAPTER CIV SUMMARY OF THE ORGANIZATION OF THE MAMMALIAN NERV- OUS SYSTEM ; SPINAL SHOCK The activity and organization of the nervous system is so complex that it has been necessary to treat each aspect of it separately and with little reference to its relation to the organization of the whole. We will now consider briefly how the various parts of the nervous system are com- pounded to form a unified machine for coordinating the activities of the body. The evolutionary development of the nervous system has indi- cated that two primitive types of nervous organization came into existence at an early stage and persist in certain parts of the mammalian body. The most primitive of these was the nerve net which was evolved by the coelenterates and which persists in the mesenteric plexus of man. The other is the segmental synaptic nervous system of the worms, which finds its representative in the spinal reflex system of the verte- brates. We have seen that these systems are not independent, but that the spinal reflex mechanism exerts a controlling influence over the peripheral nerve net through the autonomic nerves. Vertebrate evolution has brought to perfection two additional functional systems which modify and regulate the activity of the spinal reflexes, and through them, to a certain extent, the organs controlled by the peripheral nerve net. These systems are (1) the mesencephalicospinal system for the con- trol of postural tone, excited by the proprioceptive impulses which arise in the muscles, joints, and from the labyrinth and (2) the cortico- spinal system for the execution of voluntary movements initiated by im- pulses received in large part by the distance receptors of the head and conditioned by the previous associations and memories which the stimula- tion of the distance receptors calls up. The corticospinal system not only controls the activity of the spinal reflex mechanism through the connections afforded by the pyramidal tracts, but to it the system for regulation of postural tone is also subservient. The arrangement of the different levels of nervous activity in their relation to the effectors of the body is somewhat as shown in the diagram on page 964. In the intact organism we see the entire mechanism at work; in the experimental animal and in the lesions of warfare and civil life we see the activity of the residual parts of the mechanism which are left in- tact after mutilation has freed them from control by the higher centers. 963 964 CENTRAL NERVOUS SYSTEM When a lower level in the organization of the nervous system is freed from control by a superior level we observe, not only the inde- pendent activities which this level carries on in the normal animal, but certain additional activities commonly held in check by the higher centers. Injury to the nervous system consequently results in certain positive as well as negative symptoms, and these are attributed to the removal of an inhibition from the lower levels which is exerted normally by influences from higher levels now cut off by the lesion. Thus we have seen in the decerebrate animal that the removal of the control of the high- est level allows the postural tone of the extensor muscles to become greatly exaggerated. The spastic paralysis which accompanies many Cerebral Cortex Cerebellum and Midbrain SpinaJ Reflex Nerve Net i Skeletal Muscle Smooth Muscle and Glands cerebral and spinal lesions is probably a positive symptom due to a similar cause (Walshe 34 ). In the decerebrate animal many reflexes may be elicited with a certainty and to a degree unobtained in the unmutilated organism because of the removal of cerebral inhibition. The symptom! of cerebellar injury give a picture of the activity of the nervous system deprived of the normal influence of the mesencephalic regulation of tone. Voluntary movements can still be executed, but no longer with the cus- tomary smoothness or precision, because the mechanism which governs the coordination of muscular movement and tone is no longer effective. In the isolated visceral organs, and particularly in the bladder, indepen- dent activity of the peripheral nerve net is demonstrated. We have seen that these organs may function in an adequate way, but their ac- SUMMARY, MAMMALIAN NERVOUS SYSTEM; SPINAL SHOCK 965 tivities are no longer correlated to the activities of the body as a whole. The extent to which the spinal cord is capable of carrying out co- ordinated neuromuscular activity, when isolated from the higher centers of the brain is a matter of great interest. We have seen in considering the mechanisms by which reflex action is governed that the spinal cord contains arrangements capable of producing highly integrated responses. We will consequently examine the activities of the isolated spinal cord in laboratory animals, and compare them with similar conditions which are observed in man, in order to obtain an idea of the relative importance of the spinal cord and brain which show some diversity in their devel- opment. Spinal Shock and the Recovery of Reflexes in Animals In animals the spinal cord may be separated from the brain by an incision made in the cervical region. Immediately after the operation a profound condition of depression sets in, involving all the reflex arcs in the separated portion of the cord. This condition is known as spinal shock. It supervenes in all classes of animals having a spinal cord, but is much more profound in the higher than in the lower animals. As a result of this depression, the part of the body below the section exists in a limp and flaccid condition, and the application of even very strong stimuli to the skin will evoke no form of reflex movement. In the case of the lower vertebrates, such as the frog, the condition begins to pass off in from twenty minutes to half an hour, after which a stimulus applied to the skin of the foot is followed by a typical flexion movement at knee and hip, the so- called flexion reflex. In the rabbit very little reflex response is elicitable for several hours after the operation, but in a few days the reflexes return completely below the level of the section. In the dog, on which a great deal of work has been done, the involved regions of the body are pro- foundly paralyzed. The skin is in a more or less unhealthy, unnatural condition, the surface cold, the hairs ruffled; and if care is not taken, the slightest abrasion of the surface may result in a nasty ulceration. On account of the paralysis of the centers of micturition and defecation, there is also incontinence of urine and of feces. With reasonable attention, however, the dog makes a wonderful re- covery. After an interval of two weeks the hind limbs, although com- pletely paralyzed so far as voluntary movement is concerned, begin to show considerable signs of improvement. The first reflexes to return are those concerned with the deeper structures, such as the vascular reflexes, thus bringing the skin back to its normal temperature and condition. The reflexes of micturition and defecation also soon return, so that the animal no longer suffers from the continuous discharge of 966 CENTRAL NERVOUS SYSTEM urine and feces. About the same time the knee-jerk becomes elicitable. This reflex is obtained by tapping the tendon which connects the patella with the tibia, the response being a smart contraction of the extensor muscles of the knee joint. The flexion reflex also begins to reappear. This is elicited by applying a pinprick or other hurtful stimulus to the skin of a lower extremity, and when fully developed consists in a flexion of the knee and hip joints. The evident object of this move- ment is that the stimulated parts may be removed from the source of stimulation, and it is plain that all stimuli that produce the flexion reflex are such as would cause in the intact animal a sensation of pain. Such stimuli are thus classified as nocuous, and the reflex is styled a nociceptive reflex. Accompanying flexion of the stimulated limb the op- posite or contralateiral limb usually undergoes a definite extension, called the crossed extension reflex. That the nociceptive reflexes should be among the first to return after spinal transection is of considerable interest as indicating their importance in the protection of the animal from injury. They are the essential reflexes of defense, and it is con- siderably later in the recovery of the animal before reflexes dependent upon stimulation of other tactile receptors begin to show themselves. The most important of this latter group of more special reflex move- ments include the so-called scratch reflex and the extensor thrust. The scratch reflex, as its name implies, is the scratching movement of flexion and extension of the hind limb at a rate of about four contractions per second that occurs when a mechanical stimulus is applied to the flank and shoulder area of the animal. For example, if we gently draw a pencil or the fingers backward and forward among the hairs on this region of the spinal animal, the corresponding hind limb will be brought up so that the claws are approximately at the place stimulated, and the limb thus directed will undergo a series of flexions and extensions, designed evidently for the purpose of scratching the area of skin that has been stimulated. If the stimulus is a weak one, only