' BIOLOGY LIBRARY G r RffiCIPLES OP JOCFEMISTRY T! DENTS OF MEDICINE, AGRICULTURE AND RELATED SCIENCES BY SFOR1) ROBERTSON, PH.D., D.Sc. It) HIOCHEMISTRY IN THE UNIVERSITY OF ADELAIDE, SOUTH R OF BIOCHEMISTRY IN THE UNIVERSITY OF TORONTO; TRY AND PHARMACOLOGY IN THE UNIVERSITY OF CALIFORNIA ILLUSTRATED WITH 49 ENGRAVINGS LEA & FEBIGER PHILADELPHIA AND NEW YORK \ LI DEDICATED TO THE MEMORY OF MY FRIEND AND TEACHER SIR EDWARD C. STIRLING C.M.G., M.A., M.D., D.Sc. (CANTAB.), F.R.S., F.R.C.S. (ENO.) PROFESSOR OF PHYSIOLOGY IN THE UNIVERSITY OF ADELAIDE, SOUTH AUSTRALIA; DEAN OF THE FACULTY OF MEDICINE AND CONSULTING SURGEON OF THE ADELAIDE HOSPITAL; CORRESPONDING MEMBER OF THE ZOOLOGICAL SOCIETY OF LONDON AND HONORARY FELLOW OF THE ROYAL ANTHROPOLOGICAL INSTITUTE OF GREAT BRITAIN AND IRELAND IN TOKEN OF INTELLECTUAL INDEBTEDNESS AND PERSONAL AFFECTION PREFACE. IT has been the object of the author, in writing this book, to present the subject of Biochemistry in close relationship to Physiology, so that the student may perceive the intimate dependence, of these two sciences upon one another and come to regard physiological chemistry in its true light, as the foundation upon which we must ultimately build our interpretations of the functions of living matter. Emphasis has been placed upon the practical applications of the subject, and not only upon applications to the practice of medicine, but also upon applications to the industries and to general biology, for while the design of the author has been primarily to write a text- book for the use of medical students and student's intending to special- ize in biochemistry and physiology, the attempt has also been made to compile a work which will be of service to the agricultural student, the student of general biology, or the industrial chemist who is engaged in handling biological products. I am deeply indebted to my colleague, Prof. Hardolph Wasteneys, for his valuable cooperation in preparing the manuscript for the press, correcting proofs and compiling the index; and I desire to acknowledge my indebtedness to my wife for her assistance in the preparation of some of the illustrations. T. BRAILSFORD ROBERTSON. ADELAIDE, SOUTH AUSTRALIA, 1920. CONTENTS. Introduction 17 The Nature and Scope of the Subject 17 The Degree of Exactitude Attainable in Biochemistry ... .21 The Preparation Required for the Study of Biochemistry .... 24 The Subdivisions of the Subject 27 PART I. THE FOODS. CHAPTER I. THE SIGNIFICANCE OF FOODSTUFFS. The Chemical Relationship of Animals and Plants 31 The Conservation of Matter 32 The Classification of Foodstuffs 33 CHAPTER II. THE INORGANIC FOODSTUFFS. Water and Sodium Chloride . . 34 Calcium . . 40 Iron 43 Other Inorganic Foodstuffs . 49 The Complexity of our Dietary Requirements .50 CHAPTER III. THE CARBOHYDRATES; THE MONOSACCHARIDES. General Characteristics 53 The Hexoses 55 Reactions of the Carbohydrates 58 The Chemical Relationships of the Sugars 63 Certain Derivatives of Glucose 66 The Distribution of the Monosaccharides in Living Tissues 69 The Lactone Structure of Sugars 72 CONTENTS vii CHAPTER IV. THE CARBOHYDRATES; THE DISACCHARIDES, POLYSACCHARIDES AND GLUCOSIDES. The Disaccharides 76 Polysaccharides 81 Aminopolysaccharides 88 Glucosides K 89 The Carbohydrate Esters . * . 91 CHAPTER V. THE HYDRO-AROMATIC DERIVATIVES: THE CYCLOSES, CHOLESTEROL AND CHOLIC ACID. General Characteristics . . . '; 93 The Cycloses 95 Cholesterol and the Phytosterols 97 Bile Concretions; Ambergris . . . . 100 Cholesterol Esters 101 The Bile Salts and Cholic Acid 102 CHAPTER VI. THE FATS. The True Fats 107 The Characteristics of the Natural Fats 109 Waxes Ill The Phospholipins or Phosphatids 113 Glucosides of the Phospholipins 116 CHAPTER VII. THE PROTEINS AND THE AMINO-ACIDS. General Characteristics of the Proteins .......... 120 Coagulation Reactions 122 The Classification of the Proteins 124 I. The Simple Proteins 126 II. The Conjugated Proteins 128 III. The Products of Protein Hydrolysis 130 IV. The Coagulated Proteins 131 The End-produats of Protein Hydrolysis; the Amino-acids . . . . . 131 The Synthesis of Proteins 138 The Occurrence of Peptides among the Products of Protein Hydrolysis . 143 The Analysis and Characterization of Proteins by the Determination of the Amino-acid Radicals which they contain 144 CHAPTER VIII. COMPOUNDS OF THE PROTEINS. Types of Union in the Protein Molecule 148 Consequences of the Polypeptide Structure of Proteins 151 The Precipitation and Coagulation of Proteins by Inorganic Salts . . . 158 Compounds of Proteins with other Proteins 170 viii CONTENTS CHAPTER IX. THE NUCLEIC ACIDS AND THE NITROGENOUS BASES. The Decomposition-products of the Nucleic Acids . . ..... . 173 The Structure of the Nucleic Acids - 178 Amines derived from Amino-acids . ... '. -<'.' . . . . 184 The Betaines and the Vitamines . . 189 Nitrogenous Bases derived from Guanidine . . . ...... . * . 194 Nitrogenous Bases derived from the Phospholipins 196 Nitrogenous Bases forming the Active Principles of Internal Secretions . 197 CHAPTER X. THE HYDROLYZING ENZYMES. General Characteristics of the Enzymes 201 The Quantitative Relationships in Hydrolysis by Enzymes . . : : . . 207 The Influence of Temperature upon Enzymes . .... . . . . 213 The Influence of Reaction upon Hydrolyses by Enzymes . . ;.. . . . 217 The Specificity of the Hydroly zing Enzymes . . . .. . . . . . 218 The Synthetic Action of Hydroly zing Enzymes . . .221 Antienzymes . . -, . . . . . . . . . ..-.'.-'". . . . 226 CHAPTER XI. THE DIGESTION AND ASSIMILATION OF THE FOODSTUFFS. The Digestion of the Carbohydrates ... . . . . . ^ . . . . 228 The Digestion of the Fats . ...... : ., 1 .. ... . 232 The Digestion of the Proteins . . . .... .... . ... . 238 The Time- and Mass-relations of Digestion and Absorption 250 PART II. THE PROPERTIES OF PROTOPLASM. CHAPTER XII. PROPERTIES CONFERRED BY THE DIFFUSIBLE CONSTITUENTS. The Osmotic Pressure of the Tissue Fluids 255 The Osmotic Pressure of Cell Contents 263 The Composition of the Mineral Constituents of Tissue Fluids .... 268 The Neutrality of the Tissues and Tissue Fluids 271 CONTENTS i x CHAPTER XIII. PROPERTIES CONFERRED BY THE COLLOIDAL CONSTITUENTS: STRUCTURE AND CONSISTENCY. The Emulsion-structure of Protoplasm 284 The Viscosity of Protoplasm 295 Jellies and Gelatinization 298 The Osmotic Pressure of Protein Solutions 302 The Swelling of Protein Jellies '.'.. 304 CHAPTER XIV. PROPERTIES CONFERRED BY THE COLLOIDAL CONSTITUENTS: CHEMICAL AND BIOLOGICAL. Effects of Disturbance of the Inorganic Environment ' . 310 Effects of Removal of Calcium from the Tissues and Tissue Fluids . . . 314 The Mutually Antagonistic Action of Salts and Physiologically Balanced Solutions 318 The Origin of the Mutual Antagonism of Inorganic Salts 321 The Origin of Acid Secretions 327 The Selective Action of Tissues and the "Oligodynamic" Actions of Heavy Metals 328 The Biological Individuality of Tissues and Tissue Fluids 330 PART III. THE CHEMICAL CORRELATION OF THE TISSUES. CHAPTER XV. THE VEHICLES OF CHEMICAL CORRELATION: BLOOD AND LYMPH. The Composition of the Blood 335 The Coagulation of the Blood 342 The Chemistry of Hemoglobin 350 The Crystalline Forms of Hemoglobin in Relation to the Biological Indi- viduality of the Blood . 356 The Chemical Detection of Blood . . 361 The Origin and Composition of Lymph ........... 362 X CONTENTS CHAPTER XVI. EXAMPLES OF CHEMICAL CORRELATION. The Chemical Correlation of Respiratory Activities 365 The Chemical Regulation of the Circulatory System 368 The Chemical Correlation of the Processes of Digestion . . . . . .371 The Chemical Correlation of the Organs of Generation 376 The Chemical Regulation of Metabolism 381 PART IV. THE CHEMICAL PROCESSES WHICH UNDERLIE AND ACCOMPANY LIFE PHENOMENA. CHAPTER XVII. PROCESSES INFERRED FROM DIRECT OBSERVATION. The Intermediate Metabolism of the Carbohydrates: Muscular Contraction 391 The Intermediate Metabolism of the Fats; Diabetes . . . . .. ' . . 399 Oxidizing Enzymes 412 Bioluminescence 414 CHAPTER XVIII. PROCESSES INFERRED FROM INDIRECT OBSERVATION: THE ENERGY TRANSFORMATIONS IN LIVING ORGANISMS. The Influence of Temperature upon Life Processes . 417 The Influence of Light upon Life Processes 429 The Storage of Potential Energy; the Photosynthesis of Carbohydrates . 434 The Conversion of Chemical into Mechanical Energy; the Chemical Mechanics of Muscular Contraction 438 CHAPTER XIX. PROCESSES INFERRED FROM INDIRECT OBSERVATION: FERTILIZATION AND EARLY DEVELOPMENT. The Substitution of Chemical Agencies for Normal Fertilization .... 446 The Nature of the Agents which form Fertilization Membranes .... 450 The Effect of Membrane-forming Agents upon the Egg 458 The Relationship of Phospholipins to the Synthesis of Nuclear Material and the Effects of Lecithin upon Early Development 462 The Chemical Mechanics of Cell Division 466 Artificial Twin Formation and the Formation of Monstrosities 469 CONTENTS xi CHAPTER XX. PROCESSES INFERRED FROM INDIRECT OBSERVATION: GROWTH. General Characteristics of the Growth Process 471 The Influence of Race, Sex and Environment upon the Growth Process . . 484 The Substrates of Growth '. , . . . . 488 The Relationship of the Endocrine Organs to Growth . . ..-.. . 493 The Metabolic Rate and the Partition of Nutrients . . . .' . . . 500 Catalyzers of Growth . . 503 Old Age and Senescence 512 CHAPTER XXI. PROCESSES INFERRED FROM INDIRECT OBSERVATION: MEMORY AND SLEEP. Memory 521 The Fatigue Products of Nerve Centers 524 The Application of the Formula of Autocatalysis to Central Nervous Phe- nomena 527 Sleep 530 The Fading of Memory Traces " 532 PART V. THE PRODUCTS OF TISSUE ACTIVITY. CHAPTER XXII. THE WASTE PRODUCTS. The Carbonaceous Waste Products . . . 537 The Nitrogenous Waste Products 541 Conjugated Excreta 554 Aromatic Oxyacids 558 Waste Products of the Sulphur Metabolism 559 Urinary Pigments 562 The Properties and Composition of Urine 563 CONTENTS PART VI. THE ENERGY-BALANCE OF THE ORGANISM. CHAPTER XXIII. THE ANIMAL BODY AS A MACHINE. The Applicability of the Law of the Conservation of Energy to Living Organisms 567 The Isodynamic Values of the Foodstuffs 575 The Protein Requirement in the Dietary .'.-.* 578 The Normal Diet . . 582 The Calorific Requirement and the Surface Law . . \ . .... 587 The Nutrition of Children . . . . , ^ . * . . . . , . . . 590 The Energy Equivalent of Growth . . : . . .; . . .... . 592 The Outlook . 595 PRINCIPLES OF BIOCHEMISTRY. INTRODUCTION. THE NATURE AND SCOPE OF THE SUBJECT. The subject-matter of biochemistry is the application of the known principles of chemistry and physical chemistry to the study and inter- pretation of life-phenomena; of the processes, that is of digestion, assimilation, respiration, growth, reproduction, muscular contraction and the like, which combine to distinguish living from inanimate matter. From this definition it must be clear that biochemistry possesses very close affiliations with both animal and vegetable physi- ology. For physiology is the study of the way in which societies, indi- viduals, organs and cells perform their functions, and since each and every function of living matter ultimately involves or depends upon chemical changes, to this extent the study of each and every function of living matter becomes a part of the subject-matter of biochemistry. The distinction between physiology and biochemistry is in fact an arbitrary one, depending very largely upon convenience and upon the contemporary limitations of our knowledge. So long as we possess no clue whatever to the nature of the processes which underlie or accompany a life-phenomenon, the study of that phenomenon and of the method of its performance is, beyond any question, an exclusively physiological problem. But directly we take the first steps toward ascertaining the nature of the chemical phe- nomena which accompany its performance, we are taking, also, the first steps toward incorporating this problem into the subject of biochemistry. The historical growth and development of the subject have illustrated very aptly these natural applications. In the beginning, and that only one brief generation ago, biochemistry was an undifferentiated portion, a minor branch of physiology, and formed the subject of a bare half- dozen lectures delivered by the professor of physiology. Gradually the need of special training for the study of this subject, and its con- tinually increasing magnitude and practical importance have led men to make a special study of it, apart from that of the parent-subject. The labors of these men have quickly added countless phenomena to their special domain, and so important are these, and so funda- mental is the part which biochemistry now plays in medicine, agri- 2 18 PRINCIPLES- OF BIOCHEMISTRY culture and the industries, that almost everywhere the study of bio- chemistry ranks with that of anatomy, physiology and pathology as one of the studies fundamental to the understanding of medical science, or with botany, plant-physiology and bacteriology as one of the studies fundamental to the understanding of agriculture. It must not be supposed, however, that the withdrawal of biochem- istry from the parent-subject has left physiology any the poorer. Physiology has not been left merely with a residuum of undigested material, ultimately to be absorbed by the biological chemist. On the contrary, with the development of biochemistry, physiology has developed too and that to an extent unimagined by its founders. A few generations ago, physiology was a little-considered fragment of the study of anatomy, just as, one generation ago, biochemistry was a little-considered portion of the study of physiology. The same differ- entiation has separated the teaching of biochemistry from physiology as that which has separated the teaching of chemistry from that of physics. We may regard physiology as consisting for the present of the study of the applications of anatomy and physics to the elucida- tion of life-phenomena, together with the entire study of a residuum of phenomena and processes which are for the present passed by in biochemistry simply because we do not as yet possess any clue whatever to the nature of the chemical processes which underlie them. Hence, physiology is destined ultimately and at some as yet far distant date to become the study of the interpretation of life-phenomena by the aid of the principles of anatomy, gross and minute, and physics. Biochemistry is the study of the interpretation of life-phenomena by the aid of the principles and facts of chemistry. Physiology investi- gates the molar and molecular phenomena of life, biochemistry the atomic. Of course, this division is arbitrary and unreal, just as the distinc- tion between physics and chemistry is arbitrary and unreal. Nature recognizes no such classification of her phenomena. Physics merges insensibly into chemistry and in like manner physiology merges into biochemistry. An illustration of this fact has been strikingly afforded in recent times by the rapid development of physical chemistry, a whole borderland between physics and chemistry, which has undergone such extensive survey within the last generation as to demand a noteworthy degree of special training on the part of those who would attempt to master it. Even the delineation of this domain has not by any means removed all of the "debatable land," however, that lies between physics and chemistry; witness the recently discovered phenomena of radio-activity, which have opened up yet another field of investigation which is neither physics nor chemistry. And so it is with physiology and biochemistry. There is an indefinite "debatable area" between the two, and many if not most of the problems in either field require the aid of both physiology and biochemistry for their solution. NATURE AND SCOPE OF THE SUBJECT 19 The investigator of Nature cannot afford to hamper himself by arbitrary definitions and delimitations of his field. When the need arises he must be prepared to use the tools which the problem calls for, be they the tools of physics, chemistry, mathematics, anatomy, bacteriology or pathology. The teacher is somewhat more con- strained. He cannot carry his pupils too far from the center of the subject in hand, lest their lack of preparation should render them unable to follow him. Even so, however, the student of biochemistry will often have occasion to dwell, in his studies, upon certain aspects of problems which the physiologist has made peculiarly his own, and the medical student will frequently find himself studying one and the same problem in his course in biochemistry and again in his course in physiology. Nevertheless he will find that he is not merely repeating his work, not merely covering old ground, but that, on the contrary, the physiologist and the biochemist have each of them something different to say; displaying the problem in different lights and dwelling upon it in different connections. We have stated that the science of biochemistry consists in the interpretation of life-phenomena in the light of the facts and principles of chemistry. The question may here very naturally arise in the mind of the reader, How can it be possible to apply chemistry to the investi- gation of living matter? True, we can attempt to analyze living mat- ter, to separate chemical constituents from it and to identify them. But then, directly we begin to analyze living matter it ceases to be living matter. The reagents which we employ immediately "kill" it, that is to say, abruptly suspend its characteristic functions and disperse and dissolve the minute structures of protoplasm which are the physical substratum upon which its functional activities are reared. Unquestionably, an amoeba which has been boiled in hydrochloric acid may yield interesting products, but then it is no longer an amoeba, and the products which analysis yields bear only a remote relationship to those which were originally present in the living organism. To find out what is actually occurring in living matter- we must, therefore, employ methods of investigation somewhat analogous to those which the physical chemist employs in the investigation of what is actually occurring in flames. First, we study the nature of the substances which enter the flame, then we study the properties and behavior of the flame itself, always taking care to do so by the aid of instruments which do not disturb the flame, and finally we ascertain what substances the flame gives off. From these various and frag- mentary data we endeavor to reconstruct in our minds a coherent picture of the train of events as they actually occur, and this endeavor will be the more successful in proportion to the extent, the variety and exactitude of our measurements. So far as possible, then, we must bring static and not dynamic methods of mensuration to. the study of living matter, methods, that is, which do not involve the cessation of the very processes which we desire to 20 PRINCIPLES OF BIOCHEMISTRY investigate. Often, it is true, we can successfully employ destructive, dynamic methods to find out many important things. For example, in the study of digestion, we can destroy the living cells which form the lining mucous membrane of the stomach, and, having destroyed them, extract from them a substance, Pepsin, which will digest proteins, even in glass vessels in laboratory-incubators. In this way and by similar methods we can study the changes which are brought about in our foodstuffs when they enter the alimentary canal. Even in a fairly simple case such as this, however, the dynamic method does not altogether suffice. For we find that within the alimentary canal itself the foods are digested much more rapidly than we can digest them with the aid of ferments in laboratory-glassware. Some condition, other than mere warmth or mechanical agitation, some condition which we have not yet fully succeeded in imitating, very materially aids the action of these ferments in the cavity of the living alimentary canal. By such phenomena as these we are constantly being reminded that it is not by any means safe to argue directly from the behavior of dead fragments or products of living tissue, to that of living tissue itself. The results of dynamic experiments which involve the actual destruction of the living tissues which we are investigating, only afford a starting- point, therefore, or an orientation, for our guidance in a repetition of the experiment under actual living-conditions. Biochemistry, therefore, falls very naturally into two fields of study, differentiated by the methods of investigation employed. The one field, that which has until recently been the peculiar interest of the "physiological chemists," consists in the study of the crude substances which enter into the life-flame and the products which leave it. The foodstuffs and the excreta, and, incidentally, the composition of dead matter that once was living, also the study of the action and reaction of fragments of living or dead protoplasm upon the foods or upon one another, these, until comparatively recently, comprised the whole activity and interest of chemistry in the investigation of living matter. It is obvious, however, that while knowledge of these things is an essential prerequisite to the understanding of the chemical phenomena of life, yet they are far from yielding information as to the nature of life-processes themselves. It was for this reason, and with justice, that one of the greatest contributors to our knowledge in this field, G. von Bunge, exclaimed in 1894, "All processes in the organism which may be explained mechanically are no more phenomena of life than are the movements of the leaves and branches of a tree that is shaken by the storm, or the movement of the pollen that the wind wafts from the male poplar to the female." 1 We were at that time hovering upon the outskirts of the main problems, since actual penetration of them was necessarily deferred until the momentous advances of physical chemis- 1 Lchrbuch der Physiologischen und Pathologischou Chcraio, 3tc Aufl., Leipzig, 1894. EXACTITUDE ATTAINABLE IN BIOCHEMISTRY 21 try placed in our hands the necessary implements and knowledge to essay the task. Our second field of study, then, consists in an analysis of the chemical phenomena which accompany or underlie the activities of living, undis- turbed, and more or less normally functioning protoplasm, a field which until recently was almost exclusively the preoccupation of the " experi- mental biologist." Inevitably, however, these two phases of chemical inquiry, so closely affiliated, so mutually dependent, are coming to rely more and more intimately upon each other and hence are being welded more and more firmly into one. Experimental biology drawing upon the rich resources of physiological chemistry, is immensely increasing its exactitude and its certainty, while physiological chemistry, on the other hand, is rapidly widening the horizon of its inquiries in response to inspiration drawn from the field of experimental biology. In this work we will recognize no distinction between these fields, but endeavor, in so far as the limitations of our knowledge permit, to interweave them into one coherent representation of the complex tissue of chemical processes which constitutes life and its immediate consequences. THE DEGREE OF EXACTITUDE ATTAINABLE IN BIOCHEMISTRY. In the so-called "exact sciences," to wit, mechanics and physics, we have, as a rule, the power to isolate more or less completely any phenomenon or group of phenomena which we wish to study, and to guard them from disturbance by the intrusion of accidental variables. For example, it is not a difficult matter to demonstrate that a falling body experiences a constant acceleration, the most serious intrusive variable being the friction of the air, a variable which can now be very readily excluded in a variety of ways. 1 Similarly, in chemistry, it is not a difficult matter to observe the progress and equilibrium of such a reaction, as, for example, the reduction of iron oxide by hydrogen. The chemicals are procurable in pure conditions, only one reaction occurs, and it is a simple matter to exclude other chemicals and to keep the temperature and pressure of the system constant. In organic chemistry much more complex phenomena are encountered. It is the exception rather than the rule to find a reaction which proceeds evenly and without disturbance by side-reactions or secondary decom- positions. To detect regularities and establish "laws" hi such a system is a task the more complex the greater the number of adventitious variables. The difficulties which are encountered in studying organic reactions in laboratory glassware are enormously magnified in studying reactions 1 It must be remembered that the friction of the air, which to us presents no difficulty, was to our ancestors an insuperable obstacle to the measurement of gravity. In exactly the same way insurmountable obstacles which at this day defeat our ends in physiologi- cal or biochemical research will appear of trivial importance to our intellectual heirs. As a rule such obstacles merely imply that we are attacking the problem from the wrong angle. 22 PRINCIPLES OF BIOCHEMISTRY which occur in living matter. The life of any one cell consists in a multiplicity of parallel reactions, interrelated, interdependent, and interwoven into a bewildering complex. Multicellular organisms, such as ourselves, consist of millions of such cells. When the reader is reminded that the reactions in each organ or group of cells and possibly, even in each individual cell, possess an individual character of their own, and that these reactions are excessively sensitive to external agencies, the complexity of the task of unravelling the separate reactions and tracing their individual progress must be evident. It follows from the complexity of the phenomena that the regularities and relations observed by the biochemist are rarely capable of formula- tion with such precision as those which are observed by the physicist or chemist. To illustrate this fact, let us consider the difficulties attendant upon the investigation of one of our simplest problems, to wit, that of the mode of action of the protein-digesting ferment Trypsin. We have first of all to overcome the difficulty of obtaining pure protein. That obtained (and a "pure" protein in the sense that inorganic reagents may be "pure" has never been prepared), we then have the difficulty of obtaining a pure trypsin, a difficulty which has never been even partially overcome. In fact we certainly possess no pure trypsin and we have, moreover, no method of ascertaining how impure our preparations are. Not only are our preparations of trypsin impure, but they frequently contain several ferments which digest proteins, a fact which has only recently come to be appreciated. Notwithstanding all these obstacles we have found that if trypsin be allowed to act upon protein, with certain necessary precautions, a regularity may be observed in the rate of decomposition of the protein by the ferment, and this regularity may even be formulated in mathe- matical terms. We are not surprised to find, however, that the agree- ment between the formula and the experimental measurements (of quantity of protein digested) is not extremely exact. Under very favorable conditions the requirements of theory and the findings of the investigator may agree to within one per cent, of their mean value. In a purely chemical problem an agreement to within one-tenth of a per cent, is anticipated and not infrequently obtained. In physics or in astronomy an agreement to within one one-hundredth of a per cent, is not in the least exceptional. As the uncontrollable adventitious variables become fewer, it will be observed, the agreement between formulae and experimental data becomes more and more precise. Hypotheses of a more general character, not admitting of mathe- matical formulation, share in this disadvantage, and hence it arises that a larger proportion of hypotheses in biological sciences are of uncertain or very questionable validity than of those in the so-called "exact" sciences. But the difference is merely a matter of degree and tends progressively to diminish. All scientific hypotheses and "laws" are subject to a marginal inexactitude, and all human precision is relative. As our acquaintance with any field of investigation grows EXACTITUDE ATTAINABLE IN BIOCHEMISTRY 23 more extensive, the width of the margin of inexactitude diminishes, and that is all. For example, no "law" is apparently of more extensive applicability or capable of more precise mathematical formulation than Newton's law, that bodies attract one another as the inverse square of their distance apart. Yet certain astronomical data, devia- tions in the orbit of the planet Mercury, 1 point to the possibility that even this law may not be exact and that the true exponent of the distance may in truth not be 2 but 2.000,0001612. The margin of inexactitude is here represented by the minute fraction 0.0000001612, but it is here nevertheless. And so it is with all scientific hypotheses. Our laws, formulations and hypotheses are merely temporary short- hand statements of our acquaintance with the facts. As our acquaint- ance with the facts grows larger we must revise our shorthand to express our accessions of knowledge. The shorthand is not the knowledge itself. Science, in reality, consists solely in our knowledge of facts and our control of the forces of nature and not of the hypotheses which we formulate by the way in order to summarize our present state of knowledge and stimulate the imagination to fresh inquiries. Biology is, in truth, no less an "exact" science than any other, than astronomy, for example, but its hypotheses are subject to much more frequent and thorough revision than those of physics or astronomy, simply because our knowledge of the field is less and is growing more rapidly. The whole theory of the scientific method of thought has in fact been based by the great founders of science upon the assumption of the fallibility of purely intellectual operations, and hence of the untrust- worthiness of hypotheses. Newton's famous rule, " Hypotheses non fingo," while impracticable for the individual investigator, remains nevertheless true of science as a whole, of the body of exact knowledge, that is, which endures the test of time and endows mankind with the power of ruling and directing the multifarious and stupendous forces of Nature. During the centuries which have been marked by the acquire- ment of this knowledge countless hypotheses have been formed, and accepted for a while, and then abandoned as evidently absurd. But the forward march of exact knowledge has never suffered interruption and not infrequently indeed has been very much facilitated by the most obviously erroneous hypotheses. The phlogiston theory of heat is perhaps the most striking example of this kind. It was a most patently erroneous hypothesis, built up by perfectly sound reasoning based upon imperfectly understood facts. Yet for a hundred years the mere existence of this hypothesis was the greatest contemporary stimulus to the development of chemistry and it ultimately led to the establishment of the conception of the conservation of matter. As the curves of the geometrician approach and yet never actually attain their asymptote, so do we continually approach and never yet have we attained the utter truth. The merit of the scientific method 1 Cf . article on Gravitation, Encyclopedia Britannica, 1 1th edition. 24 PRINCIPLES OF BIOCHEMISTRY of thought lies in the fact that the otherwise circular speculations of humanity, ever returning unprofitably to the point from which they started, have had a thrust communicated to them which has deflected them into a perpetually widening spiral, reaching further and ever further into the infinite, promising knowledge commensurate only with the immensity of the universe, and power to which no man dare set a limit. If, then, our present conceptions in biochemistry are subject to rapid and comprehensive modification this affords no legitimate basis for scientific cynicism or indiscriminate scepticism. On the contrary it is a hopeful augury, testifying to the youth of the subject and the vast development that lies before it. No subject, indeed, promises more immediate developments of stupendous significance to man. The control of life itself, no less, is the alluring aim and destiny of the medical and biological sciences and the basis of every step in the acquirement of this control must inevitably be founded on a knowledge of the chemical processes which underlie and constitute life. We may be well content, with such a prospect before us, to resign absolute certainty to the political doctrinaire. For ourselves, dwelling amid uncertainties and hazards, advancing like bold navigators in uncharted seas, we will turn our faces toward the new and wider horizons which always lie before us. We will regard a hypothesis as an instrument of research, like a balance, a burette, or better still a compass; a guide and a stimulus to investigations, but a mere approximation to the truth which we trust will gradually approach closer and yet closer to verity as our knowledge grows in extent and proliferates in detail. THE PREPARATION REQUIRED FOR THE STUDY OF BIOCHEMISTRY. No amount of courage and enthusiasm, however, will suffice to alto- gether compensate for lack of preliminary training and acquired skill in those branches of science upon which biochemistry is founded and from which it originates. Biochemistry is in the first place and most essentially an outgrowth from organic chemistry and an acquaintance with the general principles of that science and the simpler laboratory procedures most frequently employed in it, is as essential to the understanding of biochemistry as a vocabulary of French words is to the understanding of Moliere in the original. In this work I will suppose the reader to be acquainted with organic structural formulas and the general principles according to which they are- inferred from the behavior of the substances to which they are applied. 1 The modern developments of biochemistry and particularly those which aim at the interpretation of the processes underlying the per- formance of function, involve the application of the elementary princi- 1 The reader whose previous training in this subject has been dencient may consult E. V. McCollum, Organic Chemistry for Students of Medicine, New York, 1916. PREPARATION FOR THE STUDY OF BIOCHEMISTRY 25 pies of physical chemistry and there can be ho doubt whatever that the future and most momentous developments of the subject are destined to involve physical chemistry more and more extensively. The essen- tial principles are neither numerous nor abstruse, good elementary text-books of the subject abound and the student is earnestly recom- mended, if he has not previously received training in this subject, to acquire for himself a suitable handbook of physical chemistry, 1 and to consult it frequently in the course of his studies in biochemistry. The intelligent employment of the elementary principles of physical chemistry implies a nodding acquaintance with the so-called "higher mathematics," but far more than for the mere understanding of physical chemistry, mathematics is an essential instrument in the handling of quantitative measurements of any kind whatsoever. Every branch of science is, in its youth, qualitative, and in its maturity quantitative. Even taxonomy has been converted by the discoveries of Mendel into a quantitative study involving in some instances very complex mathematical operations. Biochemistry is at the present juncture passing through a species of adolescence and emerging by very rapid stages from the qualitative into the quantitative stage of develop- ment. The student who would prepare himself for the future, there- fore, would do well to acquire such rudiments of mathematical skill as may be necessary for the elucidation of principles which . he will unquestionably be called upon to comprehend. Mathematics is in reality a symbolic language which expresses in brief terms a series of interrelated facts and considerations which would otherwise be too intolerably complex to retain simultaneously in the mind. By acquir- ing mathematical facility, therefore, the student is not augmenting the complexity of his task, but simplifying it. The applications of physical chemistry to biological problems necessitate of course an elementary knowledge of the differential calculus and the simplest methods of integration. 2 All of the work upon ferments and digestion in its quantitative and most important aspects now demands the employment of the calculus. As an example of the wider and at first sight unexpected applications of this mathe- matical technique the reader is referred to the important work of Barcroft, 3 which has marked an epoch in our understanding of the respiratory functions of the blood and which could never have yielded one tithe of the information obtained without the employment of the methods of the differential and integral calculus. A moderate familiarity with the elementary principles involved in the solution of differential equations would also upon occasion be found 1 For example, E. W. Washburn: An Introduction to the Principles of Physical Chemistry, New York, 1915. A. Findlay: Practical Physical Chemistry, London, 1914. 2 The student may consult J. Edwards: Differential Calculus for Beginners and Integral Calculus for Beginners, while for the methods of applying the calculus to the solution of scientific problems the student would do well to read Perry's Calculus for Engineers, London, 1897. 3 The Respiratory Function of the Blood, Cambridge, 1914. 26 PRINCIPLES OF BIOCHEMISTRY very useful. 1 In the treatment of quantitative data and the graphic representations thereof it is frequently necessary or desirable to apply a formula to the curves obtained or to compare them with the curve which may be deduced from theoretical premises. In this extensive field of practice a knowledge of the proper method of dealing with and minimizing the effect of accidental experimental errors is required and the employment of the method of least squares is essential if the best use is to be made of the experimental material which may be available. 2 For purposes of fitting empirical formulae to curves, eliminating ex- cessively erroneous results and interpolating probable values between values which have actually been measured, a study of the methods of interpolation and mechanical differentiation is exceedingly valuable and helpful. 3 But perhaps the most essential branch of mathematical practice in the equipment of the biochemist of the future will consist in the methods of the statistician. When we come to deal with actually living material, as we are compelled to do in order to advance our subject at all in its most significant direction, we are at once con- fronted by the problem created by the inherent variability of living things. No two animals are alike, not even may we find any two living cells which are precisely identical. In agriculture no two plots of ground are alike, no two plants are ever identical. How then, in comparing experimental animals or plants or plots of ground with "normals" or "controls" shall we ever attain to certainty of our results? It would seem that it must always be possible that the differences between any two groups of animals may merely be the pro- duct of chance selection of two groups which might have differed in the observed sense without any experimental manipulation whatsoever. This difficulty, the fundamental character of which is recognized by every biological investigator, is of course not of so much importance in those cases in which the differences for which we are looking are very large, as death contrasted with survival, decisive loss of weight con- trasted with equally decisive gain, or reduction or enhancement of normal qualities by fifty per cent, or more. But phenomena such as these are the obvious ones in any field of science, those which lie at the surface and are garnered by the earliest investigators, and they are not invariably, and in fact not usually, the phenomena upon which we ultimately come to rely for the basis of wide and fundamental generaliza- tions. Such emphatic disparities testify in themselves to the unusual- ness of the conditions invoked, and hence carry the suspicion that the response to such extreme conditions may not be a normal or at least a usual reaction of living matter to its environment. For our deeper 1 Murray: Introductory Course in Differential Equations, London, 1897. 