the initial stages of the movement may occur, such as the preliminary flexion of the leg. As we have already stated, the receptive stimulus calling forth this reflex is very specific in nature. A pinprick or rough friction of the reflex area will not produce it, neither will the application of heat, nor a single electric shock. The most adequate stimulus is one simulating as nearly as possible the con- dition which would be produced by the movement on the flank of the animal of some insect. This more or less complicated scratch reflex can of course also be elicited in animals whose spinal cord has not been cut, but we can not predict in such cases whether the reflex will occur. The brain may inhibit the reflex arc and prevent the movement. In a spinal SUMMARY, MAMMALIAN NERVOUS SYSTEM; SPINAL SHOCK 967 animal, however, the reflex always occurs provided an adequate stimulus is applied. The extensor thrust is elicited by applying pressure to the pad of the paw or the sole of the foot. It consists of a quick extension movement of the corresponding limb usually with a flexion of the opposite limb. After complete recovery from shock, the paralyzed parts of the body are capable of performing even more complex movements than those al- ready mentioned. For example, if the animal is held up with the hind legs hanging down, these will often exhibit rhythmic flexion and exten- sion movements, with the two limbs acting alternately, as they would in walking or running. This is sometimes called the mark-time reflex. Another complicated movement may be produced by placing the animal in water, when it may make the movements of swimming, but its swim- ming will not be sufficient to keep it on the surface. These swimming movements are more perfect in the spinal frog. After complete recovery from spinal shock, the hind limbs are more or less in a condition of extension contracture; the vascular and other visceral reflexes are in perfect condition, and a marked rise in blood pressure occurs when one of the sensory nerves of the hind limb is stimulated an experiment which can be performed in such animals without the administration of any anesthetic, since the animal feels no pain. In female spinal animals impregnation may occur and preg- nancy proceed in normal fashion accompanied by the usual secretion of milk. Spinal Shock and the Recovery of Reflexes in Man The potentialities of the spinal cord of man have not been fully realized until recent years when investigations by Head and Riddoch 42 on men who had been shot through the spine have shown just how complete a recovery can be made from such an injury if the patient is given proper care. In cases of complete transection of the cord there usually re- sults in man, as in the other mammals, a complete loss of reflex activity and of tone in those parts of the body innervated from segments of the cord which lie below the lesion. Occasionally the cremasteric and bulbocavernous reflexes may still be elicited. Below the lesion there is complete anesthesia, the skin is dry and readily becomes gangrenous, and the urine and feces are retained. After a period varying from one to three weeks the first reflexes reappear. These are the flexion reflexes in response to harmful stimuli. At first they can be elicited only from the part of the receptive field which normally is most sensitive, i. e., the sole of the foot, and involve those muscle groups which are normally brought into play by the weakest stimuli. Movements of the toes are conse- quently the only part of the reflex figure to display itself in the earliest stage 968 CENTRAL NERVOUS SYSTEM of recovery. Later the receptive field becomes more and more extensive and the response spreads to additional groups of muscles until the entire leg is thrown into flexion. As it does so the reciprocal inhibition of the muscles antagonistic to the flexors takes place in a normal manner, just as it does in the spinal cat or dog. Finally, however, a stage of recovery is reached in which a phenomenon occurs which is quite unlike anything seen in these animals. It is called the mass-reflex and consists in an extensive spasm of the flexor muscles of the abdomen and lower extremities which is brought about by harmful stimuli applied almost anywhere to the parts of the body affected by the injury. Accompany- ing the response there is a pronounced outburst of sweating in the af- fected regions and if the bladder is as much as half full, a reflex discharge of the urine. The mass-reflex shows that the spinal reflexes have lost their local signature and have consequently become diffuse in their dis- tribution, resembling in this regard the generalized responses of those animals such as the sea anemone (page 830), which possess a nervous system consisting of an asynaptic nerve net. In one to five weeks after the flexor responses first appear the tone of the muscles of the limbs begins to recover and may be restored to nearly, but never to equal, the state found in the normal individual. The posture of the limbs is one of slight flexion. At the same time the ten- don jerks become elicitable. These are the only types of extensor re- sponse which have been observed in cases of complete transection of the human spinal cord. During this stage of recovery the contents of the rectum and bladder are voided automatically (Head and Eiddoch 20 ' 42 ). The chief difference in the condition of the spinal man and of the spinal cat or dog consists in (1) the character of the flexor response, which in man has lost its local signature, is diffuse, and is elicited from an abnormally extensive receptive field, (2) the flexor position of the limbs at rest; and (3) the absence of extensor responses, i. e., of those responses significant in maintaining the upright position and in progres- sion. It would appear from this that in man the higher centers in the brain have taken over control of the integration of spinal reflexes to the extent of determining the local signature and limiting the spread of these responses and of governing the activities of the extensor muscles in maintaining postural tone and the extensor movements of progres- sion ; whereas in the lower mammals these phenomena may be executed by the spinal cord alone. Further evidence for this view is afforded by cases in which the human spinal cord is incompletely divided. In these individuals, although paralysis and anesthesia is complete, the reflex activities may not differ markedly from those of the lower animals. The mass-reflex is absent, flexor responses retain their local signature, and are accompanied by crossed extension in the opposite limb. The postural SUMMARY, MAMMALIAN NERVOUS SYSTEM ; SPINAL SHOCK 969 tone of the extensor muscles holds the resting limbs in slight extension. Responses comparable to the extensor thrust and the mark time reflex of the spinal animal occur. The Cause of Spinal Shock It would seem natural to suppose that the cause of the depression in reflex activity which follows transection of the spinal cord, and which is known as spinal shock, is the irritation set up directly in the tissues injured by the lesion. This irritation might be thought to have an in- hibitory influence on spinal reflexes. That this supposition is incorrect is shown by the fact that after the shock induced by a cervical tran- section of the cord has worn off, a second transection at a lower level does not cause its reappearance. The abnormal character of reflexes recovering from spinal shock does not resemble that of reflexes which are being inhibited, but rather those which have experienced fatigue. Spinal shock might be thought to be due to the disturbance in the cir- culation which follows the spinal transection, but this cannot be the case because all parts of the body would suffer alike from the fall in blood pressure, whereas only those parts of the nervous system below the lesion exhibit shock. This aboral incidence of shock is a very striking character. So slight is the effect of a spinal transection upon the higher centers that men who have been shot through the spine may not even lose conscious- ness. They are aware that sensations from the lower limbs have sud- denly been cut off, and their first impression is that they have been blown in two. Sherrington has described a monkey the cord of which was cut below the cervical region, and which immediately after the opera- tion amused itself by catching flies with the anterior extremities, whereas the posterior extremities were in a condition of the profoundest shock. The most probable explanation of shock, which is in accord with the preceding facts, is that it is due to cutting off from the spinal reflex arcs of some influence normally exerted by fibers descending from the brain. Just what this influence is, or how it facilitates reflex activity is impossible to state, but it would appear that once this influence is cut off, some time is required before the cord can acquire the power of carrying out its reflex functions without its assistance. We have seen that a parallelism exists between the depth and persistence of spinal shock and the development of the nervous system, particularly of the brain, in vertebrate evolution. This fact consequently supports the view that spinal shock is due to isolating the cord from the influence of the higher centers. Moreover those neuromuscular mechanisms are affected which are normally under control of the brain centers. Dis- turbances in the visceral activities controlled by the automatic nerv- ous system are insignificant except in the case of the discharges of 970 CENTRAL NERVOUS SYSTEM urine and feces, functions which are normally controlled by spinal reflexes and volitional activity. The centers, whose influence is cut off when spinal shock appears, lie in the brain-stem, probably in the nuclei of the pontine or mid- brain region. Consequently removal of the cerebral hemispheres does not produce anything like the severe spinal depression which occurs when the transection passes behind the level of the pons (Sherrington 37 ). KEFERENCES TO MONOGRAPHS AND ORIGINAL PAPERS ON THE NERVOUS SYSTEM Evolution of the Nervous System Barker, G. H. : The Elementary Nervous System, Philadelphia, 1919, J. B. Lippincott Co. Fundamental Properties of Nervous Tissue 2Adrian, E. D. : Brain, 1918, xli, 23. sLucas, K. : The Conduction of the Nervous Impulse, revised by E. D. Adrian, Lon- don, 1917, Longmans, Green & Co. ^Tinel, J. N. : Nerve' Wounds, English Trans., London, 1918, Bailliere, Tindall and Cox. sTashiro, S.: Am. Jour. Pliysiol., 1913, xxxii, 107. effill, A. V.: Jour. Physiol., 1912, xliii, 433. 