2 M. Merriman: Text-book on the Method of Least Squares, New York, 1891. L. Tuttle: The Theory of Measurement, Philadelphia, 1916. 8 H. L. Rice: The Theory and Practice of Interpolation, Lynn, Mass., 1899. J. Mellor: Higher Mathematics for Students of Chemistry and Physics, London, 1902. PREPARATION FOR THE STUDY OF BIOCHEMISTRY 27 understanding of life which is to come therefore, we must learn to rely with confidence upon relatively small and fluctuating disparities between groups composed of very variable material. This can be done in one way and in one way only, namely, by employing the methods of the statistician whereby we may accurately gauge the relative values of observations obtained with variable material, compute the number of observations necessary to attain a given degree of certainty or accuracy, place in their proper perspective extreme or overlapping variations in aberrant individuals and, in short, render measurements upon even such variable material as living organisms just as precise as the measurements employed in quantitative analysis. The student of biochemistry would be well-advised therefore to acquire the simple mathematical technique which is requisite for the employment of statistical methods, 1 but he should remember that this branch of mathematics above all others abounds in pitfalls for the unwary and he should be sure that he perfectly comprehends the simple fundamental principles which underlie these methods before he attempts to put them into practice. 2 If the reader should desire to gain a conception of the variety and scope of the possible applications of the statistical method to problems of biochemistry, experimental biology and agriculture, he may consult the recent work of Loeb and Wasteneys upon the applicability of the Bunsen-Roscoe law to animal heliotropism, 3 of Waynick upon the distribution of nitrifying bacteria in soils 4 and of the author upon the growth of children. 5 The Subdivisions of the Subject. In this work we will endeavor to follow up the foodstuffs from the moment when they are partaken of, to the moment when, after having circulated through the body and partaken of its life, their final products are excreted. The subject- matter is divided into six parts corresponding with various phases of the cycle of changes which the foodstuffs undergo. The subdivisions are as follows: Part I. The Foods, their properties, digestion, assimilation and conversion into living matter or into reserve-materials. The considera- tion of this phase of our subject takes us up to the point at which the foodstuffs, subjected to certain modifications, have really been converted into living protoplasm. This leads us naturally to the consideration of the second phase of our subject, namely : 1 The Student may consult G. Udney Yule: An Introduction to the Theory of Statistics, London, 1911. For tables and formulae the student may refer to C. B. Davenport: Statistical Methods, New York, 1904. 2 Probably the best introduction to the fundamental conceptions of probability which form the basis of the statistical method is contained in the classical little memoir of W. A. Whitworth entitled Choice and Chance, Cambridge, 1901. 3 Jour. Exper. Zool., 1917, 22, 187. 4 D. D. Waynick: University of California Publications in Agricultural Sciences, 1918, 3, 243. * T. Brailsford Robertson: Am. Jour. Physiol., 1915, 37, 1 and 74; 1916, 41, 535, and 547. 28 PRINCIPLES OF BIOCHEMISTRY Part II. The manner in which the properties of the foodstuffs mould and determine the properties of living protoplasm. Part III. In proceeding to consider the activities, apart from the merely passive properties of living matter, we are at once confronted with the significant fact that the multicellular organisms, like our- selves, are really immense societies composed of innumerable minute units which are the individual living cells. We have, in this society, a governing authority, the central nervous system; a postal-telegraphic system, the peripheral nervous system; a laboring class, the muscles and glandular tissue-cells; distributing agencies, the blood and lymph, and with all of these not a rigid central autocratic control, but a very considerable degree of local autonomy. Every cell is working, not by deliberate instruction, but as a part of its very specialized life. In order to avoid confusion in so vast a complex of semi-independent units, numerous cooperative mechanisms must be present to adjust supply to demand and effort to need. These mechanisms imply a certain correlation of distant .parts; for instance, between the neuro- muscular system which controls the respiratory movements, and the need of the tissues for oxygen. This correlation of different and often widely separated activities is brought about by the interaction of two distinct types of agency, nervous agencies and chemical agencies. In so far as this correlation of activities is brought about by chemical means, it will fall under consideration in this third phase of our subject. Part IV. In this part we will endeavor to attack the very kernel of our problem, that part of our studies which is destined to provide the ultimate foundation of the practice of medicine and in no small measure of agriculture also. The chemical phenomena which underlie, accom- pany or even actually constitute the living activities of cells will here be our preoccupation and we will incidentally study, so far as our fragmentary knowledge at this time permits, the changes which the foodstuffs or constituents of protoplasm undergo at the instant of their utilization for the furtherance of vital functions. Here we will find our most alluring problems and our least extensive knowledge, here is the region in which must occur the greatest conquests which lie before us and those which will exercise the most fundamental and far-reaching effect upon our own lives and the lives of those who will follow after us. Part V. In this part we will take up the study of the waste-products which ultimately result from the activities of oiir tissues; the ashes, the products of combustion and the debris which result from the daily maintenance and furtherance of life. Part VI. In this part, regarding the entire body as a chemical machine, somewhat crudely comparable to a steam-engine, we will discuss the question of the efficiency of the machine and the relation- ship of the horse-power it can develop to the nature and value of the fuel with which it is provided. It is in this connection that we will discuss data which may enable us in some measure to answer the question, what investment of particular types and mixtures of fuel will PREPARATION FOR THE STUDY OF BIOCHEMISTRY 29 return the greatest interest in the form of efficient work on the part of this very complex machine, a human being? (Given, of course the incalculable psychological asset of good-will.) This is the type of problem with which the allied governments and Germany were recently grappling and in proportion as we can contribute to its answer we are assisting not merely to guide civilization and humanity safely through the most dangerous crisis of all its long history, but also to solve a perennial problem which the War merely rendered acute a little earlier than would otherwise have been the case, the problem, namely, of correlating production and distribution with the fluctuating needs of the scattered populations of the world. The specialization of the occupations of peoples and areas which has so characterized the development of civilization in the past century carries with it inherent dangers which approach more and more near as the process of specialization extends. The specialized individual is always depen- dent upon others for his support. The specialized city or nation is dependent upon the world. The mutual dependency of peoples which our multifarious modern activities has evoked compels attention, in widely separated parts of the world, to the needs of remote and alien workers. These needs are chemical in their basis and biochemistry alone can supply us with the exact knowledge which is necessary to adjust them. PART I. THE FOODS CHAPTER I. THE SIGNIFICANCE OF FOODSTUFFS. THE CHEMICAL RELATIONSHIP OF ANIMALS AND PLANTS. In considering the nature of the foods and their elaboration into living matter, it is necessary in the first place to realize that the foods of multicellular animals such as ourselves, are, at the same time, the constituents out of which living matter is built up. This becomes evident when we recollect that the majority of our foodstuffs consists of matter that was formerly living or which is derived from matter that was formerly living. Meats and vegetables and grains are, of course, matter that w r as awhile ago alive, that is now arrested in its function and more or less rapidly decomposing into more elementary substances, but still contains, for the most part, the components of living protoplasm. Perhaps they are not linked together in precisely the way in which they are linked together in truly living matter, and perhaps the fact that this matter is no longer living is attributable to this disturbance in the linkage of its constituents. Still the con- stituents are there, and we appropriate them, modify them in some degree, and build them up into our own tissues. Other foodstuffs, such as sugar, are directly extracted from living tissues in which they form stores or reserves of energy, for example from beets or sugar-canes, and these are likewise appropriated to our own use. In this respect we differ very materially from the plants, the foods of which are in general very much more elementary than ours. Plants are actually able to build up living tissues out of substances which have in themselves no necessary connection with living protoplasm; out of mineral salts, water and carbon dioxide. For this reason it used to be thought that only plants possessed the pow r er of synthesizing the actual constituents of living matter and that we, without doing any fresh construction, simply sort out and appropriate these preformed con- stituents and thus live in a state of parasitism upon the vegetable world. 32 ,-,;. t .. .SIQtflFIQANCE OF FOODSTUFFS The discovery by Schmiedeberg and Bunge in 1876 of the synthesis of hippuric acid from benzoic acid and glycocoll in the tissues of the kidney, 1 disposed of this untenable distinction, and while we are cer- tainly to be regarded as primarily parasitic upon the vegetable and lower orders of the animal kingdom, yet we are not so unable to create constituents of living matter, as earlier investigators imagined. We know now that animal tissues can perform a multiplicity of syntheses whereby constituents of protoplasm are made which the food does not contain preformed. It will be found, however, to be a general charac- teristic of syntheses carried out in animal tissues, that the storage of energy or heat-value which is accomplished thereby is usually small, whereas in green plants syntheses are accomplished which involve the locking up, for longer or shorter periods, of very large quantities of energy; for eons as in coal deposits, or for the brief period of a single winter as in the seeds which consume their stored-up energy when they germinate in the spring. The reason for this distinction is not far to seek. The green plant has an inexhaustible reservoir of energy upon which to draw; the radiant energy of the sun; and the energy which is locked up in the starches, fats and proteins, which plants synthesize from the most elementary products of combustion, is derived in the long run from the sun. The animal has no comparable capital to draw upon, and if an animal is to perform a synthesis involving absorption of heat or energy, it can only do so at the expense of its current account, that is to say by the degradation of its own tissues or food reserves. On the whole, therefore, and with the exceptions noted, green plants are the prime conservers of energy, while the function of animals is to dissipate it again. The whole fever and bustle of life upon the earth is therefore none other than a transitory phase through which continually passes a minute fraction of the colossal outpourings of solar energy. THE CONSERVATION OF MATTER. Whatever may be the relative efficiency of different types of proto- plasm as storers of energy and creators of living matter, they are all alike subject to the law of the Conservation of Matter. 2 That is to say, although an animal or plant cell may create new chemical compounds ; new permutations and combinations of the chemical elements, it cannot create new elements. All of the carbon in its tissues must have been derived, for example, from carbon from without. If an animal gives off nitrogen in the form of urea, it must either take up fresh nitrogen from without, or else its tissues must remain permanently poorer in nitrogen. 1 Thai this synthesis occurs in some organ or tissue of the body had been recognized by Wohler as early as 1824. 2 The applicability of the law of the Conservation of Matter to living organisms was first demonstrated by its discoverer, the French chemist Lavoisier (1743-1794), and subsequently confirmed in detail by Licbig (1803-1873). CLASSIFICATION OF FOODSTUFFS 33 Now it is self-evident that we are continually voiding waste products, urea and very many other substances in the urine, carbon dioxide and water-vapor in the breath, and various items of waste in the sweat and in the feces. Furthermore, despite his rapid and continual losst of substance, when we are adult we remain tolerably constant in weight and composition, that is, if we are healthy and neither becoming emaciated nor growing fat. It follows that, as a general rule, we must be taking in from without, not only just as much total substance as we are losing daily in these various ways, but also just as much of each of the individual elements, nitrogen, carbon and so forth, as we are daily voiding. This intake of elements constitutes the act of feeding, and the forms in which we take in these elements are our Foods. THE CLASSIFICATION OF FOODSTUFFS. As has been stated, our articles of diet are more complex than those of the plants. Plants can utilize the carbon in carbon dioxide, but we, in order to replace our carbon-waste, must use some more complex compound of carbon, in fact, as our daily experience reveals, either a carbohydrate (sugars, starches, etc.), a fat, or a protein. Otherwise we inevitably suffer from carbon starvation. Plants, again, can derive nitrogen from nitrates in the soil, but we, more dependent, can only derive the nitrogen which we need from preformed protein. Mineral and other inorganic foods we only utilize to replace or provide mineral or inorganic constituents of our tissues ; we cannot utilize them directly to build up carbohydrates or fats as plants can. Our foodstuffs fall, therefore, into four main classes, to wit : 1. The Inorganic Foods, such as water and mineral salts. 2. The Carbohydrates, such as the sugars and starches. 3. The Fats. 4. The Proteins. To which must be added certain accessory articles of diet, to which frequent reference will be made, which are of vital importance to the maintenance and furtherance of life, but yet do not necessarily fall within any of the above-mentioned classes. CHAPTER II. THE INORGANIC FOODSTUFFS. WATER AND SODIUM CHLORIDE. We can readily understand how the need for the organic foodstuffs arises: the fats, carbohydrates and proteins. For they are fuels which in the course of combustion give up a certain number of heat-units which can be utilized in the performance of all the work which an animal daily accomplishes. We can also readily understand how the need for Water arises. Protoplasm consists very largely of water. Over 70 per cent, of our body-weight is water and consequently we living animals are reservoirs or sacks of water which are at the same time porous. Just as an earthenware jar containing water will gradu- ally but continuously lose the water by evaporation from the outer surface of the jar, so we also lose water continually, by evaporation from the skin and from the respiratory epithelium in the lungs, apart from the water which is daily lost in urine and which serves the purpose of flushing the excreta out of the conduits of the body. Consequently a need for water arises, a need of the cells and tissues which is expressed in our consciousness by that indefinite sensation which we call " thirst." But it is not so clear why we should require Mineral Salts. \Ve do not decompose them. They can yield us no energy. It is not at once evident why we should lose them as we cannot help losing water. Yet we do lose them daily and that daily loss must be replaced. We daily take in sodium chloride and it reappears as sodium chloride in the urine. At the end of its passage through the tissues it appears unal- tered, yet it has unquestionably performed a function and indeed many functions during its sojourn in our bodies. The probable nature of some of these functions will be more clearly apprehended at a later stage, when we take up the consideration of the relationship between the properties of living matter and those of its constituents. But having regard at present only to the beginning and the end of the cycle of processes in which the mineral salts of the diet take part as they pass through the body, the question presents itself: what is the daily loss of mineral salts and what must be the daily intake to recoup the body for this loss? The mineral a^lts which are found in our tissues are, for the most part, supplied in abundance in our diet. We do not consciously seek for them as desirable in themselves. A remarkable exception to this rule is afforded by common salt, sodium chloride, of which we feel WATER AND SODIUM CHLORIDE 35 impelled to seek an additional supply. This fact is the more remark- able because all of our ordinary articles of food contain abundance of sodium chloride, yet however much of other diet we may eat we still experience salt-hunger, a hunger which under certain conditions, may become positively distressing. In this connection it is noteworthy that a very close parallelism exists between the nature of the diet of different animals and peoples and their requirement of salt; a parallelism which was first pointed out and interpreted by the physiological chemist von Bunge. Very many of the animals whose diet is purely vegetarian experience a desire for salt. Carnivorous animals, on the contrary, such as the dog or cat, not only do not desire salt, but actually exhibit an aversion for salted food. This is very well illustrated by well-known habits of many of the wild animals. It is a fact commented upon in nearly every book of traveller's and hunter's tales, that the hoofed animals, the deer and so forth, of which the dietary is exclusively vegetable, deliberately seek for salt, in salt-pools and efflorescences, where they lick the salt, and will travel very long distances to do so. As all readers of travel and adventure know, it is at salt licks that hunters watch for such game. On the other hand, salt has never any attraction for the wild beasts of prey. This difference of behavior becomes all the more striking when we reflect upon the fact that, weight for weight, a herbivorous animal takes in with its ordinary food just about the same quantity of sodium and chlorine per day as a carnivorous animal. Each receives the same allowance of salt. Yet the herbivora experience a longing for more salt and the carnivora do not. The reason is obviously to be sought in some other difference between their contents of sodium chloride. Now one very striking difference is found between the mineral contents of vegetable and animal food. Vegetables nearly all contain a superabundance of potassium salts. Animal flesh, on the contrary, contains sodium and potassium in nearly equal proportions, so that although a herbivorous animal obtains just as much sodium chloride per day as a carnivorous animal, .yet it obtains in many cases no less than six times as much potassium as a carnivorous animal does. It is in fact a general rule, to which there are but few exceptions, that in plant-tissues potassium predominates very greatly over sodium, while in animal tissues these mineral bases are present in approximately equal proportion. Von Bunge sought to trace the origin