7Mathison, G. C. : Jour. Physiol., 1910-11, xli, 416. s Cannon and Burkett: Am. Jour. Physiol., 1913. Sensation and Sensory Localization "Head, H.: Brain, 1919, xli, 57. icHolmes, G., and Lister: Brain, 1916, xxxix, 34. uRiddoch, G. : Brain, 1917, xl, 15. "Gushing, H. : Proc. Am. Physiol. Soc., Am. Jour. Physiol., 1909. isHead, H.: Brain, 1893, et seq. i4Lennander: Keen's Surgery, v, 156. isHead, H., and Thompson: Brain, 1906, xxix, 537. leHolmes, G. : Brit. Med. Jour., 1915, ii, Nov. 27, Dec. 4, Dec. 11. Autonomic Nervous System 17 Gaskell, W. H. : The Involuntary Nervous System, London, 1916, Longmans, Green & Co. isPottenger, F. M. : Symptoms of Visceral Disease, St. Louis, 2nd ed., 1922, C. V. Mosby Co. isFearnside, E. G.: Brain, 1917, xl, 149. soHead, H., and Eiddoch, G.: Brain, 1917, xl, 188. 21 Cannon, W. B. : Bodily Changes in Pain, Hunger, Fear and Rage, New York, 1915, D. Appleton & Co. Muscle 22Forbes, A., and Rappleye, W. C.: Am. Jour. Physiol., 1917, xlii, 228. 2spratt, F. H., and Eisenberger, J. P.: Am. Jour. Physiol., 1919, xlix, 1. 24 Bayliss, W. M. : Principles of General Physiology, London, 1915, Longmans, Green & Co. ssFletcher, W. M., and Hopkins, F. G. : Jour. Physiol., 1907, xxxv 247. 26Gunn, J. A., and Underbill, J. F. : Quart. Jour. Exper. Physiol., viii, 275. SUMMARY, MAMMALIAN NERVOUS SYSTEM; SPINAL SHOCK 971 Tone and Postural Coordination 27Sherringten, G. S. : Quart. Jour. Exper. Physiol., 1909, ii, 109; Brain, 1915, xxxviii, 191. 2Lee, T. S.: Jour. Physiol., 1894, xv, 311; 1894-5, xvii, 192. ssLyon, E. P. : . Am. Jour. Physiol., 1900, iv, 77. soBlack, D.: Jour. Lab. and Clin. Med., 1916, i, 467. sigherrington, C. S.: Jour. Physiol., 1898, xxii, 319. 32Weed, L. H.: Jour. Physiol., 1914, xlviii, 205. ssThiele, H. : Jour. Physiol., 1905, xxxii, 358. 34Walshe, F. M. E.: Brain, 1919, xlii, 1. 35Holmes, G.: Brain, 1917, xl, 461. seLuciani, L. : Human Physiology, iii, (English trans.), London, 1914. Integrative Action of Nervous System 3 7Sherrington, C. S. : The Integrative Action of the Nervous System, New York, 1906. ssLoeb, J.: Forced Movements, Tropisms and Animal Conduct, Philadelphia, 1918. The Cerebrum soWatson, J. B. : Psychology from the Standpoint of a Behaviorist, Philadelphia, 1919. Psychological Review, 1916, xxiii, 89. *Bolton, J. S. : Eecent Researches on Cortical Localization and on the Function of the Cerebrum in Further Advances in Physiology, ed. by Leonard Hill, London, E. Arnold, 1909. 4iMorgulis, S.: Jour. Animal Behavior, 1914, iv, 142; ibid., 1914, iv, 362. Keflex Functions of the Spinal Cord in Man "Riddoch, G.: Brain, 1917, xl, 264. INDEX Abdominal respiration, 324 Abnormal pulses, 291 Absorption, in general, 13 from stomach, 490 ' of fats, 722 Acapnia, 306 Accessory food factors, 618 Acetoacetic acid, 715, 738 Acetone, 715, 738 Acid: buffer action, 36 excretion of, by kidneys, 47 chemistry of fatty, 718 Acid-base balance, 38, 363, 372 Acidity, actual degree of, 23 Acidosis : ammonia-urea ratio during, 650 compensated, 39 in diabetes, 715 in nephritis, 715 in starvation, 602, 603 relation to alveolar CO,, 371 relationship to breathing, 371 theory of, 38 uncompensated, 39 Acids, of urine, 558 Acromegaly, 816 Action currents in skeletal muscle, 906 Actual degree of acidity and alkalinity, 23 Adaptation, sensory, 862 Addison's disease, 772 Adenine, 667, 669 Adenosine, 671 Adrenal glands, 768 and diabetes, 708, 790 assaying content of, 779 cortex of, 776 disease of, in man, 772 medulla of, 770 sexual precocity and, 776 Adrenaline (see Epiuephrine) Adsorption, 66 compounds, 71 conditions influenced by, 68 effect of chemical forces on, 69 effect of electric changes on, 68 everyday reactions depending on, 67 of gases, 67 Afferent nerves, segmental distribution of, 866 Afferent paths: in spinal cord, 868 in brain-stem, 871 of spinal reflexes, 872 of visceral reflexes, 873 of cerebellar reflexes, 874 After discharge, 915 effect on creatinine excretion, 657 Alanine, 634, 635, 636, 639, 698 Albolene absorption, 723 Albuminuria, 552 Alkali retention, determination of, 48 Alkaline buffer, 36 Alkaline reserve, 38 Alkalosis, 373,, 380, 559. Alkaptonuria, 536 Allantoin, 668, 672, 678 Allied reflexes, simultaneous combina- tion of, 945, 946 successive combination of, 946 Allocheiria, 859 All-or-none law: of conduction, 837, 839 of contraction of skeletal muscle, 908 of contraction of heart muscle, 177 Alloxan, 668 Alveolar air: clinical investigation of, 364 estimation of gases in, 361 Fridericia method, 357 Haldane method, 357 Pearce method, 362 tension of CO 2 , 46, 361, 373 during breathing in confined space, 366 tension of oxygen, 362 Ambard's equation, 562 Amboceptor, 97 Amino acids, 634 in blood, 641 chemistry of, 634 determination of, 635 fate of, 645 groups, 636, 638 in growth, 609 in tissues, 642 in urine, 564, 654 structure of, 638, 639 Aminoacetic acid (see Glycocoll) Aminopropionic acid (see Alanine) 973 974 INDEX Ammonia : ammonia-urea ratio: influence of acidosis on, 563, 650 in disease, 661 influence of liver on, 654. as reserve alkali, 563 excretion of, 652 excretion of acid in combination with, 47 of urine, 562 Ammonium carbamate, 649 Ammonium carbonate, 649 Amylases, 81, 91, 525 Amylolysis, 525 in stomach, 489 Amylopsin, 525, 689 Anacrotic wave, pulse, 203 Anaphylactic reaction, 632, 638, 755 Anaphylaxis, 90 Anarthria, 960 Anastomosis, intestinal, 504 Anemia, 94 bloodflow in, 298 Aneurism, bloodflow in, 299 pulse in, 144, 200 Angina pectoris, fibrillation in, 196 Animal calorimeter, 572 Anions, 16, 60 Anoxemia : acid excretion in, 381 acidosis and, 380 alkalosis and, 381 alveolar CO, tension in, 374 ammonia excretion in, 381 as a stimulus to respiratory center, 380 changes in body in, 378 general effects of, 374 in mountain sickness, 415 lactic acid in relation to, 379 pneumatic cabinet and, 377 Antagonistic muscles, 915 Antagonistic reflexes, 945 Anticoagulants, 100 Antidromic impulses, 239 Antiferments in blood, 90 Antiperistalsis in cecum, 503 Antithrombin, 105, 113 Antitoxins, 70 Aiititrypsin, 91 Aortic regurgitation, pulse in, 133 Apex beat, tracing of, 289 Aphasia, motor, 960 sensory, 960 subcortical, 960 Apnea, 349, 382 nervous element in, 384 Apparatus for measuring respiratory ex- change, 589 Appetite, 506 Appetite juice, nature of, 470, 474 Arc, reflex, 832 Arginase, 81, 650 Arginine, 640, 650, 660 Aromatic sulphates, 665 Aromatic oxyacids in urine, 564 Arrhythmia of sinus, 278, 292 Arterial pressure, 124 Arteries, bloodflow in, 200 Arteriosclerosis, diastolic pressure in, 144 Aspartic acid, 640, 699 Asphyxia, 366 Assimilation limit, 685 Association neurons, 834 Astasia, cerebellar, 927 Asthenia, cerebellar, 927 Asthma, dead space in, 328 Asynergia, cerebellar, 927 Atonia, cerebellar, 926 Atophan, 684 Atr opine, effect on glands, 457 effect on heart, 231 Auditory area, 879 Auricle, pressure in, 146 propagation of beat in, 191 Auricular curve, contour of, 153 Auricular fibrillation, 196, 281, 295 Auricular flutter, 196, 281, 293 Auriculoventricular orifice, 149 bundle, 183 node, 183 Auscultatory method (of blood pres- sure), 130 Autocatalysis, 77 Autocoids, 767 Autonomic nerves, cerebral, 458 sympathetic, 458 Autonomic nervous system, 893 axon reflexes in, 898 bulbosacral outflow, 894 connector fibers of, 894 functions of, 897 general plan of construction, 893 parasympathetic, 882, 895 sacral outflow, 494 thoracicolumbar outflow, 895 Axon, 831 reflexes, 829, 898 Azelaic acid, 740 Bacillus coli communis, 534 Bacteria, in intestine, 533, 690 in stomach, 516 Bacterial digestion, 533 Balance, energy, 571 carbon, 582 material, 579 sheet of body, 579 Banting cure, 605 Basal heat production, 574 Basal ration, 610 Basophile cells, 97 ' ' Bends ' ' in caisson workers, 425 INDEX 975 Benedict's method for respiratory ex- change, 580 Benzoic acid, 664, 738 Benzoyl chloride, 663 Beriberi, 619 Beta-hydroxy butyric acid, 738 Bile, 526 and fat digestion, 721 chemistry of, 528 constituents of, 526 from gall bladder, 526 functions of, 527 pigments of, 529 salts, 528 Bilirubin, 529 Biliverdin, 529 B-imidazolylethylamine, effect on blood vessels, 253, 413 as a factor in shock, 307 Birds, removal of liver from, 652 Bladder, emptying of, 900 Blood: absorption into, 13 - amino acids in, 641 amount in body, 85 antiferments of, 90 circulation of, 124 dissociation curve of, 396 epinephrine content of, 780 fat of, estimation, 726 variations in" 727 ferments of, 90 gases of, transportation, 392 general properties of, 85 mass movement of, 296 means by which gases are carried, 403 oxidation in, 412 proteases of, 90 proteins of, 88, 89 regeneration after hemorrhage, 89 quantity of, in body, 85 refractive index of, 89 specific gravity of, 87 sugar level of, 690 regulation, 703 transfusion of, 94, 131, 194 viscosity of, 141 volume of, 138 in shock, 306 water content of, 87 Blood cell, red, fate of, 95 origin of, 93 regeneration of, 94 stroma of, 92 white, 97 Blood clotting, 99 in diseases, 111 in physiological conditions, 111 influence of calcium on, 104 influence of tissues on, 105 intravascular, 108 Blood clotting Cont 'd methods of retarding, in drawn blood, 100 negative phase of, 109 theories of, 107 time of, 101, 109 visible changes during, 99 Blood corpuscles in mountain sickness, 419 Bloodflow : clinical conditions affecting anemia, 298 cardiovascular diseases, 299 fever, 298 diseases of nervous system, 300 mass movement of, 208 measurement of, 209, 296 movement in veins, 214 normal flow, 297 variations in, 297 velocity of, 206 visceral, 212 Blood gas manometer, 395 Blood platelets, 98 Blood pressure, 124 diastolic, 129, 133 effect of hemorrhage on, 136 effect of pleural pressure on, 323 factors maintaining, 135 H-ion of blood on, 248 mean arterial, 125 measurement of, 129 in shock, 303, 304 systolic, 129 tracing, 126 Blood vessels: elasticity of, 143 tone of, 241 Body fluids, reactions of, 35 Body surface and energy production, 575 Body weight and energy production, 575 Botulism, 537 Bowman, capsule of, 541 Bradycardia, 193 Brain : circulation in, 254 vasomotor nerves, 262 volume of, 256 Breathing, in compressed air, 420 in rarefied air, 374, 415 periodic, 385, 386 Brownian movement, colloids, 58 Bruits, 158 Buffer action of blood, 388 Buffer substances, 36 Building stones of protein, 609 Bulbocavernosus reflex, 967 Bulbosacral outflow, 894 Butyric acid, 737 976 INDEX Cadaverine, 002 Caffeine, 668, 681 Caisson disease, 420 cause of, 421 decompression of workers, 424 prevention, 422 symptoms, 420 working conditions in, 425 Calcium ion, influence on clotting, 104 influence on heart, 167 Calcium rigor, 167 Calomel electrode, 30 Calorie, 571 Calorie requirement, 625 Calorie test of labyrinth, 920 Calorimeter, 571 animal, 572 Benedict, 573 bomb, 573 hand, 296 respiration, 572 Eussel-Sage, 573 Calorimetry, direct, 572, 580 indirect, 580 Caloro-receptors, 855 Canalization, 844 Canals, semicircular, 918 removal of, 918 Cannabin, 611 Capillary analysis of colloids, 57 Capillary circulation, 251 during muscular contraction, 252 Krogh on, 251 Carbamino reaction, 635 Carbohydrates, absorption of, 689 assimilation limits, 685 digestion of, 689 and growth, 617 metabolism of, 685 production from protein, 699 saturation limit, 685 Carbon balance, 582 Carbon dioxide, combining power, 42 effect on respiratory center, 366, 368 estimation in blood, 403 output, 585 volume percentage in blood, 404 Carbon dioxide tension, 354 in alveolar air, after exercise, 435 estimation of, 357, 361 in mountain sickness, 416 in periodic breathing, 388, 389 in arterial blood, 354 in venous blood, 359 Carbonic acid (see Carbon dioxide) Carboxyl group, 634 Cardiac decompensation, 219 dead space in, 328 Cardiac depressor nerve, 243 Cardiac muscle, physiologic characteris- tics of, 176 Cardiac pouch (stomach), 487 Cardiac sphincter, 482 Cardiorenal disease, bloodflow in, 299 energy output in, 578 Cardiopneumatic movements, 154 Cardiograms, 276, 289 Cardiovascular disease, bloodflow in, 299 Casein, 521, 611 Caseinogen, 521 Castration, 821 Catabolism, 571 Catalase, 91 Catalysts, 73 Cations, 16 Catalytic power, 23 Caval pocket, 784 Coelenterates, nervous system of, 828 Cellulose, digestion of, 533 Centers : diabetic, 704 motor, 885 sense, auditory, 879 visual, 880 sensory, 878 word centers, 960 Cephalin, 720 Cereals and growth, 615 Cerebellar ataxia, 927 Cerebellum : ablation of, 926 clinical observations, 926 functions of, 926 lobes of, 929 localization of function of, 929 Cerebral circulation, 254 Cerebral compression, 258 Cerebral cortex, stimulation of, 885 Cerebral localization, 886, 878 Cerebral vessels, ligation of, 254 Cerebrin, 720 Cerebrospinal fluid, 121, 255 Cerebrum, functions of, 951, 958, 963 relation to distance receptors, 952 relation to spinal reflexes, 951 CH method of expressing, 27 / Chemo-receptors, 855 Cheyne-Stokes breathing, 385, 390 Chlorides, urine, 565 Chalone, 767 Chlorophyl, 530 Cholesterol, 528, 688, 720 estimation of, 726 Choline, 720 Chorda tympani, 236, 239, 412, 458 Chromaffin cells, 771 Chromatolysis, 847 Chromatine, 670 Chromosomes, 670 Chyme, 490, 516 Circle of Willis, 254 INDEX 977 Circulation of blood: control of, 221 influence of gravity on, 248 mass movement of blood, 208 through the heart, 267 through the liver, 265 through the lungs, 264 time of, 206, 214 Circulation time, 206, 214 Clinical application, circulation, 270 respiration, 364, 393 Clotting of blood (see Blood clotting) Coagulative ferments,, 82 Cod-liver oil, nutritive value, 735 Coefficient of oxidation, 408 Coefficient of solubility of gases, 354 Coefficient of utilization, 410 Cold, sensation of, 860 Cold spots, 860 Collaterals, 831 Colloids: Brownian movement, 58 capillary analysis, 57 characteristic properties of, 51 diffusibility of, 52 dispersion means, 55 dispersoid, 55 electric properties of, 56 osmotic pressure, 58 electrophoresis, 57 external phase, 55 gelatinization, .62 heterogeneous, 52 homogeneous, 52 imbibition, 63 internal phase, 55 isoelectric point, 65 lyophobe, 61 mutual precipitation of colloids, 57 osmotic pressure of, 142 size of colloid particles, 54 suspensions, 54 suspensoids and emulsoids, action of electrolytes on, 64 Tyndall phenomenon, 52 Compensated acidoses, 39 Compensatory movements of eyes, 918 Complemental air, 317 Compressed air sickness, 420 cause of symptoms, 421 prevention of, 422, 424 treatment of, 424 Concentration cell, 30 Concentration point, auricles, 185 Concept, 952, 959 Conditioned reflexes, 466, 954 Conduction : all or none law, 837, 839 between neurons, 841 energy of, 838 in nerve trunk, 827 in reflex arc, 841 Conduction Cont 'd polarity in, 841 refractory period in, 839 Conductivity, determination of, 17 equivalent, 19 molecular, 19 specific, 17 Conductivity cell, 18 Conglutin, 612 Construction of autonomic nervous sys- tem, 893 Contraction of skeletal muscle: isometric, 904 isotonic, 904 tonic, 904 tetanic, 905, 906 chemistry of, 910 Contracture, extension, 967 Cooking, 630 Coronary circulation, 267 Coronary vessels, vasomotor nerves, 268 Corpus luteum, 822 functions of, 822 Corpuscles of blood, red, 92 white, 97 Cortico-spinal system, 963 Coughing, 317, 351 Cranial cavity, pressure in, 258 Creatine, 647, 656 chemistry of, 656 estimation of, 657 in disease, 661 metabolism of, 658 origin of, 660 Creatinine, 647 chemistry of, 656 coefficient, 658 estimation, 657 in urine, .563 metabolism, 658 of blood in disease, 683 origin of, 660 Cremasteric reflex, 967 Cretinism, 795 Critical concentration, 8 Crossed extension reflex, 966 Cuorin, 720 Curare, effect on myoneural junction, 845 Current of action, of heart, 188 of skeletal muscle, 906 Cyanosis, 378, 444 Cysteine, 639 Cystine, 611, 629, 639 Cystosine, 667 Cytases, 497 Dalmatian dog, purine metabolism of, 673, 679 Dalton's law, 353 Dead space, 319, 327 Deafness, 880 978 INDEX Deamidization, deaminization, 535 Deaminizing enzyme, 671 Decerebrate rigidity, 925 Decolorization of liquids by charcoal, 67 Decompensation of heart, 220 Decompression of caisson workers, 424 Defecation, 504 Defects of branches of A-V bundle, 284 Defibrinated blood, 102 Degeneration of nerve fibers, 846 Degeneration, successive, 849 Deglutition, 479 Delayed conduction, 282, 291 Delirium cordis, 196 Denervated iris, 784 Dendrites, 831 Depression of freezing point, 10 of urine, 557 Depressor nerve, 243, 244, 245 Depressor substances, 253, 415 Dessert, physiological value of, 472 Detoxication compounds, 662 Detoxication process, 535 Dextrins, 525, 689 Dextrose (see Glucose) Diabetes : acidosis in, 715 and the ductless glands, 710 assimilation limits in, 685 blood examination in, 691 * blood fat in, 726 center, diabetic, 704 early diagnoses of, 685, 692 energy output in, 578 experimental, 704 fat metabolism in, 715 insipidus, 815 ketosis, 715 pancreatic, 712 nervous, in man, 706 permanent, 707 phlorhizin, 697 postprandial hyperglycemia, 691 renal, 693 starvation treatment in, 716 treatment of, 623, 686 Diabetic acidosis, 715 Diabetic center, 704 Diabetic gangrene, 300 Dialuric acid, 678 Dialysate, 53 Dialysis, 12 method, colloids, 52 Diaphragm, action of, 337, 338 physiology of, 341 Diastasis, 219 Diastolic filling of heart, 153, 217 Diastolic pressure, 129, 133 measurement of, in man, 129 Dicrotic notch, 203 wave, 203 Diet at different ages, 627 of different communities, 626 Dietetics, 625 Differential