of the craving which herbiv- orous animals experience for salt to the excess of potassium in the diet. Suppose that a salt of potassium, say potassium citrate, gains entrance into the blood by having been ingested with the food. On arriving in the blood-stream, the potassium citrate meets with an excess of sodium chloride, for in the blood-plasma, or fluid part of the blood, sodium predominates very greatly over potassium. Of course a certain degree of interchange of ions will take place. A proportion of the potassium 36 INORGANIC FOODSTUFFS citrate will react with sodium chloride to form potassium chloride and sodium citrate, in accordance with the equation: CHzCOOK CH 2 COONa I /OH I /OH C + 3NaCl ' C + 3KC1 I \COOK \COONa CHzCOOK CH 2 COONa and an excess of sodium citrate appears in the blood together with an unusual excess of potassium chloride. Of course a similar interchange would take place, with analogous results, if the salt ingested were potassium tartrate, malate or any other of the organic salts of potas- sium which are so frequently abundant in vegetable tissues. Now it is a function of the kidneys, as the reader will soon come to appreciate very fully, to keep the composition of the blood very nearly constant. They act, in fact, with the utmost precision, picking out and rejecting abnormal or excessive constituents. The composition of the blood cannot vary beyond the slightest extent without the supervention of grave disturbances involving all the tissues of the body. As a result of the ingestion of potassium citrate, tartrate, malate or other potassium salts which are found in vegetables, we have seen that a new salt of sodium is formed in the blood-plasma, to wit, sodium citrate, tartrate, malate or what not. This abnormal constituent is straightway picked out and eliminated by the kidneys, together with as much as possible of the excess of potassium chloride, and thus as a result of the ingestion of potassium salts the blood is robbed of both sodium and chlorine. This theoretical deduction can very readily be illustrated experi- mentally. Von Bunge collected his urine from day to day and measured the diurnal excretion of sodium. He then simply added 18 grammes of K 2 O, in the form of citrate or phosphate, to his daily diet. The twenty-four-hour excretion of sodium (estimated as Na 2 O) immediately increased by 8 grammes. Now 18 grammes of K 2 O is not at all an unusual amount to ingest along with a vegetable diet. If one were to satisfy one's protein requirements with potatoes, as many Irish peasants do, for example, one would obtain no less than 40 grammes of K 2 O per diem. One result of subsisting upon a vegetable diet, therefore, is a con- tinual abstraction of sodium and chlorine from the blood. Now the blood resists most strongly any alteration in its composition. The reader will come to appreciate more and more clearly as this work progresses, how intimately the most fundamental activities of the body are dependent for their continuance upon the unalterable composition of the blood. The slightest alteration even in the ratio of sodium to potassium in the blood would work havoc with our tissue-activities. Hence the blood must recoup itself, and it can only recoup itself by WATER AND SODIUM CHLORIDE 37 abstracting sodium and chlorine from the tissues. Hence the tissues, in consequence of a vegetable diet, are robbed of sodium chloride. They experience salt-hunger, a want which finds psychological expres- sion in an indefinable longing for things which taste salt. That the desire for salt which so many herbivorous animals experi- ence is really attributable to the nature of their diet is remarkably illustrated by the habits of various human races. Von Bunge has collected together by exhaustive inquiries from travellers, explorers, and works of travel, a quantity of information regarding the consump- tion of salt among different peoples. Only to cite a few among very numerous instances: Country people, in Europe at all events where habits have become fixed by centuries of adherence to the soil, eat more vegetables and less animal food than the dwellers in cities. For instance in France, where the collection of internal revenue upon salt facilitates the acquirement of statistical data, it has been found that the consumption of salt per head is three times as great in the country districts as in the cities. Then there are whole tribes of nomads in various parts of the world who are hunters, such as certain tribes of the old North American Indians, some Arabian and Siberian tribes and the Bushmen of South Africa. These people live, or used to live exclusively upon a flesh-diet and they never taste salt. In fact, as a rule, they find salt very disagreeable and consider the use of it by Europeans ridiculous. Not only is this the case with tribes who have lived for generations upon a flesh-diet, but it applies also to Europeans who visit them and adopt their diet. Thus one traveller informed von Bunge that while he lived among the Tunguses, an exclusively carnivorous tribe which dwells in Siberia, he lived entirely upon reindeer-flesh and game, and never experienced the slightest desire for salt or inconvenience from the lack of it. Very different was the experience of the Scotch explorer, Mungo Park, when travelling among the negro tribes of West Africa. These people live upon a mixed diet containing a very high proportion of vegetables. Salt is very rare in their country, and, as the vegetable diet causes a longing for salt, Park states that among the natives, to say that a man eats salt with his meals was equivalent to saying that he was rich. In Park's own words: "In the districts of the interior salt is the greatest of all delicacies. It strikes a European very strangely to observe a child sucking a piece of rock-salt as if it were sugar. I have frequently seen this done. I myself have found the scarcity of this natural product very trying. Constant vegetable food causes a painful longing for salt that is quite indescribable. On the coast of Sierra Leone the desire for salt is so keen among the negroes that they gave away wives, children, and everything that was dear to them, in return for it." Hunting tribes, therefore, who subsist on flesh, experience no need for salt and never eat it even when it is easy to obtain. Agricultural tribes, on the contrary, experience a keen desire for salt. A peculiar 38 INORGANIC FOODSTUFFS confirmation of von Bunge's interpretation of this phenomenon is afforded by the custom of one tribe to which von Bunge refers, the negro inhabitants of a region in the neighborhood of Khartoum. These natives manufacture or formerly manufactured a salt of their own, by igniting the ash of a plant belonging to Salsola or salt-wort group. As has been stated, the majority of plants contain a much larger proportion of potassium than of sodium. The plants of the Salsola group are quite peculiar in the respect that their ash contains a much higher proportion of sodium than of potassium. The employ- ment of this particular plant-ash among all the others that might have been tried can hardly be considered accidental, in other words it must have been found to satisfy a desire not equally readily satisfied by the ashes of other plants. The relation of a need for salt to the partaking of a vegetable diet has had several peculiar historical consequences. For example, in the Mosaic Law, the Jews are expressly commanded to present their vegetable offerings to the Deity accompanied by salt. In Greek and Roman times, sacrificial animals were offered up to the Gods without salt, but the fruits of the earth with salt. The effect of eating salt with our food is therefore, to widen the circle of palatable foods, We all know how insipid potatoes taste without salt. That is probably attributable to their high content of potassium, unusual even in plants. By adding salt to our diet we are able to render potatoes palatable, and so with many more foodstuffs of vegetable origin. So far, all of the facts which we have cited are in excellent harmony with the view that a diet containing an excess of potassium salts gives rise to a necessity and a desire for common salt. Not 'every animal appears to experience this desire, however, for rabbits and hares, for example, live on a diet containing an excess of potassium salts and yet do not seek for salt and do not appear to experience any inconvenience from lack of it. Domestic herbivorous animals will live without incon- venience on a purely vegetable diet without salt indefinitely, although they will eat salt when it is offered them and unmistakably find it gratifying. None of these live on a diet so excessively rich in potassium as potatoes, for example, but nevertheless there is no question but that they must ingest a large excess of potassium salts. Yet the blood-plasma of such animals remains of the usual composition, containing an excess of sodium over potassium. Here we meet for the first time with a phenomenon which is of very general occurrence in living matter, namely the phenomenon of selec- tive assimilation by tissues. Living tissues, as we shall have occasion to note many times, are not mere passive recipients of whatever may be contained in the fluids which bathe them. They choose and select suitable ingredients in suitable proportions and reject unsuitable or excessive ingredients. A remarkable illustration of this is afforded by an experiment of Landsteiner's. He fed young rabbits upon meadow WATER AND SODIUM CHLORIDE 39 hay exclusively for three and a half months. At the same time a similar batch of animals was fed exclusively upon cow's milk. Now these two diets contained very different relative amounts of sodium and potassium, hay being much richer in potassium than in sodium, and milk richer in sodium than in potassium. Yet at the end of the period the composition of the blood obtained from the two groups, as regards sodium and potassium, was identical. The tissues, not only the epithelium of the kidney but that of the intestine as well, actively choose the constituents which they will reject or absorb respectively. In just the same way a plant, living in water rich in sodium and poor in potassium, will nevertheless pick the potassium out and build it up into tissues which are rich in potassium and poor in sodium. But this power of selection is limited, and in extreme cases, as, for example, a diet so rich in potassium as potatoes, some aid is required, and sodium and chlorine in the form of common salt must be added to the dietary. From the standpoint of physical chemistry it is of course evident that selective absorption of mineral salts by the epithelium of the intestine or their selective elimination by the kidneys must involve the performance of work; the expenditure of energy. For the osmotic pressures of the various salts in the solutions bathing the cells would tend to drive them into the absorbing or excreting tissues in pro- portion to their concentration and if, on the contrary, they appear on the other side of these epithelial tissues in emphatic disproportion to their original concentrations, the process of assimilation or excretion must have involved the overcoming of the forces of Osmotic Pressure. The energy necessary to achieve this can only be derived from the combustion of other foodstuffs or constituents of tissues which are thus robbed of the supplies available for carrying on the other activities of the body. Selective absorption or excretion implies work, therefore, and anything which relieves the tissues in any measure of the necessity of exercising selection sets free a certain number of heat-units for other uses or, in other words, improves the utilization of other foodstuffs. The gratification and frequent improvement in nutrition which accompanies the administration of salt to herbivorous animals may thus originate in relief of the tissues from the strain and burden of selection and the liberation of foodstuffs for the maintenance of other tissue-activities which is in effect, equivalent to the addition of a certain amount of food to the accustomed dietary. The beneficial effects of salt may therefore, and in the long run, reside not so much in the actual sodium and chlorine administered as in the additional carbo- hydrate; fat, or protein which is thus rendered available for the main- tenance and upbuilding of the body. It is probably for some such reason as this that the total mineral- requirements of the body vary exceedingly with the dietary upon which an animal is subsisting.' Especially is this the case when the require- ment on a normal mixed diet is contrasted with that which obtains when the diet is limited in such a way as to provide only those proteins 40 INORGANIC FOODSTUFFS of vegetable origin which are most remote in their composition from the proteins of animal tissue. In such a diet a large proportion of the nitro- gen is wasted because, as we shall see in a subsequent chapter, the amino-acids into which the protein splits up on digestion are present in the wrong proportion and have to be resorted and selected in very different proportions in order to build up proteins of the animal type. It has been found that an animal subsisting on a diet of this kind suffers not only a large wastage of nitrogen, necessitating the consumption of a large quantity of food to maintain nitrogenous equilibrium, but also a large wastage of mineral constituents, so that it cannot be maintained in health or nutritive equilibrium without the addition to the diet of a considerable excess of mineral substances over the amount which would be required by an animal subsisting on a more varied diet. CALCIUM. During the early months of the growth of a suckling infant or animal, lime is very rapidly being absorbed and utilized by the tissues for the formation of bones. This calcium is totally derived from milk. Now the lime in milk is present therein in two forms, namely, in the form of calcium phosphate and in the form of a bulky, indiffusible compound with one of the proteins of milk, casein. The calcium phosphate is, of course ionized, but the calcium caseinate, on the contrary, does not yield calcium ions in solution. When we add acids to milk, or when owing to the action of bacteria upon the milk-sugar which it contains lactic acid is produced in the milk, it assumes the curdled appearance which we are accustomed to associate with "sour milk." This appearance is due to the separa- tion of free Casein, uncombined with calcium, which has been abstracted from the calcium caseinate by the acid. Free casein is insoluble in water or very dilute acids and hence is precipitated in curds or flocculi, while the calcium is now present in the "sour" milk in the form of the calcuim salt of the acid which has been added. Precisely the same thing happens when milk which has been ingested by the suckling comes into contact with the hydrochloric acid which is contained in the gastric juice. Free casein ; more or less modified by partial digestion, is precipitated and calcium is set free as calcium chloride. There has been a good deal of discussion in the past as to whether the two forms of lime in milk are equally readily utilized by the suckling. In view of the above-mentioned facts there would appear to be no very good reason for distinguishing between them, since in the stomach, where absorption begins, both forms of calcium are reduced to a common level by the conversion of the calcium caseinate into the ionized and diffusible chloride. Notwithstanding this fact it has been frequently argued that the CALCIUM 41 calcium which is combined with casein in milk is of superior nutritive value to that which is present in the milk from the beginning in the form of diffusible inorganic salts of lime. An experiment which used to be frequently quoted in support of this view was that of Lunin's; who fed six mice upon a mixture of casem, fat and cane-sugar plus the inorganic salts contained in milk. These animals lived respectively twenty, twenty-three, twenty-nine, thirty and thirty-one days; where- as two mice of the same age fed entirely upon whole cow's milk for a period of seventy-five days remained in good health at the end of the experiment. In the first experiment the inorganic bases were all com- bined with inorganic acids to form diffusible and ionizable salts, where- as in the second experiment the lime, at least, was combined with casein. Hence, it was argued, lime in the inorganic form did not fulfil the necessary requirements of the animals. This experiment might easily have been seen from the first to be inconclusive, for natural milk and an artificial mixture such as that prepared by Lunin must obviously differ in many particulars besides the single particular of the diffusibility of the calcium. But in the light of our more recent accessions of knowledge concerning the nutri- tion of animals it has become quite clear that Lunin's experiment bears a very different interpretation to that which was originally put upon it. We know now, thanks to researches which will be detailed in a later part of the work, that besides a sufficiency of proteins, fat and carbo- hydrates, any diet which is to maintain animals in health for a con- siderable period must contain other essential constituents which are present in milk or in animal tissues in minute amounts. These con- stituents fall into two distinct classes, at least, and possibly as our knowledge increases will be found to be more numerous and more diverse in their chemical characteristics than we at present realize. The two classes of these "accessory foodstuffs'* which are at present recognized, however, are in the first place the vitamines, which are nitrogenous, water-soluble substances and in the second place a group of substances which are commonly found associated with animal fats, but are generally absent from vegetable fats. Thus Hopkins has found that if animals be fed for a considerable period on milk-salts, casein and milk-sugar they will not survive, while the addition of a small amount of butter suffices to render the diet adequate for the needs of the animals. In the light of these facts it will readily be seen that Lunin's experi- ment does riot bear on the question of calcium-nutrition at all, but rather on the question of accessory .organic foodstuffs. Furthermore, recent experiments have shown that the cane-sugar employed by Lunin in his artificial mixture is not by any means a sufficient substitute for milk-sugar in the dietary of young animals. There is thus no evidence whatever that the two forms of lime in milk are not equally available and useful to the suckling, as we should 42 INORGANIC FOODSTUFFS expect them to be from the fact that they are alike diffusible and ionizable very shortly after they arrive within the stomach. These considerations have an important bearing upon the practical Question of the modification of cow's milk for infant-feeding. It is me common practice to add lime-water (calcium hydroxide solution) to milk for young infants for two purposes; in the first place in order to delay the acidification and consequent "curdling" of the milk by the hydrochloric acid in the stomach. This results in deferring the floc- culation of the casein until it has undergone partial digestion by the rennin and pepsin in the gastric juice, when the flocculi which are formed are finer and more gelatinous and therefore more easily pene- trable by digestive juices than they are if curdling occurs without pre- liminary digestion. In the second place the lime-water is added with a view to increasing the supply of lime to the infant and thus assisting the growth of bony tissues, teeth, etc. From the latter point of view this practice has been decried in some quarters, on the ground that lime which is not organically combined is not so readily assimilated and utilized as calcium which is in organic combination. We have seen that there is no experimental justification for this distinction, and even if there were, the lime-water which is added to milk immediately combines in considerable proportion with the casein to form a com- pound of exactly the same type, only richer in calcium, as that which is found in normal milk, so that the greater part of the calcium thus administered does in fact reach the stomach in a state of organic combination. It is, of course, quite