manometer, 394 Diffusion, 12 Digestibility of foods, 630 Digestion, by pancreatic juice, 523 in intestine, 523 in stomach, 515 mechanism of, 478 Digestive glands: control of, hormone, 460 nervous, 458 general physiology of, 453 microscopic changes during activity, 423 Disaccharides, 689 Dispersion medium, colloids, 55 Dispersoid, colloids, 55 Dissociation, 16, 17 Dissociation constant, 19, 401 Dissociation curve : of blood, 396 of hemoglobin, 397 influence of salts on, 398 influence of H-ion concentration on, 339 influence of temperature on, 399 Dissociation of sensation, 868 Dissociation hypothesis, applications of, 21 Dissociation, rate of, 399 Distance receptors, relation to cerebrum 952 Diuresis, 552 and pituitrin, 811 Diuretics, 552 Diver's palsy, 420 Douglas method, 489 Dropped beat, 282, 291 Du Bois formula, 576 Ductless glands, 766 in diabetes, 710 Dudgeon's sphygmograph, 201 Dyspnea, 330, 366 Dystrophia adiposo-gonitalis, 818 B Earth-worm, nervous system of, 830 Eck fistula, 651 Eclampsia, 654 Edema, 63, 120 Edestin and growth, 611 Effectors, 829 independent, 828 Efferent pathways: in brain and cord, 888 to viscera, 893 Effort syndrome, 441, 442 Einthoven's triangle, 271, 272 El.istin, digestion of, 520 Electric conductivity, 16 Electric currents, development of, INDEX 979 Electric properties of colloids, 56 Electrocardiograms, 159, 270 normal, 273 standardization of, 271 ventricular complex, 274 waves of, 272 P-wave, 189, 273 T-wave, 225, 273, 278 Electrocardiograph, 271 Electrocution, cause of death in, 195 Electrolytes, 16 action of, on colloids, 64 Electrolytic solution pressure, 29 Electrophoresis of colloids, 57 Electrostatic attraction, 29 Emboli, 108 Emetics, 484 Emotional glycosuria, 706 Emphysema, 328, 330, 341 Empyema, 341 Emulsions, 719 Emulsoids, colloids, 61 Endocrine organs, 766 Endoenzyme, 71 Endogenous metabolism, 649, 656 of purines, 676 Energy balance, 571 Energy output, and age, 597 and body weight, 575 and disease, 578 and muscular work, 586 and sex, 577 and surface area, 575 and temperature, 586 in starvation, 602 Enterokinase, 477, 523 Enzymes, 72 action of temperature on, 75 amylases, 82 and catalysis, 73 antienzymes, 82 arginase, 82 coagulative ferments, 83 conditions of activity, 83 endoenzymes, 72 glyoxylase, 83 invertases, 82 Upases, 82 nature of, 73 oxidases, 83 peculiarities of, 81 peroxidases, 83 properties of, 94 proteases, 81 reversibility of action of, 25, 78 specific action of, 74 types of, 80 urease, 83 velocity constant, 75 Epilepsy, Jacksonian, 883, 887 Epinephrine, 241, 502, 536, 773, 902 and diabetes, 708 emergency hypothesis of, 786 Epinephrine Cont 'd estimation of, 779, 785 methods of determining, 779, 785 physiological action of, 774 r ever give action of, 777 secretion of, in fright, 786 variation in action of, 779 Equilibrium, nitrogen, 605 Equivalent, conductivity, 19 Erepsin, 520, 524, 525 Ergastoplasm, 455 Ergot, 536 Ergotoxine, 236, 538, 776, 897 Erythrocytes, 92 fate of, 95 regeneration of, 94 Escapement, 223 Esophagus, during swallowing, 480 inhibition of, 482 peristaltic wave in, 482 Esters, 718 Ester value, 719 Ethereal sulphates, 535, 665 Ethylamine, 536, 662 Excelsin, 612 Excitation, 827 Exogenous metabolism, 649 Exophthalmic goiter, 799 energy output in, 578 Excretion of acid combined with am- monia, 47 Excretion of urine, 541 Extension contracture, 967 Extensor thrust, 967 Exteroceptors, 917 Extrasystole, 278, 292 Eyes, movements of, 887 Facilitation, 843 Factor safety, in diet, 629 Fatigue level, 911 Fatigue of muscle, 910, 911 Fatigue of reflexes, 845 Fats: absorption of, 722 chemical theory, 723 mechanistic theory, 723 and growth, 617 blood, 726, 727 destination of, 729 determination, 726 during absorption, 728 during fasting, 728 variations in, 727 chemistry of, 721 depot fat, 729, 730 destination of, 731 desaturation of, 734, 740 digestion of, 722 fat dust, 726 liver fat, 729, 731 980 INDEX Fats Cont'd metabolism of, 718, 726, 736 tissue fat, 729, 735 transportation to liver, 732 Fatty acids, 718 acid number, 719 breakdown of, 737 ester value, 719 formation from carbohydrates, 730, 736 in liver in disease, 733 iodine value, 719 melting point, 719 Keichert-Meissl value, 719 saponification value, 719 Feces, 533, 555 Ferments (see Enzymes) Ferments in blood, 90 Fever, bloodflow in, 298 body changes in, 750 causes of, 748 cold-bath treatment, 299 purine excretion during, 680 Fibers, anterior root, 238 connector, 894 internuncial, 894 postganglionic, 894 preganglionic, 894 Fibrillation, auricular, 196, 281, 295 ventricular, 195 Fibrin, 100 fibrin needles, 100 source of, 102 Fibrin ferment (see Thrombin), 103 Fibrinogen, 89, 102, 104, 112 Filtration, 13 Final common path, 945 Fistula, biliary, 526 gastric, 468 salivary, 466 Flexion-reflex, 966 Flutter, auricular, 269, 196, 281, 293 Food : .accessory factors of, 618, 630 cooking, importance of, 630 effect of, on circulation, 247 effect on creatinine excretion, 657 laxative qualities, 631 palatability, 630 Food factors, accessory, 618, 630 Food factors of growth, 608 Foodstuffs, rate of leaving stomach, 492 Forced breathing, 341 Formaldehyde titration, amino acids, 521 Formation of solid surface films, 67 Free nerve termination, 854 Freezing point, constant, 10 Freezing point, depression of, 10 Fridericia's method for alveolar air, 3.17, 358 Fructose, 698 Fundus of stomach, 485 G Gallstones, 528 Galvanometer, string, 187, 270 Ganglia, 831 Gas in stomach, 496 Gas laws, 3, 353 Gases, adsorption of, 67 coefficient of solubility, 354 estimation of, 361 partial pressure of, 353 solution of, 353 tension of, 353 transportation in blood, 392 Gaskell's clamp, 175 Gastric contents, regurgitation of, 483 Gastric digestion, 515 rate of, 521 Gastric fistula, 468 Gastric juice, quantity secreted, 474 strength of, 475 Gastric secretion, 467 hormone control of, 472 local stimulation of, 472 nervous control of, 469 Gastric tube, 487 Gastric ulcer, 490 Gastrin, 474, 490 Gastroenterostomy, 494 Gastrointestinal contents, reaction of, 539 Gelatinization, 62 Glands, changes during activity, 453, 456 electric changes, 457 normal conditions of activity, 465 oxygen consumption of, 408, 411, 456 respiration of, 408, 411 Globulin, 611 Gliadin, 612 Glomerulus, 541 Gluconeogenesis, 694, 708, 712 direct method, 695 indirect method, 696 in normal animals, 699 Glucose : ' fate of absorbed, 694 glucose to nitrogen ratio, 696 injections, intravenous, 687 subcutaneous, 688 parenteral assimilation, 688 tolerance for, 688 utilization of, in tissues, 708 Glutamic apid (see Glutaminic acid) Glutaminic acid, 640, 699 Glutein, 611 Glutelin, 611 Glycol aldehyde, 697 Glycerol, 697 Glycoholic acid, 528, 664 Glycine, ' 494, 639 Glyeininj 611 Glycocoll, 637, 639, 664, 699, 738 INDEX 981 Glyeogen, 694 fate of, 701 sources of, 694 Glycogenase, 694 Glycogenolysis, 701 hormone, 707 nervous, 704: postmortem, 702 Glycolaldehyde, 697 Glycolysis, 702 Glyconeogenesis (see Gluconeogenesis) Glycosuria, alimentary, 690 emotional, 702 postprandial, 691 relation to sugar of blood, 692 y renal, 693 Glycuronates, 665 Glycuronic acid, 665, 666 Glyoxal, 664 Glyoxylase, 82, 698 Glyoxylic acid, 664 G-N-ratio, 696 Goiter, exophthalmic, 799 Gonads, 821 of female, 822 of male, 821 Gout, 681, 683 etiology of, 683 guanine, 673 uric acid excretion in, 681 Gram molecule, 3, 5 Gram molecular solution, 22 Gravity, on circulation, 248 compensation for, 249 Growth, 608 accessory factors, 618 basal ration, 610 carbohydrates and, 583 curves of, 610, 611 curves of inhibition, 614 fats and, 617 inorganic salts and, 618 lysine and, 612 proteins and, 609 trypanophanc and, 612 vitamines, 618 Guanidine, 640, 656, 804 Guanine, 667 gout, 673 Guanosine, 671 Giinsberg reagent, 521 Gustatory area, 879 H Haldane-Barcroft apparatus, 45 Haldane gas apparatus, 593 Haldane's method for alveolar air, 357 Hallucinations, 883 Heart : action of, 145 auricular curve, 153 diastole of, 146 isometric period in, 150 Heart Cont 'd law of, 216 minute volume of, 218 muscle, properties, 176 nutrition of, 161 opening and closing of valves, 154 output of, 216 in relation to venous inflow, 216, 217 oxygen requirements of, 408, 411 oxygen supply of, 166 perfusion of outside body, 161 postsphygmic period, 150 presphygmic period, 150 pressure in, 146 pumping action of, 135, 145 resuscitation in situ, 165 rhythmic power in, 170, 174 sounds of, 156 systole of, 146 tone of, 218, 219 utilization of glucose in, 713 vagus control of, cold blooded, 222 vagus control of, mammalian, 225 vagus terminations in, 230 ventricular curve, 151 work of, 213 Heart beat: arrhythmia of, 278, 292 disorders of, 219, 278, 291 myogenic hypothesis of, 170, 171 neurogenic hypothesis of, 170, 172 origin of, in cold-blooded animals, 170 origin of, in mammalian, 182, 189 pace maker of, 174 propagation of, 191 reserve power, 220 sympathetic control of, 232 ultimum moriens, 185 vagus control of, 222, 225 Heart block, 174, 282, 291 effect of vagus on, 224 Heart disease, vital capacity of lungs in, 330 Heart-lung preparation, 163 Heat production and age and sex, 577 and body weight, 575 surface, 576 disease, 578 sensation of, 861 Heat spots, 860 Heat value of foods, 571 Hematin, 530 Hematocrit, 7 Hematoporphyrin, 530 Hemiplegia, 889 Hemodromograph, 207 Hemoglobin, 92 dissociation constant, 401 dissociation, curve