another question whether administration of lime beyond a certain daily amount is of any value in assisting the growth of bony tissues. Experience in connection with other articles of diet conclusively shows us that in many instances the effective administration of foodstuffs is limited by the ability of the tissues to utilize and elaborate them, any supply in excess of this being rejected and wasted. Defective development of bony tissues may be sometimes attributable to deficiency of lime in the diet, but it is probably more often due to inability of the bone-producing tissues to utilize the lime which is presented to them. This, however, obviously constitutes no objection to the addition of lime-water to the milk of an infant; it merely indicates a reason why this procedure by itself may often be insufficient to correct. faulty or deferred development of the calcareous tissues. The lime-requirement of the adult is very greatly increased in the female by activity of the mammary glands. Thus from 0.3 to 0.5 gramme of calcium oxide per hundred pounds of body-weight per day is sufficient to supply the minimum needs of a pig or goat which is not yielding milk, but a milch-goat requires an additional 1 to 2 grammes of calcium oxide per day for every pound of milk it yields. Insufficiency of lime in the diet under such circumstances results in actual withdrawal of lime from the skeleton, a condition which when it becomes sufficiently IRON 43 acute to cause softening and bending of the bones is known as Osteo- malacia. It is not to be inferred, however, that osteomalacia is always due to deficiency of calcium in the diet. It may be due as indicated above to physiological disturbances or nutritional deficiencies leading- to faulty utilization of the calcium which the dietary affords. Calcium is excreted, in part by the kidneys and in part by the intestinal mucosa. A high proportion of soluble phosphates in the diet tends to increase the output of calcium in the feces, probably owing to the formation of calcium phosphate which is insoluble in the alkaline fluids of the intestine. Just as potassium salts increase the output of sodium in the urine, so, and for similar reasons, do magnesium salts increase the output of calcium in the urine. IRON. Iron is an essential constituent of the red pigment of the blood, Hemoglobin. Since hemoglobin is the carrier of oxygen from the lungs to the tissue-cells, it is obvious that iron in this, if in no other capacity, plays a vital part in the economy of the body, but, in addition to the hemoglobin-iron, iron is also found, and not necessarily asso- ciated with hemoglobin, in other parts of the body. Thus the liver contains about 0.02 per cent, of iron calculated on the basis of the fresh, undried organ washed free from blood. The muscles contain appreciable quantities of iron, especially heart-muscle, which contains about 0.01 per cent, of the fresh, undried weight. In smaller quantities iron is found elsewhere in the body, regularly accompanying Nucleins and Nucleoproteins wherever they are found. The iron-content of the adult is subject, like that of other tissue- constituents, to daily losses. Experiments with starving individuals (and it is under conditions of starvation that the body is most economi- cal of its resources) show that the nominal daily loss of iron in the feces is from seven to eight milligrammes, while in addition to this a daily loss of about one milligramme occurs through the kidneys. In all, then, it is probable that about ten milligrammes of iron, or about one three-hundredth of the total hemoglobin-iron in the body is lost per day. This loss must be replaced from the diet. Under certain pathological conditions, or conditions of malnutrition, a loss of hemoglobin occurs from the blood and the patient is said to have become "anemic." This loss of hemoglobin may and on the other hand may not be accompanied by a diminution in the number of red blood-corpuscles. As might be anticipated, the result of this condition is suboxidation in the tissues with consequent symptoms which are sometimes of the severest gravity. These are very well illustrated by the chlorosis, or "green sickness" which very frequently overtakes girls at the age of puberty. From periods of remote antiquity antedating by many centuries our knowledge of the chemical compo- sition and significance of hemoglobin, this disease has been combated 44 INORGANIC FOODSTUFFS by the administration of inorganic salts of iron, and often with beneficial effect. For long it was thought, without any question, that the salts of iron so administered were absorbed and that the beneficial effect of the medicament was due to the replacement of the iron in the blood by the iron so administered. Doubt was thrown upon this explanation by the discovery that iron is eliminated from the body in the feces. Doses of inorganic salts of iron, administered to healthy individuals, were recovered apparently unaltered in the feces, and from this fact the erroneous conclusion was drawn that inorganic salts of iron are not absorbed. The beneficial effects of iron in anemia were either denied, a denial in which practising physicians declined to share, or else accounted for by the irritant action of the salts of iron upon the epithe- lium of the intestinal tract. A mild irritation has a well-known " tonic" effect which is rather difficult to define in precise terms; but which is frequently manifested, not only by increased activity of the tissues which are stimulated, but also of other and sometimes distant tissue. The beneficial effects of iron were therefore attributed to increased activity of the tissues resulting in increased assimilation and utilization of the organically combined iron in the diet and not to direct assimi- lation of the iron administered as a medicament. Much has been done to clear up this question by the employment of microchemical tests to trace the course of iron through the intestine. When mice are fed upon milk alone for a considerable period, on placing the alimentary canal of these animals in ammonia and ammonium sul- phide the characteristic precipitate of iron sulphide does not appear, or at the most there is only a very slight green coloration. Now milk is one of the articles of diet which is poorest in iron, cow's milk con- taining only about 2.3 mg. of iron per 100 grammes of dry substance. Very different results are obtained if the mice are fed upon milk to which inorganic salts of iron have been added. In the stomach there is little if any reaction for iron, while in the duodenum there is a marked green coloration. If the tissues of the intestine are examined under the micro- scope, little granules of iron are found imbedded in the protoplasm of the intestinal epithelium, and leukocytes are found laden with minute particles of iron. In the jejunum, however, and in the ileum, very little iron is found, while in the cecum and large intestine a strong iron-test is once more obtained. Coming from the intestinal canal, especially from the duodenum, the lymphatics may be seen filled with cells containing iron. The liver and spleen give much stronger tests for iron than those of the mice fed upon milk alone. There can be no question, therefore, but that the inorganic iron- salts thus administered are absorbed. Part of the iron appears to be conducted by way of the lymphatics to the thoracic duct and the blood- stream. Part is unquestionably conducted by the portal vein to the liver, which is a storehouse of iron as it is of many other things . The absorption takes place mainly in the duodenum; the excretion of waste IRON 45 iron occurring, on the contrary, in the cecum and large intestine, although part of the small intestine may also participate in this function. It is one thing to show that inorganic salts of iron are absorbed and' it is another to show that they may be utilized in the building up of hemoglobin. The iron in hemoglobin is very firmly and intimately combined, and cannot be detected by the reagents ordinarily employed for this purpose, such as ammonium sulphide or potassium ferro- cyanide. In fact the iron in hemoglobin resists the action of boiling, concentrated potassium hydroxide and boiling hydrochloric acid. Only by dissolving the hematin radical (which is the iron-containing moiety of the hemoglobin molecule) in concentrated sulphuric acid is the iron split off and the hematin changed into iron-free hematin, or Hematoporphyrin. Most of the iron in our diet is in the form of hemoglobin or other organic compounds of iron from which free ionic iron is not readily split off. The yolk of eggs is very rich in iron, as might be anticipated from the fact that the yolk of an egg must contain all of the constit- uents necessary to form the hemoglobin of the developing embryo. The iron-compound in yolks of eggs is not hemoglobin, but some antecedent of hemoglobin. On extracting the yolk of a hen's egg with alcohol or ether, none of the iron goes into the extract. The residue, which contains all of the iron, is a mixture of proteins and nucleo- proteins. The iron cannot be extracted from this residue by alcohol and hydrochloric acid, although inorganic salts of iron readily yield up iron to these reagents. During the digestion of iron-containing protein by Pepsin in the stomach, the part containing iron does not go into solution and its digestion is not accomplished until it reaches the small intestine and comes in contact with the digestive fluid secreted by the pancreas. It is not digestible by pepsin and in this and in other respects corresponds in its behavior to the class of bodies which the reader will later learn to recognize as nucleins. The ordinary tests for iron are given by this substance, to which von Bunge gave the name "Hematogen," but not so readily as by inorganic salts of iron. On adding ammonium sulphide to an ammoniacal solution of this nuclein a greenish coloration is produced, which only slowly changes to black on standing. In other words ionized iron is at first only present in traces and is slowly split off from the compound under the prolonged influence of the reagents. The compound thus behaves in a manner very like that of the protein salts of the heavy metals, for instance casein salts of silver, mercury and so forth to which the reader's atten- tion will be directed in a later chapter. There is little reason to doubt that hematogen is simply a protein salt of iron in which the protein is acting the part of a weak acid, or else a double salt of protein and an inorganic salt of iron. Protein compounds of this type yield no metal- ions in solution, or at the most, only traces of them. Since compounds such as these are the only^forms in which we 46 INORGANIC FOODSTUFFS normally receive iron in our diet, for we only partake of inorganic salts of iron as a therapeutic measure, there can be no question but that we can absorb, assimilate and utilize the iron contained in organic, non-ionized compounds. It will be recollected that the iron in hemoglobin or hematin does not yield the ammonium-sulphide test for iron. On administering hematin or hemoglobin to mice which have undergone iron-starvation, however, and applying the iron-sulphide test to various parts of the intestine, we ascertain the remarkable fact that the duodenum and the cecum yield the iron-test just as they do when inorganic iron is admin- istered. In other words, the iron in the process of digestion in the duodenum has become loosened from its combination in the hematin radical and set free as an inorganic or at least an ionized salt of iron. Since the iron, immediately subsequent to absorption, appears in the same condition whether administered in the ionic form or not, there would appear to be no very good reason for supposing that inorganic salts of iron are not utilized to nearly as great an extent as the organic salts of iron. The most specific disadvantage which attends the use of inorganic salts of iron is their irritating or corrosive action upon the intestinal epithelium, a corrosive action which, like that of mercury salts, is probably to be attributed to the formation of insoluble protein salts of the metal within the epithelial cells. This leads to the dis- ruption of the gelatinous structures of the cells and their conversion into granules or flocculi which, no longer being held together by the cohesiveness of a jelly, fall apart with consequent disintegration of the cells. Many individuals who display an " idiosyncrasy" or exceptional sensitiveness to intestinal irritation are very severely affected by this corrosive action of iron-salts and for this reason the general employment of non-ionized organic compounds of iron in therapeutics, such as hemoglobin or hematogen, is much to be preferred. With the exception of the disadvantages arising from the corrosive action of inorganic salts of iron, therefore, the ionized and unionized compounds would appear, so far as the above-cited evidence goes, to be equally useful sources of iron in the diet. There are certain impor- tant facts, however, which would appear at first sight to bear out the contention that inorganic salts of iron, notwithstanding their absorp- tion, are not utilizable for the synthesis of hemoglobin. We have seen that milk contains a very low percentage of iron in comparison with other foods, especially in comparison with green vegetables, certain fruits such as apples, and flesh. If sucklings are kept beyond the normal period of lactation exclusively upon a milk diet, they become anemic from lack of iron. If we compare rabbits which have been allowed to change to a diet of green vegetables after the normal period of lactation, with those which have been brought up upon an exclusive milk-diet, we find that the former contain much more hemoglobin than the latter. But the remarkable fact is that if we add inorganic salts of iron to the milk-diet the total hemoglobin in the animals is not IRON 47 increased, although they grow much more rapidly than the similarly fed animals which do not receive iron. This would appear to indicate that inorganic salts of iron are utilizable for certain purposes in the body connected with the growth of the animals, but not for the building up of hemoglobin. This conclusion, however, would be premature. Recent acquisitions to our knowledge of the structure of the hematin moiety of the hemoglobin molecule have shown that it contains a particular molecular grouping, namely, the Pyrrole Group : __c c II II c c \/ N which there is every reason for supposing cannot be synthesized by animals but must be obtained by them preformed, that is to say from the tissues of plants or from the tissues of animals which acquired it from plants. This pyrrole grouping is contained in small amounts in the majority of proteins and it forms a very important component of Chlorophyll, the green coloring-matter of plants which, as we shall see, is very closely related, chemically, to hemoglobin. It is not improb- able, therefore, that inorganic iron-salts added to an exclusive milk- diet are not utilized for building up hemoglobin simply for the reason that other component parts of the hemoglobin molecule, as essential as iron itself, are either lacking altogether in the milk-diet or present therein in insufficient amount to subserve the needs of the blood-form- ing tissues and those of the other tissues of the body as well. We will return to this question in later chapters in connection with the chemistry of hemoglobin, and again in connection with the general problems of growth and nutrition. The percentages of iron which are contained in several common articles of food are enumerated in the following table: IRON-CONTENT OF FOODS IN PER CENT. OF EDIBLE PORTION, AFTER SHERMAN. 1 Food. Egg-white . Iron (Fe). 0001 Food. Potatoes Iron (Fe). 0013 Butter .... Whole milk . . . . . 0.0002 00024 Cheese . Dates 0.0013 0030 Apples .... Carrots Lettuce Cornmeal . White bread . Asparagus Cabbage Fish . . . 0.0003 . . . 0.0006 . . . 0.0007 . . . 0.0009 . . . 0.0009 . . . 0.0010 . . . 0.0011 0.0008-0.0013 Eggs ., -. Meat Spinach . Oatmeal Barley . . Egg-yolk Blood . . ...... 0.0030 . . . . 0.0023-0.0033 0.0036 . .... 0.0038 0.0041 0.0086 0.0526 It will be noted that the iron-content of spinach is very high. Spin- ach is also very rich in chlorophyll, as its deep green color indicates, and thus contains a large proportion of another essential constituent 1 Chemistry of Food and Nutrition, New York, 1918. 48 INORGANIC FOODSTUFFS of hemoglobin, the pyrrole radical. Chlorophyll, it is true, is indigest- ible by the digestive juices, but it is split up by the bacteria which inhabit the intestine, and in this way a portion of the pyrrole which it contains may possibly be rendered available for assimilation from the intestine and utilization by the tissues. It will be recollected that if iron is administered to young animals which are undergoing iron-starvation by being kept upon an exclusive milk-diet, their growth is markedly accelerated despite the fact that the iron is not utilized to build up hemoglobin. This effect is of sig- nificance, inasmuch as it indicates that iron subserves other important functions in the body besides that of entering into the composition of the oxygen-carrying pigment of the blood. We are reminded of the prevalence of iron in nuclear elements, and led to suspect that iron plays some essential part in the functions of the nuclei. It is a note- worthy fact, however, that if iron be added, in similar amounts to those employedjn the above-cited experiments, to an abundant and mixed diet, containing a normal sufficiency of iron, this acceleration of growth is not observed. Evidently beyond a certain diurnal allowance the tissues of the growing animal are not able to utilize iron for the purposes which result in the acceleration of growth. Here we meet again with a phenomenon to which reference was made in connection with the utili- zation of calcium. The ability of the tissues to profitably utilize the materials brought to them sets a definite limit to the amount of a food- stuff which it is of any avail to consume. It is doubtless for this reason that iron, whether in the organic or the inorganic form, is with- out effect in accelerating the rebuilding of hemoglobin after hemor- rhage. The blood-forming tissues are able to manufacture so much hemoglobin per diem and the supply of more raw materials than they can "work up" in a day is useless. The ultimate reason for this phenomenon, which is of such general occurrence in life-phenomena, resides undoubtedly in the multifarious variety of the chemical processes which underlie and accompany vital activities. In every detail of change which accompanies the per- formance of any function by living tissues not merely one chemical reaction is involved but a whole series of interwoven reactions following and depending upon one another. Now in any series of chemical changes of which the second utilizes some product of the first, the third some product of the second, and so forth, it is always the specifically slowest reaction which "sets the pace" for those which succeed it. No matter how quickly raw materials may be supplied, this "master- reaction" can proceed only at a certain maximum speed and succeeding reactions must wait for its products before they can seize and elaborate them. Provided then, that any article of diet be supplied in sufficiency to maintain at top speed the "master-reaction" of the series of pro- cesses intojwhich it enters, excess of this particular item in the dietary is mere wastagejand casts an unnecessary strain upon the organs of elimination. OTHER INORGANIC FOODSTUFFS 49 Insufficient hemoglobin content of the blood, therefore, and any other type of maldevelopment and malnutrition may originate in either of two ways, namely, through inadequacy of the diet, or through imperfect utilization of substances which are present in abundance in the dietary. Certain mild types of anemia, probably belong to the former category and the consensus of opinion of the physicians is that these are favorably affected by administration of iron. In other types of anemia, in which the utilization of iron is defective or in which, as in the anemia of hemorrhage, the lack of hemoglobin is due to loss or destruction after it has been manufactured, we cannot expect therapeutic administration of iron to be followed by equally favorable results. OTHER INORGANIC FOODSTUFFS. The remaining inorganic constituents of the body will be but briefly considered at this point, some of them falling under review in other connections in later chapters. While tjie majority of them probably play important or even essential parts in our bodily economy, we have as yet only succeeded in a few instances in obtaining a clue to the nature of these functions. Among the metals other than those which we have considered, Magnesium is, from a quantitative point of view, the most important. Magnesium is found in small quantities in all animal and plant cells, and in milk. There appears to be a rather definite relation- ship or proportionality between the magnesium and the calcium contents of the tissues, and from the fact that a trifling excess of magnesium, when introduced into the circulation, causes profound disturbances such as glycosuria, we may conclude that magnesium has powerful physiological actions and that in consequence even the amounts which normally occur in tissues are not devoid of physiological significance. It is stated that traces of Lithium are normally found in animal tissues, and it is a much-discussed question whether or not a minute trace of Arsenic is a normal constituent of human tissues, the gravity of the discussion being attributable, of course, to the medicolegal significance of the question. The consensus of opinion appears to be, however, that arsenic is found in human tissues only after the administration of drugs containing arsenic or in districts where arsenic occurs in considerable amounts in the soil and water. Among non-metallic inorganic constituents of the body, Chlorine plays a leading part, in the alkali chlorides of the blood and tissues and in the hydrochloric acid in the gastric juice. It is derived from chlorides in the food. Fluorine occurs in small amounts in milk (0.00003 per cent.) and is a normal constituent of bones and teeth; it is unquestionably not devoid of significance in the formation of these tissues. 