of, 396,398,399 estimation of, 93 rate of dissociation, 399 relationship to bile pigments, 530 specific oxygen capacity of, 392 transportation of 2 by, 392 982 INDEX Hemolysis, 7, 9(3 Hemolytic jaundice, 94 Hemophilia, 113 Hcmopoietic activities of bone marrow, 94 Hemorrhage, 95, 138 immediate effects of, 138 recovery from, 89, 139 Hemorrhagic diseases, 113 Henle, loop of, 541 Hepatic artery, flow in, 265 Heterocyclic compounds, 640 Hexone bases, 638 Hexoses, 685 Hibernating animal, metabolism of, 581 Hibernation, breathing during, 389 Higher functions of cerebrum, 951, 958, 963 H ion or hydrogen ion, 168 H-ion concentration, 22 after hemorrhage, 143 catalytic power of, 23 determination of, 31 of intestinal contents, 539 law of mass action and, 26 method of expressing, 27 method of measurement : electric method, 29 indicator method, 32 standard solutions for, 34 H-ion concentration in blood: effect on dissociation curve, 398, 401 effect on respiratory center, 352 Hippuric acid, 564, 663, 738 Hirudin, 101 Histamine, 536 as a factor in shock, 307 effect upon capillaries, 253, 413 Histidine, 639, 641, 656 Homogentisic acid, 536, 565 Hordein, 612 Hormones, 3, 766 in control of circulation, 221 respiratory, 352,, 366 Howell theory (blood clotting), 107 Hunger, 506 Hunger contractions : alcoholic beverages and, 512 control of, 511 during starvation, 510 in esophagus, 509 inhibition of, 511 in stomach, 506 nerve centers and, 513 remote effects of, 509 rhythmic, 506 splanchnic nerve and, 511 vagus nerve and, 511 Hiirthle manometer, 128, 147 Hydrocephalus, 255, 263 Hydrochloric acid, amount of, 516 and emptying of stomach, 491, 494 functions of, 516 source of, 517 Hydrogen ion (see H ion) Hyperacidity, 495 Hyperglycemia, in pancreatic diabetes. 712 postprandial, 691 splanchnic, 704 Hypernephroma, 769 Hyperpituitarism, 816 Hyperpnea, 366, 371, 377 Hyperthyroidism, 798 Hypertonic solution, 6 Hypopituitarism, 817 Hypothyroidism, 797 Hypotonic solution, 6 Hypoxanthine, 667, 671 Ignition juice, 473 Ileocolic sphincter, 501, 503 Imbibition, 63 Imidazole and growth, 639, 656 Imidazole ring, 656 Imidazolylethylamine, 253, 413, 461, 536 Immediate induction, 947 Impulses, nature of, 837 Indican, 565, 665 Indicator method, list of indicators, 33 Indole, 535, 639, 665 Indoxyl sulphate of potassium, 665 Induction, immediate, 947 successive, 949 Inhibition, 843 reciprocal, 914, 937 Inhibitory effects of autonomic nerves, 896 Innervation, double of visceral organs, 896 Inorganic constituents of urine, 565 Inorganic salts and growth, 618 Inosine, 670, 671 Inosinic acid, 668 Inspiration, negative pressure during, 322 Insulin, 714 Integration of allied reflexes, 945, 946 Intercostal muscles, 336 Interganglionic connectives, 832 Internal respiration, 391 Interstitial cells of ovary, 822 Intestinal bacteria, 533, 690 Intestinal ballast, 631 Intestinal juice, control of, 477 Intestinal muscle : metabolic gradient of, 500 rhythmicity of, 499 segmenting movements of, 497 Intestinal obstruction, 505, 538 Intestinal secretions, 476 INDEX 983 Intestine : absorption from, 13 anastomosis of, 504 bacterial digestion in, 533 digestion in, 523 law of, 501 movements of: large, 503 clinical conditions affecting, 504 small, 497 nature of, 501 nervous control of, 501 Intracardiac pressure curves, 146, 151 Intracranial pressure, 258 Intragastric pressure, 488 Intrapleural pressure, 321 Intrapulmonic pressure, 316 Intra vitam anticoagulants, 101 Intravascular clotting, 108 Inulin, 696 Invertase, 82, 526, 689 Iodine value of fats, 719 lodothyrine, 798 lonization, 16 Irradiation on to respiratory center, 430 Iris, denervated, 784 Isoelectric point, 64 Isoleucine, 639 Isomaltose, 79 Isometric period, 150 Isotonic solution, 6 J Jacksonian epilepsy, 883, 887 Jugular pulse tracing, 285 Juice, gastric, 467, 516 intestinal, 476 pancreatic, 476, 523 K Keith and Flack, conducting tissue in heart, 185 Keith, Kowntree, and Geraghty method, 85 Kent, bundle of, 183 Ketosis, 715 Kidney, oxygen requirements of, 408, 412 removal of, 655 structure of, 541 Knee-jerk, 966 L Labyrinth, 918 clinical tests of, 920 Lactalbumin, 611 Lactam, 682 Lactase, 525, 689 Lactic acid, 413, 639, 689, 697, 708 effect on respiratory center, 379 in relation to anoxemia, 379 produced by exercise, 431, 435 Lactim, 682 Language, 958 Laws of gases, 353 of mass action, 23 applied measurement of H-ion concen- tration, 26 Lead poisoning, 684 Lecithin, 720 estimation of, 726 in bile, 532 in blood, 726, 728 Leech extract, 101 Legumelin, 612 Legumin, 612 Leucine, 640, 698 Leucemia, 681 Leucocytes, 97 sensitizing of, 70 transitorial, 98 Levulose, 698 Levy and Eowntree method, 41 Leydig cells, 821 Limulus, heartbeat of, 173 Lipase, 25, 91, 522, 525, 719 Lipemla, 728 Lipoids of blood, 728 List of indicators, 33 Litten's diaphragm phenomenon, 338 Liver : circulation through, 265 disease of, 654 glycogen in, 695 metabolism of fats in, 731 perfusion of, 652 removal of, 651 urea formation in, 651 Local irritants, 248, 253 Localization : one dimensional, 861 two dimensional, 861 three dimensional, 861 Locke solution, 168 Loven reflex, 248 Lungs, circulation through, 264 mode of expansion of, 342 Lymph : absorption into, 13 electric conductivity, 16 filtration in, 118 formation and circulation, 115 formation of, 15 Lymph spaces, 115 Lymphagogues, 119 Lymphatics, 115 Lymphocytes, 97 Lyophobe colloids, 61 Lysine, 629, 640 Lysine and growth, 612, 614, 616 M Maintenance, diets for, 61G Maltase, 525, 689 Maltose, 525, 689 984 INDEX Manometer : blood-gas differential, 39;") Hurthle, 128, 147 mercury, 125 optical, 147 spring, 128 valved mercury, 152 Mark-time reflex, 967 Mass action, 23 Mass action and H-ion concentration, 26 Mass movements of blood, 208 measurement of, 296 Mass-reflex, 968 Mastication, 478 Mechanics of respiration, 316 Mechanism of urine excretion, 544 Megacaryocytes, 104 Melting point, fats, 719 Mercury manometer, 125 Mesencephalico-spinal system, 963 Metabolism : calculations, 596 endogenous, 649 exogenous, 649 general, 570 in starvation, 600 normal, 604 of carbohydrates, 685 of central nervous system, 851 of fats, 718 of nerve fibres, 856 of proteins, 632 of purines, 676 special, 570 Methyl glyoxal, 698 Methyl group, 634 Methyl purines, 668 Methylation, 660 Methylglyoxal, 698 Mett's method, 521 Microcytes, 95 Microtonometer, 356 Mid-capacity of lungs, 328 Milk, clotting of, 521 Miniature stomach, 468 Minimal air, 317 Mononuclear leucocytes, 97 Monoplegia, 887 Monosaccharides, 689 Morphogenetic, 767 Morawitz theory, blood clotting, 108 Motor areas: representation of function in, 886 stimulation of, 885 Mountain sickness, 378, 415 acid and ammonia excretion in, 420 acid-base equilibrium in, 416 adaptation to, 418 alveolar CO 2 in, 416 blood corpuscles in, 419 symptoms of, 417 Movements, of intestine, 497 of stomach, 485 Municipal food statistics, 628 Muscarine, action on heart, 232 Muscle, cardiac, properties of, 176 refractory period, 178 respiration in, 408, 409 staircase phenomenon (treppe), 177 skeletal, 177 respiration in, 394 Muscular exercise, 248, 574 circulatory changes during, 427 effect on metabolism, 586 effect on respiration, 427 H-ion during, 431, 432 purines during, 480 redistribution of blood during, 433 respiratory changes during, 427 temperature of blood during, 433 Mutual precipitation of colloids, 57 Myenteric reflex, 830 Myogenic hypothesis of heartbeat, 171 Myoneural junction, 845 effect of curare on, 845 effect of epinephrine on, 845 Myxedema, 796 energy output in, 578 N Narcotics and blood fat, 727 Necrosis of liver, 654 Negative heart pulse, 154 Negative pressure in ventricle, 152 Nephelometer, 726 Nephrectomy, 655 Nephritis, 683, 684 acidosis in, 715 urea retention in, 562 Nerves : degeneration of, 846 regeneration of, 846 specific properties of, 859 vasodilator, 239 Nerve cell body, function of, 846 Nerve fiber: conduction in, 837 degeneration of, 846 direction of conduction in, 837 fatigue of, 841 isolation of conduction in, 837 metabolism of, 850 regeneration of, 846 Nerve net, 830 Nervi erigentes, 239, 895 Nervous control : of gastric secretion, 469 of ileocolic sphincter, 503 of intestinal glands, 477 of intestinal movements, 501 of pancreas, 462 of respiration, 344 of salivary glands, 458 of stomach movements, 492 Nervous conduction, polarity in, 830, 841 INDEX 985 Nervous impulse: conduction of, 836 rhythm of, 841 Nervous diabetes, 704 in man, 706 Nervous system : autonomic, 893 bulbar outflow, 894 enteral, 895 internuncial fibres, 894 oculo-molor, 895 sacral outflow, 894 thoracico-lumbar outflow, 895 evolution of, 827 influence on excretion of urine, 563 integration of, 945, 951 metabolism of, 851 nutrition of, 846, 849 Network, nerve, 830 Neurogenic hypothesis, of heart, 172 Neurons, 830 association, 834 effector, 894 internuncial, 894 Neuroid transmission, 828 Neurolemma, 848 Neutrality, regulation of, 36 Nicotine : action on vagus, 231 Nitrogen : action on synapse, 238, 844, 895 excretion of, premortal rise, 600 in starvation, 600 undetermined, urine, 647, 662 Nitrogen balance, 605 Nitrogenous constituents of urine, 560 Nitrogenous equilibrium, 605 Nitrogenous metabolites, in starvation, 602 Nociceptives : reflex, 966 Noeud vital, 344 Nonelectrolytes, 16 Nonthreshold substances, 546 Normal acid, 22 Normoblasts, 94 Noxious stimuli, 862 