4 50 INORGANIC FOODSTUFFS Silicon is a constant constituent of hair and feathers, no less than 40 per cent, of the ash of hair consisting of SiO 2 . This is doubt- less derived from silicates in the vegetable portion of our diet, silicon playing an important part in communicating rigidity to many plant-tissues. According to Drechsel the silicon in feathers exists therein in a state of organic combination, as the silicate of a hydro- aromatic alcohol closely related to cholesterol. Phosphorus is, of course, an element of prime importance in the life-economy, in the form of the phosphoric acid radical in phospho- proteins such as casein and in the form of complex substituted phosphoric acids, as Nucleic Acid and the glycero-phosphoric acid radical of the phosphorus-containing fats or phospholipins. This phosphorus is derived from the phosphates, phosphoproteins, nucleins and phospholipins in the diet. There is some room for question whether animal tissues utilize the inorganic phosphates in the diet for the building up of the nucleins and phospholipins. A fact which seems to indicate that animals do not depend upon inorganic phosphates for the production of these substances is that mice will grow normally and reproduce on a diet containing a high proportion of aluminum hydrate, although this results in the formation of the insoluble alumi- num phosphate from any inorganic phosphates which may be present in the alimentary canal, and its elimination, without absorption in the feces. Sulphur also plays an exceedingly important role, but in the form of the complex amino-acid cystine, which is a decomposition-product of many proteins, rather than in the form of free sulphates or sulphides. Iodine is a normal constituent of the Thyroid and plays an essential part in the important functions of this gland. We will consider the nature of the organic combination in which it occurs and its significance in the bodily economy in a later chapter. It has been repeatedly stated that iodine is found in other tissues of the body, notably in the pituitary gland, but more recent analyses have shown that in the absence of iodide-medication, iodine is not found in normal animal tissues other than the thyroid. Iodine is an important con- stituent of seaweed, from the ash of which a quantity of the iodine of commerce is derived. The relatively high concentration of iodine in the tissues of these marine plants is of especial interest because the iodine content of sea-water is exceedingly low. This constitutes therefore an interesting case of the Selective Absorption by living tissues to which reference was made in connection with the proportion of sodium to potassium in the blood and tissues of animals. THE COMPLEXITY OF OUR DIETARY REQUIREMENTS. It is to be hoped that the recital of the above category of the inorganic constituents of our body, present, several of them, in the most incon- siderable traces, will have the effect of making the reader pause ere he COMPLEXITY OF OUR DIETARY REQUIREMENTS 51 embraces any of the dietary fads and "systems" which are so prevalent in this uninformed and loquacious period of our social evolution. The average man or woman hesitates to pronounce an opinion on the motive machinery of steamships or aeroplanes or on the fuel-requirements of a Diesel engine, but regarding that infinitely more complex engine, a human being, the average individual deems himself fully informed and all that is required to make numerous converts to any dietetic fad is a considerable degree of self-assurance. So complex are the requirements of the animal economy; so little do we know the parts that these several requirements play and their delicate adjustments to one another, that we are totally unable at this stage of our knowledge to enumerate the constituents of any restricted dietary which shall certainly and for prolonged periods of time, convey to the subject all that he requires for the orderly functioning of his body. In medical practice it is, of course, necessary to occasionally prescribe a limited and specified diet for a definite period in order to combat certain conditions or maladies, but to do so for lengthy periods of time, especially for growing infants and children, is to simply assume a knowledge which we do not possess. The problem of the dietary requirements, as we have seen, is complex enough when we consider only the inorganic foodstuffs; but when we add to these the organic requirements of the body the complexity of the problem of nutrition is multiplied a hundredfold, and we are as yet hopelessly in the dark respecting the source and function of a multitude of constituents of the body and of the degree to which they may be essential. Our knowl- edge in this field is rapidly extending, perhaps more rapidly at present than in any other field of biochemistry, but even at the present rate of accession of knowledge, the complete knowledge essential for enumer- ation in detail of all the dietary requisites of a human being is very far distant indeed. The knowledge that we do possess, however, enables us in certain particular instances, as, for example, in the Weir Mitchell treatment of certain nervous disorders, or the Allen treatment of diabetes, to accomplish very decisive therapeutic results by restricted dietaries prescribed for limited periods, in conjunction with hygienic measures and adequate biochemical and clinical observation and control. The very success of such measures in any particular instance carries with it the danger of converting an ignorant patient into a fanatical diet- faddist who, upon recovery of health, proceeds to convert, first his acquaintances and then, if he has the opportunity, a wider public, to the health doctrine which he has evolved out of the temporary measures of the physician. This is no doubt the origin of many of the dietary and hygienic eccentricities to which certain genuine or imaginary invalids devote themselves. No small part of this perverted activity could probably be stifled at its birth, if the physician who is prescribing dietary or hygienic measures were to make a practise of explaining as thoroughly and simply as he is able, to the patient and his immediate 52 INORGANIC FOODSTUFFS associates" the precise object of the measures advocated, their t porary character, and the fact that they are applicable only to particular case in point, and not to humanity in general, irrespeci of age, sex, health or disease. We will take up the question of the dietary requirements of the b< in several subsequent chapters and in a variety of connections. T above remarks will, however, be found to apply only the more forcit ! with the expansion of our acquaintance with the complexity and vark of the problems of nutrition. REFERENCES. GENERAL: Albu-Neuberg: Physiologic und Pathologie des Mineralstoffwechsels. Berlin , 19 Osborne and Mendel: Jour. Biol. Chem., 1918, 34, p. 131; 1913, 15, p. 311. C. negie Inst. of Washington, Pub. 156, 1911, Pt. II. McCollum and Davis: Jour. Biol. Chem., 1913, 14, p. xl; 1915, 21, p. 615. SODIUM AND POTASSIUM: von Bunge: Text-Book of Physiological and Pathological Chemistry, trans, by Starling, E. A. Philadelphia, 1902. CALCIUM: Lunin: Zeit. physiol. Chem., 1881, 5, p. 31. Hopkins: Jour. Physiol., 1912, 44, p. 425. Stehle: Jour. Biol. Chem., 1917, 31, p. 461. Givens and Mendel: Ibid., 1917,\31, p. 421 (which see for Bibliography). Givens, M. H.: Ibid., 1917, 31, pp. 435 and 441; 1918, 31, p. 119; 1918, 35, p. 241, IRON: von Bunge: Cited above. Abderhalden: Zeit. Biol., 1899, 39, pp. 113 and 483. Zeit. physiol. Chem., 1902 34, p. 500. Macallum, A. B.: Jour. Physiol., 1894, 16, p. 268. Lapicque: Arch. Physiol. Norm, et Path., 1895, 7, p. 280. Kunkel: Pfliiger's Arch., 1895, 61, p. 595. Hooper and Whipple: Am. Jour. Physiol., 1917, 45, p. 573. Whipple and Hooper: Ibid., 1917, 45, p. 576. CHAPTER III. THE CARBOHYDRATES; THE MONOSACCHARIDES. GENERAL CHARACTERISTICS. The Carbohydrates are extremely abundant in nature, and play an exceedingly important part in the life-cycle. In vegetable tissues they are of importance, not only as foodstuffs and reserve materials, but also as structural materials. For example, the walls of plant-cells are usually composed of cellulose, a complex carbohydrate. In the animal economy the carbohydrates are chiefly of importance as food and reserve-materials and afford a very important source of kinetic energy to our tissues. The carbohydrates owe their name to the fact that all of them contain carbon and in all of them, moreover, the proportion of hydrogen to oxygen is the same as it is in water, namely, 2 to 1. This is not a very satisfactory definition of the group, however, since many substances are known which correspond to such a definition and yet are most distinctly not carbohydrates. In more exact terms it may be said that carbohydrates are aldehyde and ketone derivatives of the polyatomic ilcohols. The majority of the naturally occurring simple sugars :ontain six atoms of carbon and are termed Hexoses, although some Contain five atoms of carbon and are termed Pentoses. From the simple ftonosaccharides, more complex sugars, the Disaccharides, are formed >y the combination of two molecules of monosaccharide with the Jimination of a molecule of water. More complex carbohydrates till, the starches and dextrines, collectively termed the Polysaccharides, ,re derived from the monosaccharides by the combination of a variable lumber of sugar molecules, with the elimination of a corresponding lumber of molecules of water. It is only within comparatively recent times that the artificial ynthesis of sugar has been accomplished, but within the brief period >f thirty years nearly all of the natural sugars have been synthesized, ,nd the light which the consequent accessions to our chemical knowl- dge have thrown upon the function and transformations of the carbo- tydrates in living organisms is so great, that today we are in a position o interpret countless phenomena which were entirely obscure before hese discoveries had been made. The first sugar to be synthesized was Glycerose, which was prepared >y Emil Fischer in 1890. This sugar, which contained, however, only hree atoms of carbon (formula (CH 2 OH) 2 CO was prepared by the entle oxidation of the triatomic alcohol, glycerol (C 3 H 5 (OH) 3 . This ynthesis is particularly interesting because it establishes a connection etween the carbohydrates and the fats, since all of the naturally ccurring fats contain a glycerol radical. From this sugar it was found 54 CARBOHYDRATES MONOSACCHATMJ2ES -^ X . possible to prepare a sugar containing six atoms of carbon in the mole- cule, by the action of dilute alkali. At the same time Fischer succeeded in synthesizing a hexose (that is to say, a six carbon atom sugar) from its elements, by the polymerization of formaldehyde (HCHO), in accordance with the equation: 6HCHO = C 6 Hi 2 O 6 and this sugar was found to be identical with that which had been synthesized from glycerose. Examination of this new sugar showed, however, that it differed in a very important property from the naturally occurring hexoses, fruit- sugar, glucose, or mannose. These sugars, when in solution, rotate the plane of polarization of a beam of polarized light to the right or to the left. The synthetic sugar did not rotate the plane of polarized light, and hence a special name was given to it, Across. The reason for the optical inactivity of acrose was found to lie in the fact that it is a mixture of equal parts of optical antipodes, the one rotating the plane of polarized light to the right, and the other to the left in equal degree. As a matter of fact acrose can be decomposed by appropriate measures into optically active constituents, and according to the conditions which accompany the transformation we obtain fruit-sugar, mannose, or glucose. It is a remarkable fact that nearly all natural products which are derived from living material are possessed in some degree of Optical Activity. This was at first thought to be a peculiarity of substances formed by living organisms and to point to the operation within living tissues of some force peculiar to living matter. We now understand that the optical activity of the constituents of living matter is due to the circumstance of their synthesis in the presence or through the agency of optically asymmetric catalyzers. The exact conditions upon which this property of optical activity depends were first made clear by Le Bel and Van't Hoff in 1874. Previously to this Pasteur had expressed the opinion, based upon his fundamental observations on the differing crystal-forms of the right- handed and left-handed varieties of tartaric acid, that the optical activity of certain molecules must be attributable to a certain degree of asymmetry of the molecule. This asymmetry, in the case of carbon compounds, Van't Hoff was able to trace to the carbon atom. If we imagine the four valencies of a carbon atom to be pointing toward the four apices of a tetrahedron, of which the center is the carbon atom, the following arrangements of four different masses are possible: HEXOSES 55 The difference between these arrangements resembles that between an image and its reflection in a mirror; the diagrams cannot be super- imposed upon one another so that the corresponding parts will coincide, except by inverting one of the diagrams, and thereby converting it into the other, its mirror-image. Now it would appear that when a carbon atom is united by its valencies to four different masses, either of the above arrangements is possible, the one yielding a dextrorotatory and the other a levorotatory compound. An optically inactive body is produced either by a mixture of equal numbers of the two forms of molecules or by "internal racemization," i. e., by the presence within the molecule of two equally active carbon atoms rotating the plane of polarized light in opposed directions. This being the case, the number of possible optical isomers of a substance which contains two asymmetric carbon atoms is four, since either of the two possible varieties, levo- and dextro- of the first asym- metric atom may be combined with either of the two possible varieties of the remaining atoms. Similarly the number of possible optical isomers of a substance which contains three asymmetric carbon atoms is eight, since any of the four possible arrangements about the first two atoms may be combined with either of the two possible arrange- ments about the third atom, and, in general, the number of possible optical isomers of a substance which contains n asymmetric carbon atoms is 2 n . THE HEXOSES. The relationships which have been described above are very well illustrated among the hexoses. A large number of sugars are known which possess the formula C 6 H]2O 6 . The structural formulae of these sugars have been elucidated by Fischer and others, and it has been shown that a number of these possess a structure 1 which can be repre- sented by the general formula: CHO *CHOH *CHOH I *CHOH *CHOH CH 2 OH It will be observed that the four carbon atoms which are distin- guished by asterisks are asymmetric, because they are each united with four different masses. For example, take the second carbon atom from the top of the diagrammatic formula. It is united with the following groups: -CHO, -H, -OH and -C 4 H 5 (OH)4. According to the rule which is enunciated above, there must be 2 4 = 16 possible optical isomers of this compound. 1 Or are readily convertible into substances possessing such a structure, cf. below: 56 CA RBOH YDRA TESM ON OS A CCHA RIDES This will be rendered clearer by the accompanying diagram, which illustrates the structure of the sixteen possible stereo-isomers of any compound which contains four asymmetric carbon atoms. Designat- ing a dextrorotatory carbon by the symbol + and a levorotatory carbon by the symbol it will be seen that each carbon is dextrorotatory in eight isomers, and levorotatory in eight others. It is also evident that provided the end-groups attached respectively to the first and fourth asymmetric carbons are identical, the isomer number 11 is identical with the isomer number 5, 12 with 6, 13 with 7 and so forth. 11 12 13 14 15 16 10 This is what actually occurs in the corresponding polyatomic alcohols, in which the CHO group of the sugar is replaced by the group CH 2 OH. In the hexoses, of which glucose is a representative, the two end- groups are, of course, different and hence no two possible isomers are identical. There are, therefore, 16 possible sugars or hexoses of the aldehyde type, possessing the above formula. We may represent them as follows, using the prefixes d- and 1- to signify dextro- and levo- rotatory respectively. CHO I H C OH H C OH HO C H HO C H CH 2 OH 1-mannose. CHO HO C H I H C OH HO C H H C OH s OHsOH 1-idosc. CHO HO C H HO C H H C OH H C OH CH 2 OH d-mannose. CHO H C OH HO C H I H C OH HO C H I CH 2 OH d-idoso. CHO HO C H H C OH HO C H HO C H I CH 2 OH 1-glucose. CHO H C OH H C OH HO C H H C OH CH 2 OH l-galose. CHO H C OH HO C H H C OH H C OH CH 2 OH d-glucose. CHO I HO C H I HO C H I H C OH HO C OH I CH 2 OH d-galose. CHO I HO C H I H C OH I H C OH HO C H CH 2 OH 1 galactose. CHO HO C H HO C H I HO C H HO C H CH 2 OH 1-allose. HEXOSES J CHO CHO 1 H C OH 1 HO C H HO C H H C OH 1 H C OH H C OH H C OH CH 2 OH HO C H 1 CH 2 OH d-galactose. 1-talose. CHO 1 CHO 1 H C OH H C OH H C OH HO C H H C OH HO C H H C OH CH 2 OH HO C H 1 CH 2 OH d-allose. 1-altrose. 