Nuclease, 671 Nucleic acid, 669, 720 Nuclein ferments, 91 Nucleins, 669 Nucleoside, 670 Nucleotide, 670 Nystagmus, 920 Nutrition, 608 of nervous tissue, 846, 849 Obesity, Banting cure for, 605 Oleic acid, 718 Olein, 719 Olfactory area, 879 Oncometer, 235 Opsonins, 70 Optic thalamus, sensory center of, 876 Organs, loss of weight during starva- tion, 602 perfusion of, 652 Ornithine, 650, 664 Ornithuric acid, 664 Orthopnea, 329, 335 Oscillatory method of blood pressure, 132 Osmometer, 5 Osmosis, 4 Osmotic pressure, 4, 10 and formation of lymph, 13 and hemolysis, 7 and plasmolysis, 8 measurement by depression of freez- ing point, 11 in physiologic mechanisms, 13 in production of urine by kidneys, 14 of transfusates, 142 Ovary, 822 Ovalbumin, as food, 611 Ovovitellin, as food, 611 Oxidases, 82 Oxidation of blood, 400 Oxybutyric acid, 650, 715, 740 Oxygen : coefficient of oxidation, 408 coefficient of utilization, 410 determination of, 596 estimation in blood, 403 requirements of tissues, 408 tension in alveolar air, 361 tension in arterial blood, 354, 356 therapeutic value of, 445 transportation by blood, 392 volume percentage in blood, 403 Oxygen insufficiency, (see anoxemia) Oxygen supply of heart, 163, 166 Oxyproteic acid, 662 Pacchionian body, 256 Pain: referred, 858 sensation of, 862 transmission in cord, 868 sense, 862 Palatability, 630 Palmitic acid, 718, 736 Pancreas : hormone control of, 460 histologic changes of, 464 oxygen requirements, 396 nervous control of, 462 sugar metabolism and, 710 Pancreatic diabetes, 712 Pancreatic digestion, 523 986 INDEX Pancreatic juice, 476 and fat digestion, 721 secretion of, 460 Pancreatin, 524 Parasympatlietic system, 895 Parathyroids, 800 disease of, 800 injury of, 800 removal of, 800 Paralysis, flaccid, 924 Paroxysmal tachycardia, 281, 293 Partial dissociation, 283 Partial pressure of gases, 353 Past pointing, 920 Pelvic nerve, 895 P'elargonic acid, 740 Pentose, 670, 696 Pepsin, action of, 519 products of, 520 Pepsinogen, 519 Peptides, 636 Peptone, 106, 520 Perfusion, of kidney, 664 of liver, 652 Perfusion fluid, of heart, 166 Perfusion of heart, 161 Periodic breathing, causes of, 385, 386 types of, 385 Peripheral resistance, 135, 234 as cause of shock, 304 Peristalsis : in esophagus, 481 in large intestine, 503 in small intestine, 497 in stomach, 486, 489 Peristaltic rush, 500, 505 Peristaltic wave, 499 Pernicious anemia, energy output in, 578 P'eroxidases, 82 PH, 27 Phagocytes, 98 Phenaceturic acid, 738 Phenol, 534 Phenolacetic acid, 536 Phenolphthalein, 516, 558 Phenylacetic acid, 664, 738 Phenylalanine, 639 PTienyl group, 639 Phlorhizin, 695, 696 Phono-receptors, 855 Phosphates, excretion of, 47 Phosphate solutions for H-ion, 34 Phosphates of urine, 566 Phospholipins, 720 in bile, 532 Photo-receptors, 855 Phrenic center, 345 isolation of, 345 PTiysiochemical basis, 1 Physiological processes depending on ad- sorption, 70 Pigments, absorption of, 117 Pilocarpine, action on heart, 232 Pilomotor fibers, 897 Pineal gland, 820 Pituitary gland, 806 anterior lobe of, 808 disease of, 816 functions of, 807 pars intermedia of, 815 posterior lobe, 809 Pitot's tubes, 201 Pituitrin, effect on vessels, 811 effect on carbohydrate metabolism, 814 effect on kidney, 812 effect on milk secretion, 813 Plasma, 100 Plasmolysis, 8 Plastic tonus, 905 Platelets, of blood, 98, 107 Plethora, 87 Plethysmograph, 209, 235, 320 Pleurisy, 341 Plexus of Auerbach and Meissner, 500 Pneumothorax, 322 P'oikilocytes, 95 Polarity, in conduction, 841 in reflex arc, 841 Polygraph, 286 Polynuclear cells, 97 Polypeptides, 520, 636 Poly phosphoric acid, 670 Polysaccharides, 522 Polysphygmograms, 285 Portal vein, bloodflow in, 265 Postdicrotic wave, pulse, 203 Postprandial hyperglycemia, 691 Postsphygmic period, 150 Postural co-ordination, 914 central control of, 924 Posture of body, 917 Potassium, microchemical test for, 4.~>n Potassium ions, on heart, 167 Potential acidity of urine, 559 Precipitins, 632 Predicrotic wave, pulse, 203 Premature beats, 278, 292 Premortal rise, 600 Presphygmic period, 150 Pressor impulses, 243, 244, 245 Pressure : intragrastric, 488 intrapleural, 321 effect of, in blood pressure, 323 intrapulmonic, 316 negative, 322 osmotic, 10 Pressure pulse, 129 Principle of Willard Gibbs, 66 Proline, 640 P'roprioceptors, 914 clinical tests for, 920 Prosecretin, 461 Proteases, 90 Protein sparers, 636 INDEX 987 Proteinases, 81 Proteins : as colloids, 64 bacterial digestion of, 535 chemistry of, 633 metabolism of, 631, 647 end products, 647 minimum requirement, 606, 629 of blood, 88 relative value of, for growth, 646 salting out of, 61 Proteose, 520 Prothrombin, 104, 107 , 112 Psychopathology, 960 Ptomaines, 536, 662 P'tyalin, 525, 689 Pulmonary circulation, 264 Pulmonary ventilation, 367 Pulses, 198 abnormal, 291 alternans, 181 bigeminus, 181 contour of wave, 200 length of wave, 199 palpable, 202 pressure, 129 pulse curves, 202 pulse waves, 198, 199, 200, 202 rate of transmission, 199 velocity, 200 venous, central, 205, 285 venous, peripheral, 205 Purkinje fibers, 184 Purine bodies (see Purines) Purines: chemistry of, 563, 647, 667, 674 endogenous, 676, 677 exogenous, 674 metabolism of, 667 in starvation, 603 synthesis of, 677 in urine, 563 Putrefaction, intestinal, 535, 565 Putrescine, 662 Pyloric canal, 485 Pyloric sphincter, control of, 490 Pyloric vestibule, 485 Pyrimidine bases, 669, 670 P'yruvic acid, 635 E Eami communicantes, 238 Raynaud's disease, bloodflow in, 300 Reaction of urine, 558 Reactions depending on adsorption, 67 Reactions of body fluids, 35 Receptor-effector system, 829 Receptors, 829, 854, 933 chemo, 855 distance, 952 extero, 917 evolution of, 854 of skin, 859 Receptors -Cont 'd phono, 855 photo, 855 proprio, 914 tango, 855 temperature, 861 touch, 860 Reciprocal inhibition, 915, 937 action of strychnine on, 941 action of tetanus toxin on, 941 Reciprocal innervation of blood vessels, 247 Red blood corpuscles, origin of, 93 Reduction of blood, 400 Referred pain, 858 Reflex, 832 conditioned, 466, 954 unconditioned, 466 Reflex arc, 832 integration within, 933 polarity of, 841 Reflex conduction: canalization, 844 facilitation, 843 induction, 843 inhibition, 843 summation, 843 Reflexes : abdominal, 892 Achilles tendon, 892 allied, 945 simultaneous combination of, 946 successive combination of, 946 antagonistic, 945 axon, 829, 898 bulbocavernosus, 967 conditioned, 854 cremasteric, 967 crossed extension, 966 extensor thrust, 967 flexion, 966 genital, 892 mark-time, 967 mass-reflex, 968 myenteric, 830 nociceptive, 966 palmar, 892 plantar, 892 proprioceptive, 914 pupillary, 892 scapular, 892 scratch, 966 triceps, 892 unconditioned, 466 Reflex fatigue, 948 Reflex figure, 941 spread of, 943 Refractive index, blood, 89 Refractory period, 839, 934 Refractrometric methods, 89 Regeneration of erythrocytes, 94 of nerve fibres, 846 Regulation of neutrality, 36 988 INDEX Regurgitation of gastric contents, 483 Reichart-Meissl value of fats, 719 Eenal diabetes, 693 Renal function, theories of, 545 Renal threshold for sugar, 692 Rennin, 521 Reserve alkalinity, measurements of, in- direct methods, 41, 46 measurement of, titration methods, 41 Residual air, 317, 328 Respiration : abdominal, 324 beyond the lungs, 391 during muscular exercise, 427 external, 391 in compressed air, 420 in rarefied air, 415 internal, 391 mechanics of, 316 movements of diaphragm in, 337 movements of ribs in, 332 Respiration calorimeter, 572 Respiratory center, 344 afferent impulses to, 348, 350 automaticity of, 346 hormone control of, 352, 366 reflex control of, 348 sensitivity to alveolar CO,, 371 stimulation by CO 2 , 366, 3~67 subsidiary, 345 Respiratory changes in muscular exer- cise, 427 Respiratory exchange: according to body weight, 585 and body temperature, 586 and muscular exercise, 586 and temperature of environment, 586 clinical method for determining, 589 in diabetes, 709 in tissues, 408, 412 Respiratory hormone, nature of, 366 Respiratory movements, 322, 325 Respiratory passages, pressure of air in, 316 Respiratory quotient, 582 in diabetes, 709 influence of diet on, 582 influence of metabolism on, 584 influence of muscular activity on, 435 Respiratory tracings, 320 Respiratory valves, Pearce 's, 589 Reticulated erythroblasts, 94 Reversible action of enzymes, 25 Ribs, movements of, 332 musculature of, 336 undulatory movements of, 334 Right lateral connection, heart, 185 Romberg's sign, 921 Rotation test of labyrinth, 920 Rhythmic segmentation, 497 Sacral outflow, 894 Salicylates, 681 Saline injection, effect on blood pres sure, 139 Saliva, control of secretion, nervous, 458 psychic, 466 normal secretion, 466 Salt, dietetic value, 618 Salted blood, 101 Salting of proteins, 61 Saponification, 719 Sarcosine, 656 Saturation limits, 685, 687 Scratch reflex, 966 Scurvy, 622 Sea anemone, nervous system of, 828 Second wind, 438 Secretory fibers, varieties of, 459 Secretion, 460 chemical nature of, 461 mechanism of action of, 455 Secretion (see under various glands) general considerations, 453 Segmentation movements, 497 Semicircular canals, 918 destruction of, 918 eye movements and, 918 Semilunar valves, 150, 155 Semipermeable membrane, 4 Sensation : afferent paths of, 866 distribution of, 863 local sign of, 856 quality of, 855 Sense, temperature, 861 touch, 860 pain, 862 Sensibility : cutaneous, 859 deep, 859 Sensory adaptation, 862 Sensory area of cutaneous and deep sensibility, 878 Sensory centers, 876 of brain, 876 of cerebral cortex, 876 of optic thalamus, 878 Serine, 639 Serum albumin, 88 Serum globulin, 88 Sex, effect on creatinine excretion, 658 effect on energy output, 577 Sham feeding, 469 Shell shock, 302 Shock, 301 action of heart in, 305 anesthetic, 302 blood pressure in, 303 experimental investigations,, 304 gravity, 301 INDEX 989 Shock Cont 'd hemorrhagic, 302 histamine as a factor in, 307 nervous, 302 prognosis of, 311 oligemia in, 306 recovery from, 312 secondary symptoms of, 310 shell, 302 spinal, 302, 924, 965 surgical, 303 toxemia as a cause of, 309 trauma as a cause of, 309 treatment of, 311 vasomotor control in, 304, 305 Sinoauricular node, 185, 278, 293 Sinus arrhythmia, 292 Sinus bradycardia, 292 Skatole, 535, 665 in urine, 565 Skeletal muscle, respiration in, 408, 409 Skin, receptors of, 859 Smooth muscle: contraction of, 912 fatigue of, 912 metabolism of, 912 rhythmicity of, 913 Soap, 718 Sodium ions, 166 Solution of gases, 353 Solutions : gas laws and, 3 gram molecular, 5, 22 hypertonic, hypotonic, and isotonic, 6 nature of, 3 Sb'rensen method for estimating amino groups, 635 Sounds, cardiac, 156 recording of, 158 Specific conductivity, 17 Specific dynamic action, 574 Specific gravity of urine, 557 Sphingomyelin, 720 Sphygmic period-, 290 Sphygmograph, Dudgeon's, 201 Spinal cord: section of, 849 in laboratory animals, 965 in man, 967 hemisection of, 870 sensory pathways in, 868 successive degeneration in, 849 Spinal reflexes, 965 Spinal shock, 924, 965 cause of, 969 in animals, 965 in man, 967 Spirometer, 556 Splanchnic circulation in shock, 305, 306 Splanchnic nerve, 238, 704 Sponges, nervous system of, 828 Spot finding, 861 Stalagmometer, 66 Standard of neutrality, 26 Standard solutions, preparation of, 34 Stannius' ligature, 176 Starvation, 600 acidosis during, 602, 603 cause of death, 604 effect of creatinine excretion, 658 energy output during, 602 excretion of nitrogen, 600 loss of weight, 602 nitrogenous metabolism, 602 purines during, 603 secretion of gastric juice during, 511 sensations during, 510 sulphur during, 603 treatment of diabetes, 716 Statistical method, in diet control, 626 Stearic acid, 718 Stilling, "Summer cells" of, 769 Stomach : arrangement of food in, 489 digestion in, 515 emptying of, 491 effect of pathological conditions on, 494 rate of, 492 gas in, 496 miniature, 468 movements of, 485 effect on food, 488 tonus rhythm of, 506 Stroma of red cell, 92 Stromuhr, 207 Strychnine, action on reciprocal inhibi- tion, 941 action of, on synapse, 844 Subarachnoid space, 116, 255 Subcostal angle, 338 Subcostal borders, 338 Subdural space, 116 Submicrons, 55 Successive degeneration, 949 Sugar, storage of, 694 Sugar level in blood, 690 Sugar metabolism (see Carbohydrates), 685 relation of pancreas to, 710 Sulphates, ethereal, 535, 665 Sulphates, of urine, 566 Sulphur, excretion of, 648 in starvation, 603 Summation, 934 in conduction, 843 in reflexes, 842 Superior laryngeal nerve, influence on respiration, 351 Supplemental air, 317 Surface area, and energy output, 575 Surface tension, measurement of, 65 Surgical shock, 303 Survival period, 615 Suspensions, 52 Suspensoids, colloids, 61 990 INDEX Swallowing, 479 center, 481 of liquid food, 482 nervous control of, 481 sounds produced by, 483 x-ray during, 483 Sympathetic control of heart, 232 afferent, 228 Sympathetic nerve, 458 Sympathetic system, 895 Synapse, 830, 841 Synaptic fatigue in shock, 311 Synaptic resistance, 842 Syntonin, 520 Systolic index, 134 Systolic pressure, 129 measurement of, in man, 129 Tabes dorsalis, 300 Tachycardia, paroxysmal, 293 Tango-receptors, 855 Taurine, 528 Taurocholic acid, 528 Temperature : effect on dissociation curve, 399 effect on metabolism, 586 sensation of, 861 transmission in cord, 868 Tendon jerks, 921 Tension of CO 2 in venous blood, 359 of gases in alveolar air, 46, 356, 373 Testicles, 821 Tetanus, 905, 906 in stomach, 507 Tetanus toxin, action on reciprocal inhi- bition, 941 Tetany, 802 calcium deficiency in, 804 cause of, 804 electrical excitability in, 803 experimental, 800 guanidine metabolism in, 804 symptoms of, 802 Theinc, 668 Theobromine, 668 Thirst, 514 Thoracic operculum, 333 Thoracicolumbar outflow, 895 Threshold : of receptors, 855 of touch, 861 Thrombin, 103 Thrombogen, 107 Thrombokinases, 107 Thromboplastin, 107, 112 Thrombosis, 108 Thrombus formation, 113 Thymic acid, 682 Thymine, 669 Thymus, 824 Thyroid gland, 791 disease of, 795 removal of, 794 Thyroidectomy, 794 Thyroxin, 798 Thymus, 824 Tidal air, 317 Tissot method, 580, 591 Tissue fluid, 116 Tissue juice, 117 Tissues: amino acids in, 641 influence of, on clotting, 105 oxygen requirements of, 408, 412 utilization of glucose by, 708 Titrable. acidity and alkalinity, 22 Tonometer, 356, 393 Tone, 905 inhibition of, 925 influence of brain on, 924 of heart, 220 reflex adjustment of, 914 Tonus, 905 plastic, 905 Tonus rhythm, of stomach, 506 Torcular herophili, 259 Touch : localization, 861 sense, 860 Toxins, 70 Transfusion of blood, 142, 179 Trephining, 263 Treppe, 178, 910, 911 Trichlorlactamide, 668 Trimethylamine, 629, 662 True colloidal solutions, 52 Trypsin, 461, 463, 638 action of, 523 Trypsinogen, 461, 463 Tryptic digestion, products of, 524 Tryptophane, 629, 633, 640, 665 and growth, 614, 615 Tubules, uriniferous, function of, 549 Tumors and diet, 616 Turbidity of colloids, 52 Tyndall phenomenon, colloids, 52 Tyrodes solution, 168 Tyrosine, 640, 665, 698, 773 Uncoinpensated acidosis, 39 Unconditioned reflex, 466 Undetermined nitrogen, 629, 647, 6(52 Undulatory movement of ribs, 334 INDEX 991 Urea, 561, 643, 650 in blood, 645 during disease, 683 excretion of, 561, 649 retention of, in nephritis, 562 Urease, 82, 645 Uric acid, 563, 564, 667, 681 amount of, 556 chemical nature of, 667, 671 endogenous excretion, 680 in disease, 683 metabolism of, 667, 676 of blood, 681 salts of, 564 synthesis of, 677 under drugs, 681 Uric acid diathesis, 667 . Uricase, 673 Uricemia, 683 Uricolytic index, 674 Urine : acids of, 563, 564 amino acid, 564 amount of, 556 aromatic oxyacids of, 564 chlorides of, 565 composition, 560 crcatinine of, 563 depression of freezing point of, 557 excretion of, 544 H-ioii concentration of, 558 in disease, 567 hippuric acid, 564 homogentisic acid, 565 inorganic constituents of, 565 nitrogenous constituents ofj 560 normal organic salts of, 560 phosphates, 566 physical processes involved in produc- tion of, 14 purine bodies of, 563 rate of excretion, 675 reaction of, 558 skatole, 565 solid constituents of, 560 specific gravity of, 556 sulphates of, 566 total potential acidity of, 550 urea of, 561 Uriniferous tubule, 541 Urobilin, 529 Urobilinogen, 530 Utilization limit, 688 V Vagus : control of heart, 222 impulses, afferent, 227 Vagus center, effect of nicotine on, 231 location of, 227 tonicity of 226 Vagus nerve, influence on respiration, 348 Valine, 640, 641 Valves, cardiac, mechanism of, 154 auriculoventricular, 154 semilunar, 155 Valvular disease, 220 Van Slyke method for acidosis, 42, 43 Van Slyke method for amino groups, 636 Vascular reflex, 297 Varicose veins, 215 Vasoconstriction, 236 Vasoconstrictor fibers, 237 methods of detecting, 236 of extremities, 238 of head, 238 of viscera, 238 origin of, 237 Vasodilator fibers, 239 methods for detecting, 236 origin of, 239 Vasomotor center: afferent impulses, 243, 244 chief center, 240 effect of H-ion of blood on, 242 hormone control of, 242 subsidiary centers, 240 Vasomotor fibers, 236 origin of, 237 Vasotonic impulses, 241 Veins, disappearance of pulse in, 205 Velocity constant, enzymes, 75 Velocity, mean lineal, 206 pulse, 200 Venous blood, tension of CO 2 in, 359 Venous inflow, 216 Venous outflow, 235 Venous pulse tracing, 285 Venous sinuses, intracranial, 255 Ventilation : chemical conditions of air and, 754 physical conditions of air and, 757 physiological principles of, 754 susceptibility to infection and, 759 Ventilation of lungs, 367 Ventricle, curves of pressure in, 146, 148, 151 Ventricles : conductivity tissue of, 182 fibrillation, 195 spread of beat in, 192, 194 Ventricular hypertrophies, 284 Vignin, 612 Viscera, blood supply of, 254 Visceral bloodflow, 212 Viscosity of blood, 141 Visual area, 880 Visuo-motor areas, 886 Vital activity, 14 992 INDEX Vital capacity, 317, 329 in disease, 329 Vital theory of urine excretion, 545 Vitamines, 618 Vividiffusion, 641 Vomiting, 483 W Water content of blood, 87 Water hammer, in blood pressure meas- urement, 134 Wheatstone bridge, 18 White crescentic line, 231 Wiggers manometer, 147 Willard Gibbs, principle of, 66 Word blindness, 861 Word centers, 860 Word deafness, 861 Xanthine, 667, 671 Xanthine oxidase, 671 Xanthosine, 671 X-rays, in study of stomach, 469 movements of stomach seen by aid of 485, 489 Zein, inadequacy for growth, 612 Zymogen granules, 454, 455, 464 UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL LIBRARY THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW Books not returned on time are subject to a fine of 50c per volume after the third day overdue, increasing to $1.00 per volume after the sixth day. Books not in de- mand may be renewed if application is made before expi- ration of loan period. fl D D :" - 194H OC I 2 2m-4,'36