57 CHO HO C H HO C H HO C H I H C OH CH 2 OH d-talose. CHO HO C H I H C OH H C OH 1 I CH 2 OH d-altrose. The various sugars have been prepared synthetically and their con- stitutional formulse confirmed. Thirteen of them are laboratory products, and only three of them are met with in nature, to wit: d- glucose, d- mannose and d- galactose. In addition to these hexoses of the aldehyde type, or Aldoses, another hexose of quite a different type is of very common occurrence in nature, namely fruit-sugar or Fructose. Unlike all of the hexoses considered above, fructose is a sugar of the ketone type, or ketose. Its structure may be represented by the formula : CH 2 OH CO HO C H H C OH H C OH CH 2 OH Fructose exists in a dextrorotatory and a levorotatory form, the one being the mirror-image of the other. We customarily distinguish between dextro- and levorotatory forms by the prefixes employed above, d- and 1-; as, for example, d-glucose and 1-glucose. The form of fructose which is represented in the formula given is the levorotary form, but it is, nevertheless, termed d-fructose, because of its close relationship to d-glucose, which will be apparent on comparing the two f ormulse : 58 CARBOHYDRATES MONOSACCHARIDES OHO CH 2 OH | I H C OH CO | I HO C H HO C H H C OH H C OH | I H C OH H C OH | I CH 2 OH CH 2 OH 4-glucose d-fructose The mirror-image, which is in reality dextrorotatory, is therefore termed l-fructose. The levorotation of d-fructose has led to its being very generally designated levulose, by which name we will hereafter fre- quently refer to it. Another ketose which occurs in nature is d-Sorbinose: HO which is formed when the juice of the mountain-ash is exposed to air, by the oxidizing action of a ferment upon the alcohol sorbitol which is contained in the juice. REACTIONS OF THE CARBOHYDRATES. The aldehyde and ketone, or potentially 1 aldehyde and ketone struc- ture of the sugars renders them peculiarly liable to oxidation. Like other aldehydes and ketones, they reduce metallic oxides in alkaline solution; thus they reduce cupric to cuprous oxide, upon which fact Fehling's method of sugar-estimation is based, and they reduce silver salts in ammoniacal solution, leading to the formation of a silver mirror. Other reactions which are characteristic of the sugar-group are the following : On heating a solution of sugar in concentrated sodium or potassium hydroxide, the liquid turns dark brown (Moore's test). If to about 0.5 c.c. of a dilute aqueous solution of glucose are added a few drops of a ten per cent, alcoholic (acetone free) solution of cHnaphthol, and 1 c.c. of concentrated sulphuric acid be cau- tiously run into the lower part of the tube, so that the lighter solu- tion floats upon the top of the heavy acid, the zone of contact becomes reddish violet (Molisch's test). This reaction is due to the formation i Cf. below. REACTIONS OF THE CARBOHYDRATES 59 of furfurol from the sugar by the concentrated acid. The furfurol then reacts with the ct-naphthol yielding a colored product. If sugar be heated to a considerable degree the mass partially carbonizes and turns deep brown. Numerous products are formed to which the collective name of caramel is given. Caramel has distinc- tively colloidal properties and very high coloring-power, upon which depends its use in the artificial coloring of beverages. The sugars themselves are very soluble and on that account are frequently difficult to characterize and to purify. They form, how- ever, insoluble or sparingly soluble compounds with Phenylhydrazine, which are of great service in characterizing the various sugars, enabling us to identify them in many cases with a considerable degree of cer- tainty. If an aldose, or sugar, that is, of the aldehyde type, be acted upon by phenylhydrazine in the presence of excess of acetic acid, the following reaction occurs: CHO CH(OH) CH(OH) I CH(OH) CH(OH) 2(OH) C 6 H 6 NH.NH 2 CH:N.NHC 6 H 6 I CH(OH) = CH(OH) CH(OH) CH(OH) CH 2 (OH) Phenylhydrazone. H 2 O The Phenylhydrazones are, for the most part, readily soluble; so that this stage of the reaction is easily overlooked, since the -subse- quent secondary reactions which are about to be described produce sparingly soluble substances. Mannose is, however, an exception to this rule, the phenylhydrazone being very sparingly soluble and readily detected and isolated. Other hydrazines such as methylphenyl- hydrazine, benzylphenylhydrazine, and diphenylhydrazines also react with sugars to form hydrazones which are in some cases sparingly soluble and can readily be separated and purified by repeated recrystal- lization, and identified by their melting-points. If excess of phenyl hydrazine be employed, the remainder of the reagent which is not used up in converting the sugar into a hydrazone acts as an oxidizing agent, converting a CH(OH) group into a CO group, thus: CH:N.NHC 6 H 6 I CH(OH) CH(OH) CH(OH) CH(OH) CH 2 OH Hydrazone + CH:N.NHC 6 H 6 I CO CH(OH) I + C 6 H 5 NH.NH 2 = CH(OH) CH(OH) CH 2 OH phenylhydrazine = oxidation product C 6 H 6 NH 2 + NHs aniline + ammonia. 60 CARBOHYDRATES MONOSACCHARIDES This oxidation-product subsequently reacts with yet another mole- cule of phenylhydrazine, with the formation of an Osazone: CH:N.NHC 6 H 5 CH:N.NHC 6 H 5 I 1 CO C:N.NHC 6 H 5 1 1 CH(OH) CH(OH) 1 1 CH(OH) + C 6 H 5 NH.NH? = CH(OH) + H 2 O I 1 CH(OH) CH(OH) i CH 2 OH CH 2 OH oxidation-produc t + phenylhydrazine = osazone + water Glucose, mannose and fructose all yield the same osazone. In the case of fructose the reactions described above simply take place in the reverse order, thus: CH 2 (OH) CH 2 (OH) 1 1 CO C :N.NHC 6 H 6 1 1 CH(OH) CH(OH) 1 1 CH(OH) + C 6 H 5 NH.NH 2 = CH(OH + H 2 O .1 1 CH(OH) CH(OH) 1 I CH 2 (OH) CH 2 (OH) ketose + phenylhydrazine = hydrazone + water CH 2 OH i CHO i C:N.NHC 6 H 6 C:N.NHC 6 H 6 1 I CH(OH) CH(OH) 1 I CH(OH) + C 6 H 6 NH.NH 2 = CH(OH) + C 6 H 5 NH 2 + NH 3 1 | CH(OH) CH(OH) I | CH 2 (OH) CH 2 (OH) hydrazone + phenylhydrazine = oxidation-product + anilin + ammonia CHO CH:N.NHC 6 H 6 1 | C:N.NHC 6 H 6 C:N.NHC 6 H 6 I | CH(OH) CH(OH) \ | CH(OH) + C 6 H 5 NH.NH 2 = CH(OH) + H 2 O 1 | CH(OH) CH(OH) 1 I CH 2 (OH) CH 2 (OH) oxidation-product + phenylhydrazine = osazone + water REACTIONS OF THE CARBOHYDRATES 61 The osazones as a class are characterized by their relatively slight solubility in water. They form yellow needle-shaped crystals and the shapes of the individual crystals and the way in which they group c. FIG. 1. Osazone crystals. A, phenylglucosazone; B, phenylmaltosazone ; C, phenyl- lactosazone. (After Halliburton.) together are to some extent characteristic for each osazone. The melting-points of the osazones are not very definite, depending some- vhat upon the mode in which the heat is applied, but they are, as a ule, sufficiently definite to serve as a means of identification. 62 CARBOHYDRATES MONOSACCHARIDES When the Hexoses are heated for prolonged periods with dilute mineral acids (excepting nitric acid) they yield Levulinic Acid (acetyl propionic acid) and formic acid, besides "humin substances" containing a higher proportion of carbon than the carbohydrates. The reaction producing levulinic acid proceeds as follows: C 6 Hi 2 O 6 = CH 3 CO.CH 2 .CH 2 .COOH + H.COOH + H 2 O hexose = levulinic acid + formic acid + water. When nitric acid is employed Saccharic Acid is produced (see below) . Levulinic acid is soluble in water, alcohol and ether and forms a colorless viscous liquid which boils at 250 C. and yields with silver nitrate a crystalline salt with the formula CH3.CO.(CH 2 ) 2 COOAg. The Pentoses do not yield levulinic acid on boiling with mineral acids ; on the contrary, they yield Furfurol HC CH II II HC C.CHO \ / o which may be collected by distillation and detected by the aid of aniline acetate paper, which is colored red by furfurol. This reaction may also be used for the estimation of pentose since the yield of fur- furol is quantitative. For this purpose the furfurol is distilled and bisulphite added to the distillate, when the usual bisulphite compound with aldehydes is formed and the unconsumed bisulphite is estimated by titration with iodine. Or the furfurol may be converted into the phloroglucide by addition of phloroglucin and the yield of this com- pound determined gravimetrically. It should be very carefully borne in mind, however, that Glucuronic Acid and its compounds (see below) also yield furfurol on treatment with dilute acids so that before deciding that pentoses are present their identity should be established by the formation of osazones and the absence of glucuronic acid. The pentoses also yield the following reactions: The Orcin Reaction. To a small amount of the solution is added an equal volume of a solution of orcin in concentrated hydrochloric acid. On heating, the solution turns reddish-blue and then bluish-green. If pentoses are present in abundance, a green precipitate will be obtained on cooling and standing. On shaking up the mixture with amyl alcohol the green coloration passes over into this solvent. This reaction is also yielded by glucuronic acid. The Phloroglucin Reaction. This reaction is carried out in the same manner as the above, phloroglucin being used in the place of orcin. The mixture turns red on heating and becomes cloudy on cooling On shaking with amyl alcohol the red color passes over into the amyl. alcohol layer. This reaction is also yielded by glucuronic acid. Selivanoff's Reaction. The Ketoses may be distinguished from the Aldoses by Selivanoff's reaction, as follows: REACTIONS OF THE CARBOHYDRATES 63 To a few cubic centimeters of solution is added an equal volume of twenty per cent, solution of hydrochloric acid containing a small pro- portion of resorcinol. The liquid turns red on heating and a red sub- stance is gradually deposited which is soluble in alcohol. This reaction depends upon the formation of oxymethyl-f urf urol from the ketose by heating with acids. If the acid be too concentrated or the mixture boiled for more than about twenty seconds, the hexoses will also yield a small proportion of oxymethyl-f urf urol and will in consequence yield the same reaction. It is necessary therefore to avoid a higher con- centration of hydrochloric acid in the final mixture than about twelve per cent., and to boil only for a period of less than twenty seconds. This danger of confusion with the aldoses may be avoided by employing glacial acetic acid containing a small proportion of hydrochloric acid, instead of concentrated hydrochloric acid, as the solvent for the resorcinol. The Chemical Relationships of the Sugars. Each sugar being an aldehyde or ketose, or at any rate potentially an aldehyde or ketose, corresponds to an alcohol from which it is derived by oxidation. Re- duction of the sugars, therefore, results in the formation of alcohols. Glucose yields Sorbitol, mannose yields Mannitol and galactose yields Dulcitol. The following are the formulae which illustrate the structure of these alcohols and their derivation from the corresponding sugars. CHO CH 2 OH CHO CH 2 OH I I I I HC OH H C OH HO C H HO C H I II I HO C H HO C H HO C H HO C H + H 2 = + H 2 = | H C OH H C OH H C OH H C OH 1 II I H C OH H C OH H C OH H C OH I II I CH 2 OH CH 2 OH CH 2 OH CH 2 OH d-glucose. d-sorbitol. d-mannose. d-mannitol. CHO CH 2 OH H C OH H C OH HO C H HO C H I + H 2 = | HO C H HO C H H C OH H C OH CH 2 OH CH 2 OH! d-galactose. d-dulcitol. All of these alcohols occur in plants, mannitol especially being widely distributed. They have a sweet taste, but they are not fermentable by yeast. Just as the sugars, being potential aldehydes or .ketoses, are con- 64 CARBOHYDRA TESMONOSACCHARIDES verted by reduction into Alcohols, so, by oxidation, they are converted into acids. Glucose yields three different 6-carbon atom acids on oxidation. Two of these acids are monobasic and the third is dibasic. CH 2 OH Glucose. COOH I H C OH HO C H H C OH H C OH CH 2 OH Gluconic acid. CHO H C OH HO C H H C OH H C OH I COOH Glucuronic acid. COOH I H C OH HO C H I H C OH I H C OH I COOH Saccharic acid. From mannose the dibasic acid which is obtained is Manno-sac- charic Acid. From galactose the dibasic acid which results from oxidation is Mucic Acid. The ketoses, including levulose, behave quite differently on oxidation. The aldoses, on being oxidized yield acids containing the same number of carbon atoms as the original sugar. The ketoses, on the contrary, break down on oxidation and yield compounds containing fewer carbon atoms than the original sugar. Similar relationships subsist among the sugars which contain fewer than six carbon atoms. Thus we have: BIOSES. Alcohol. CH 2 OH I CH 2 OH Glycol. Sugar. CHO CH 2 OH Glycolose. Monobasic acid. COOH I CH 2 OH Gly collie acid. Dibasic acid. COOH COOH Oxalic acid. TRIOSES. Alcohol. CH 2 OH CHOH CH 2 OH Glycerol. CH 2 OH I CHOH CHOH CH 2 OH Erythritol. Sugar. CHO CHOH CH 2 OH Glycerose. Monobasic acid. COOH CHOH CH 2 OH Glyceric acid. TETROSES. CHO COOH CHOH I CHOH I CH 2 OH Erythrose. CHOH CHOH CH 2 OH Erythric acid. Dibasic acid. COOH CHOH I COOH Tartronic acid. COOH I CHOH CHOH COOH Tartaric acid. Similarly, the pentose Arabinose corresponds to the alcohol Arabitol and to the acids Araboric and Trioxyglutaric, while the pentose Xylose corresponds to the alcohol Xylitol. OF THE CARBOHYDRATES 65 By appropriate methods it is possible to convert sugars into others containing more carbon atoms and vice versa. Thus the aldoses combine directly, with Hydrocyanic Acid with the formation of Nitriles, in accordance with the equation: C 5 Hn0 5 .CHO + HCN = C 6 HuO 6 CH(OH).CN These nitriles^on hydrolysis, yield acids containing one carbon atom more than the original carbohydrates, thus: C5HnO 5 .CH(OH).CN + 2H 2 O = C 5 H U O 5 CH(OH)COOH + NH 3 Reduction of these acids, by means of sodium amalgam, yields the corresponding aldose with one carbon atom more than the original sugar. In this way glucose has been prepared from arabinose, and seven- and even nine-atom sugars have also been prepared, by successive steps, starting with glucose. The conversion of a sugar containing more, into one containing fewer carbon atoms may be accomplished by converting the sugar by gentle oxidation into the corresponding (monobasic) acid, and then subjecting the calcium salt of this acid to further oxidation, with the result that the carboxyl-group is decomposed into carbon dioxide and water, and a sugar containing one less carbon atom than the original sugar is formed : CHO COOH I i CH(OH) CH(OH) A COH I I if I CH(OH) CH(OH) CH(OH) I I t/l CH(OH) CH(OH) - CH(OH) + CO 2 + H 2 O I I .1 CH(OH) CH(OH) CH(OH) i I ! CH 2 (OH) CH 2 (OH) CH 2 (OH) Aldo-hexose Aldonic acid Aldo-pentose This reaction is of very great interest to the biochemist because the conversion of a carboxyl- group into CO 2 and H 2 is known to be readily accomplished by bacterial action and probably also by animal tissues. TJie possibility is thus indicated that pentoses in the tissues may be derivable from glucose, a possibility, the significance of which will be apparent at a later stage. Not only is it possible to convert a hexose into a pentose and vice versa, but it is also possible to convert one hexose into another. It was found by Lobry de Bruyn that in the presence of alkalies, glucose, mannose or levulose in aqueous solution yields a mixture of the three sugars. More concentrated alkali brings about more pronounced decomposition, as is evidenced by the formation of lactic acid and other hydroxy-acids in Moore's test for carbohydrates. The production of lactic acid from glucose by the action of alkalies is a phenomenon of 5 MONOS^^f. 66 CARBOHYDRATES MONOSAlTTIARIDES great importance in the light of the fact that the decomposition of glucose, or glycogen which is an anhydride of glucose, in muscular tissue leads to the formation of lactic acid. The action of alkalies upon glucose led to the suspicion that if am- monia were employed amino-derivatives of hydroxy-acids, such as are found among the constituent radicals of the proteins, might possibly be formed, and it was found by Windaus and Knoop that, as a matter of fact, ammonia, acting upon glucose, mannosiMJtalose, sorbose, arabinose, xylose, rhamnose or lactose yields Methyl^^oxaline : CH 3 C NH >CH HC N/ No other amino-products of this decomposition were identified, but this one is of extraordinary interest, because of the very great impor- tance and variety of roles played by the Iminazole ring in physiological phenomena. While it is very doubtful whether the synthesis of this ring is possible for animal tissues to accomplish, and in fact there is much evidence tending to show that it is not, yet it is, of course, unques- tionably accomplished by vegetable tissues, since the iminazole ring in the form of the amino-acid Histidine and in the purine-base moiety of the Nucleic Acids, is an invariable and essential constituent of living matter. On heating hexoses in concentrated solution with amino-acids (glyco- coll, alanine, leucine, tyrosine or glutamic acid) the mixture darkens with the formation of "Humin Substances" which are very deeply colored. At the same time carbon dioxide is discharged from the mixture. It is believed that the carbon dioxide is released from the carboxyl-group of the amino-acid which unites with the aldehyde- group of the sugar to form cyclic compounds. Similar substances are formed (from tryptophane) when proteins are hydrolyzed by strong acids in the presence of carbohydrates. Certain Derivatives of Glucose. Two derivatives of glucose, Glucuronic Acid and Glucosamin, claim our attention at this juncture, because they are both of profound physiological importance. We have seen that on oxidation, glucose yields, first two monobasic acids and thereafter, on continued oxidation, each of these monobasic acids yields the same dibasic acid. One of the monobasic acids is glucuronic acid, the dibasic acid is saccharic acid. The connection between glucose, glucuronic acid and saccharic acid can be seen at a glance from their formulae: REACTIONS OF THE CARBOHYDRATES 67 CHO CHO COOH I I I H C OH H C OH H C OH I I I HO C H HO C H HO C H H C OH H C OH H C OH H C OH H C OH H C OH I I I CH 2 OH COOH COOH d-glucose. d-glucuronic acid. d-saccharic acid. Glucuronic acid is therefore at the same time an acid and an alde- hyde. On boiling its solution or on prolonged standing it is trans- formed into a crystalline lactone which is represented by the formula: CHO H c OH Glucuronic acid yields the pentose reactions with orcin or phloro- glucin and hydrochloric acid (see p. 62) and also the following reaction: Naphtho-resorcinol Reaction. A small amount of naphtho-resorcinol is dissolved in concentrated hydrochloric acid and to this reagent is added an equal volume of a solution of glucuronic acid. A violet-blue coloration results which is soluble in ether. This reaction is not specific for glucuronic acid, being given by many ketose and aldehyde acids. It is, however, useful for the purpose of distinguishing between glu- curonic acid and the pentoses. Glucuronic acid does not occur in the free condition in animal tissues, nor has it as yet been identified in plants. In the form of ester-like compounds, however, it is found in many plants, notably in Scutellaria, and esters of glucuronic acid are found in^many parts of the body, in the blood, the liver and in urine. The normal forms in which it is found in urine are Phenyl-, Indoxyl- and Skatoxyl-glucuronic acids. Indoxyl and skatoxyl are highly toxic products of intestinal putrefac- tion; the compounds which they form with glucuronic acid are, however, harmless. Under ordinary conditions, glucose is readily oxidized in the body to carbon dioxide and water, passing through intermediate stages of which lactic acid is one. But in the presence of some toxic agents it appears that the oxidation of glucose is arrested at the formation of glucuronic acid, which combines with the toxic substance, the compound being 68 CA RBOH YDRA TESMONOSA CCHARIDES eliminated as such. It is not known whether or not glucuronic acid is a normal intermediate product of glucose oxidation in the animal body, or whether it is only formed under the exceptional condition of the presence of certain toxic bodies. Large quantities of glucuronic acid, in these ester-like combinations, appear in the urine when certain drugs are introduced into the system. The following is a partial list of the drugs which are eliminated in this manner : Isopropyl alcohol chloral Methyl propyl alcohol butyl chloral Methyl ethyl carbinol bromal Tertiary butyl alcohol dichloracetone Tertiary amyl alcohol a and /3 napthol Benzol turpentine Nitrobenzol camphor Aniline borneol Phenol menthol Resorcinol pinene Thymol antipyrine The elimination of these drugs in this manner constitutes a pitfall for the unwary who may seek, after the administration of such drugs as these to a patient, to investigate the urine for the presence of sugar therein by the phenylhydrazine test, or its clinical modification known as Cipollina's test. For glucuronic acid forms an osazone which may easily be mistaken for glucosazone. The distinction may very readily be made, however ; owing to the fact that the osazone of glucuronic acid is decomposed by heating while that of glucose is not. If the precaution be taken, therefore, of heating the precipitate on a boiling water-bath for half an hour before examining it, no confusion of the osazone of glucuronic acid with glucosazone is possible, for the osazone of glucuronic acid is decomposed by this procedure and redissolves, while the osazone of glucose remains unaltered. Glucuronic acid is therefore to be regarded as a protective agent which guards the organism against the deleterious action of certain substances introduced from without or, in some cases, from within the body. Sometimes the glucuronic acid accomplishes its protective function by combining directly with the toxic substance, rendering it harmless until in the course of time it is eliminated; in other instances the toxic substance unde^oes some degree of change and elaboration before it is paired with glucuronic acid. Thus chloral hydrate and butyl chloral undergo reduction before they couple with the glucuronic acid; o-nitro toluol, on the contrary, is oxidized to nitrobenzyl-alcohol before it pairs with glucuronic acid. Other substances undergo hydration or both hydration and oxidation before they can couple with the glucuronic acid. Glucuronic acid is possibly of importane not only as a carrier of toxic substances out of the body, but also as a connecting-link between the hexoses and the pentoses. It will be recollected that when a mono- MONOSACCHARIDES IN LIVING TISSUES 69 basic acid derivative of an aldohexose is acted upon by oxidizing agents, the carboxyl group is eliminated in the form of carbon dioxide and water, and the corresponding aldo-pentose is formed. When d-glucu- ronic acid is subjected to intense putrefaction, it undergoes an analogous change, with the production of 1-xylose, thus : CHO CHO I I H C OH . H C OH I I HO C H HO C H + CO 2 H C OH H C OH H C OH CH 2 OH COOH Glucosamin, on the other hand, affords a connecting link between the carbohydrates and the hydroxy-amino-acids. It is readily obtained in considerable quantities from the exoskeletons of Crustacea, as for example from the shells of lobsters, by boiling with concentrated hydrochloric acid. It also occurs in fungi and it is a constituent of the Mucins and Mucoids; sticky glutinous proteins which are found in mucous secretions and elsewhere. The formula of glucosamin is: CHO H C NH 2 HO C H H C OH H C OH CH 2 OH In the true mucins, but not in the mucoids the glucosamin radical is probably acetylated, and acetyl glucosamin, in common with other acetyl derivatives of hydroxy-amino-acids, yields Ehrlich's Reaction, namely a pink color when its solution is mixed and warmed or allowed to stand with an equal volume of a two per cent, solution of paradi- methylaminobenzaldehyde in hydrochloric acid of specific gravity 1.09. The mucins also yield this reaction. THE DISTRIBUTION OF THE MONOSACCHARIDES IN LIVING TISSUES. As has been stated, a pentose, d-Ribose is a normal constituent of the nucleoproteins. The following, after Grund, is the percentage 70 CARBOHYDRATES MONOSACCHARIDES of pentoses calculated on the basis of the dry tissue, which is present in various parts of the mammalian body: Pancreas 2.48 Liver 0.56 Thymus 0.56 Submaxillary gland 0.53 Thyroid gland 0.50 Kidneys 0.49 Spleen 0.46 Brain 0.22 Muscle 0.11 The structural formula of d-ribose may be represented as: CHO I H C OH I H C OH H C OH CH 2 OH d-ribose. it is levorotatory, the prefix d- being employed to denote its relationship to d-altose and d-altrose. In certain very exceptional cases a pentose is formed in the urine. The disease which leads to this elimination of pentoses is known as Pentosuria, in contradistinction to Glycosuria, the very much more common elimination of -glucose. Only a few cases of pentosuria have been observed, but it is an extremely ' noteworthy fact ' that the pentose which is eliminated in this disease would appear to be almost invariably optically inactive, although the pentose, 1-ribose, which is normally found in the tissues is, of course, optically active. Not only this, but the pentose in the urine is not ribose but Arabinose, CHO CHO I I HO C H H C OH I I H C OH HO C H H C OH HO C H I I CH 2 OH CH 2 OH d-arabinose. 1-arabinose. which would seem to point to its derivation from glucose rather than from the decomposition of nucleo-proteins, for it will be remembered that arabinose may be derived from glucose by the oxidation of the calcium salt of gluconic acid (xylose being the corresponding pentose resulting from the oxidation of glucuronic acid). However this may be, the pentose elimination in these cases is independent of the pentose- MONOSACCHARIDES IN LIVING TISSUES 7l content of the food and may occur when the combustion of carbo- hydrates in the tissues would appear to be otherwise normal. The pentoses are widely distributed in the vegetable kingdom, chiefly in the form of polysaccharides, bearing the same relation to the pentoses as starch and glycogen do to the hexoses. These polysac- charides, which are complex anhydrides of the monosaccharides, are known as Pentosans. The following table shows the percentage of pentosan, in terms of pentose, found in the dry substance of various vegetable foods: Meadow hay . , . . . . . '. . . . . ... . . . 21.64 Rape cake . .... 11.50 Oil-seed cake ...>.... . 9.07 Bruised barley . . . . . . . . . . . .... 7.96 Rice flour ;...-. .. : . . . 5.73 Sesame cake . . . ,. . . . . 3.87 Table turnip . ., . . .'...'. . . . 1.13 Spinach . . . . . ^ 1.02 With regard to the distribution of the hexoses; Levulose is not often found in the animal kingdom. In honey it occurs together with glucose and is immediately derived from the juices of flowers, but it is a question whether it is ever normally found in animal tissues. It is occasionally found in the urine, and is then derived directly from the levulose absorbed from the intestine; it may be regarded as a sign either of excessive overindulgence in sweets or honey or else, if this origin can be excluded, as a sign of overactivity of the pituitary gland, which as we shall see later on, lowers the limit of tolerance for all forms of sugar. A urine which yields evidence of the' presence of a reducing sugar should therefore always be tested for levulose by Selivanoff's test (see p. 62) before a provisional diagnosis of diabetes is decided upon. In vegetable tissues levulose is widely distributed, especially com- bined with glucose to form cane-sugar. It is also found in the form of a complex anhydride, or polysaccharide, Inulin in the tubers of dahlias and in the sweet potato. Grape-sugar, or d-glucose, is the most important of all the mono- saccharides in the animal economy. It is the central figure in the carbohydrate metabolism. Polysaccharides are broken down to glucose before assimilation, and again before utilization as a source of energy, or transportation from one part of the body to another. It is the circulating form of carbohydrate, Glycogen and other poly- saccharides being the storage-forms. In view of these facts the absurdity will be apparent of the effort which was made in certain circles in the United States, a few years ago, to represent glucose and glucose-syrups as deleterious articles of food. Provided they contain no other 'constituents which are harmful such preparations are merely solutions of the only carbohydrate which is to any important extent a normal and invariable constituent of the blood. 72 CARBOHYDRA TESMONOSACCHARIDES Normal urine contains minute traces of glucose, and sometimes larger amounts, especially after a meal which is very rich in carbo- hydrates. Such glycosuria is known as Alimentary Glycosuria and is devoid of significance unless it occurs too frequently and readily, in which case it may possibly indicate disturbance of the functions of the pituitary gland. In certain pathological conditions or under experi- mental conditions much profound and serious glycosurias may occur. These will fall under consideration in a later chapter. Galactose is found in important quantities in two places in the animal kingdom, namely combined with glucose to form milk-sugar or Lactose; and in the form of glucoside-like compounds, the Cere- brosides, which are found in the brain. THE LACTONE-STRUCTURE OF SUGARS. Before proceeding to the consideration of the disaccharides, it is important to review some recent accessions to our knowledge of the sugars which have led us to reconsider in some degree the structural formulae by means of which we have hitherto represented them. It is necessary to enter thus deeply into the subject of the configuration of the sugar molecule because a clear understanding of these questions has already fundamentally contributed to our knowledge of the mode of action of ferments, and is unquestionably destined to do so even to a greater degree than heretofore. ' In considering the enzymatic hydrol- ysis and synthesis of the disaccharides we shall have occasion to refer very frequently to the facts which are about to be described. It will be recollected that in compounds containing only four asymmetric carbon atoms, such as we have been assuming the hexoses to be, only sixteen stereo-isomers are possible. Now, as a matter of fact, it has long been known to sugar-chemists that the optical rotatory power of solutions of d-glucose is not a constant quantity. The optical rotatory power of fresh solutions changes gradually, sometimes increasing, but more usually falling, until a constant value is ultimately reached. This constant value is the same for all glucose solutions which have attained equilibrium, but the initial rotatory power of fresh solution may be as much as twice as great as the final constant rotatory power. This phenomenon is variously known as Mutaro- tation, Multirotation and Birotation. Analogous phenomena in other solutions are generally attributed to the presence of two or more different, optically active substances, of different rotatory power and convertible into one another. Adopting this point of view, Emil Fischer first suggested, in explanation of the phenomenon of mutarotation, that the glucose undergoes hydration in solution, with the formation of an alcohol of lower rotatory power, thus: LACTONE-STRUCTURE OF SUGARS 73 CHO CH 2 (OH) I I CH(OH) CH(OH) CH(OH) CH(OH) + H 2 CH(OH) CH(OH) CH(OH) CH(OH) CH 2 (OH) CH 2 (OH) This view, which never had any experimental support, was rendered unnecessary and untenable by the discovery of the fact that two different forms of d-glucose are obtainable, isomers of one another but differing in rotatory power. The one form, a-d-glucose, crystallizes out at ordinary temperatures from seventy per cent, alcohol, and has a molecular rotation of ()D + 110; the other, /3-d-glucose, crystal- lises out from solutions in water at temperatures above 98 C., and has a molecular rotation of ()D + 19. It appears that there are indeed twostereo-isomeric forms of d-glucose, which would be impossible were there onlv four asvmmetric carbon atoms in the molecule, as the formula CHO - CH(OH) - CH(OH) - CH(OH) - CH(OH) - CH 2 (OH) requires. The glucose molecule must, in fact, contain not less than Five asymmetrical carbon atoms. This conclusion, first suggested by Simon, was verified by Armstrong in the following way: Two methylated d-glucoses are known, formed from glucose by the replacement of a hydrogen by a methyl group. The structures of these two methyl glucosides are believed to be respectively: CH 3 O CH HC O CH 3 HCOH \ HCOH N HOCH ' / HOCH HC/ HC HCOH H U' COH CH 2 OH CH 2 OH a-methyl-d -glucoside /3-methyl-d-glucoside Each of these glucosides can be hydrolyzed by an appropriate ferment. Now it is observed that a glucose of high rotatory power is produced in the hydrolysis of the a-methyl glucoside, while on adding a drop of ammonia to the solution the rotation rapidly falls to the equilibrium-value of the rotatory power of ordinary glucose. On the other hand, when the 0-methyl glucoside is hydrolyzed, a glucose of low rotatory power is produced, and on adding a drop of ammonia to 74 CARBOHYDRATES MONOSACCHARIDES the solution the rotatory power rapidly rises to the equlibrium-value of the rotatory power of ordinary glucose. From these observations it appears that the true formula for d- glucose is either: HO C H H C OH HCOH \0 HOCH HC/ HCOH CH 2 OH a-d-glucose. HCOH \ HOCH / HC/ HCOH CH 2 OH p-d-glucose. of which the former is the a (highly rotating) form, and the latter the )8 form of low rotatory power. In solution, an equilibrium is finally attained between the two forms, and the attainment of this equilibrium is much accelerated by an alkaline reaction. The rotatory power of the pure a form is ()D + 110; that of the pure (3 form () D + 19. The rotatory power of an equilibrated solution of the mixed glucoses is (a) D + 52.5. From these figures it is a simple sum in proportion to calculate that in a ten per cent, solution of glucose, about thirty- seven per cent, is of the a form and about sixty-three of the form at equilibrium. We see that glucose contains, therefore, not four but five asymmetri- cal carbon atoms, a fact which is not revealed by a study of long- standing or equilibrated solutions and was therefore very naturally overlooked in the first attempts to attach a structural formula to individual hexoses. If this be true of the other hexoses as well, however, then there must exist not 2 4 = 16 stereo-isomers of glucose, but 2 s = 32. As a matter of fact, we find that many of the sugars exhibit mutaro- tation, for instance d-glucose, d-galactose, d-mannose, d-fructose, 1-arabinose, l-xylose, and some of the disaccharides. There is little room for doubt that the structural formulae of each of these sugars are analogous to the formulae for glucose which are depicted above. Since the hexaldoses all give the aldehyde reactions, that is, reduce metallic oxides in alkaline solution, and unite with phenylhydrazine by means of an aldehyde group, we must suppose that in the presence of these reagents the oxide grouping is broken down and the aldehyde group regained. This fact is very readily understood if we suppose that every solution of glucose contains a trace of the aldehyde form, in equilibrium with the oxide forms. A reagent such as a metallic oxide or phenylhydrazine reacts with the trace of aldehyde form and thus LACTONE-STRUCTURE OF SUGARS 75 removes it from the solution; the oxide form is then no longer in equilibrium and therefore regenerates the aldehyde form in the process of regaining the equilibrium which has been disturbed. This fresh supply of the aldehyde form in its turn reacts and is removed from the solution, and so the process repeats itself until all of the sugar is used up. At the same time this view of the structure of the sugar enables us to understand why it is that although they give most of the aldehyde reactions, yet they give them much less energetically than the typical aldehydes. We may also ascribe to the same source the fact that the sugars do not react in stoichiometrical proportions with metallic oxides; in the proportions, that is, which would be expected if a molecule of sugar reacted quantitatively with a molecule of metallic oxide. We cannot predict, by merely writing down chemical equations, how much of any metallic oxide under given circumstances will be reduced by a given amount of sugar. Instead, for every concentration of sugar employed and for every circumstance of the reaction, we have to estimate afresh, and by direct measurement, the reducing power of the sugar. These measurements are commonly expressed in tables which denote the relationship of reduced cupric oxide (or other metallic oxide) to the quantity of sugar present in the solution investigated. But such tables are empirically established and are therefore reliable only if the cir- cumstances of concentration, reaction, temperature and so forth are exactly the same as those which prevailed in the estimations from which the tables were computed. REFERENCES. GENERAL: Armstrong: The Simple Carbohydrates and the Glucosides. London. 2d edition. Levene and Jacobs: Ber. d. d. Chem. Ges., 1910, 43, p. 3141. PENTOSES: Levene and Jacobs: Ber. d. d. Chem. Ges., 1908, 41, p. 2703; 1909, 42, pp. 1198, 2102 and 3247; 1911, 44, p. 746. Grund: Zeit. f. physiol. Chem., 1902, 35, p. 111. Bendix and Ebstein: Zeit. allg. Physiol., 1902, 2, p. 1. Clark, E. B.: Jour. Biol. Chem., 1917, 31, p. 605. PENTOSURIA: Garrod, A. E.: Inborn Errors of Metabolism. Oxford University Press, 1909. DECOMPOSITION OF SUGARS: Neuberg, A.: Oppenheimer's Handbuch der Biochemie, Erganzungsband, 1913, p. 569. Levene and Meyer: Jour. Biol. Chem., 1912, 11, p. 361; 1912, 12, p. 265; 1913, 14, p. 149; 1913, 15, p. 65. AMINO-SUGARS: Levene: Jour. Biol. Chem., 1917, 31, p. 609. CHAPTER IV. THE CARBOHYDRATES: THE DISACCHARIDES, POLY- SACCHARIDES AND GLUCOSIDES. THE DISACCHARIDES. The disaccharides are carbohydrates which contain twelve carbon atoms, and are formed from two molecules of hexose with the elimi- nation of water in accordance with the equation: C 6 Hi 2 O 6 + C 6 H 12 O 6 = Ci 2 H 2 2On + H 2 O. The majority of the disaccharides reduce Fehling's solution (i. e., cupric oxide in alkaline solution), react with phenylhydrazine to form hydrazones and osazones, and exhibit mutarotation in solution. They therefore contain a potentially aldehyde or ketone group or groups, and an oxide linkage analogous to that in glucose. Certain of them are exceptions to this rule, however, one of the most marked excep- tions being cane-sugar, which is formed by the union of one molecule of glucose with one of fructose (levulose), and which does not reduce Fehling's solution nor react with phenylhydrazine, nor display muta- rotation in solution. The disaccharides are merely special instances of a very large group of compounds which are generically termed Glucosides, or compounds of sugars with other bodies, the point of union being the aldehyde group of the sugar. A typical glucoside, for example, is Amygdalin, found in cherry-stones and in almonds, which on hydrolysis yields glucose, hydrocyanic acid and benzaldehyde. The nucleic acids are glucosides. Glucosides which yield galactose on hydrolysis are found in the tissues of the brain. The disaccharides are glucosides in which both constituents of the molecule are sugars. The most important disaccharides from the point of view of animal biochemistry are cane-sugar or Sucrose, Maltose, Isomaltose, Lactose and Isolactose. All of these excepting sucrose contain one potentially active aldehyde group; that is, they reduce Fehling's solution, form osazones and exhibit mutarotation. Cane-sugar is the ordinary sugar of commerce and occurs widely distributed in the vegetable kingdom, where it acts as a reserve-material, that is, as a store of nutriment to be broken up into utilizable material and consumed when needed. It occurs especially in the sugar-cane, in the sap of certain palms and of the sugar maple, the birch and the carob tree. Ripe fruits and many leaves contain considerable amounts DISACCHARIDES 77 of this sugar, while one of the most important sources of sugar is the root of the sugar-beet, a variety which has originated by selection from the common beet (Beta maratima). Cane-sugar was not known in Europe until its introduction from the tropical parts of Asia where the sugar-cane has been grown from time immemorial. The possibility of extracting cane-sugar from beets was not realized until it was pointed out by the German chemist Marggraff in 1760. Hence the large con- sumption of sugar now obtaining among European peoples is a recently acquired habit. It is, of course, of enormous nutritive importance as it results in reducing by an equivalent amount the requirement of starch and other polysaccharides. It also enables us, when sugar from the cane is used, to utilize tropical areas for the production of carbo- hydrate foodstuffs and set free greater areas of the temperate regions for the cultivation of polysaccharides and proteins (grains, meat and dairy products) for which the tropical areas of the world are not suit- able. The consumption of sugar from the cane is therefore economically preferable to the consumption of sugar from the beet. Cane-sugar does not reduce Fehling's solution nor does it exhibit mutarotation. It is neither potentially nor actually an aldehyde or a ketone. It is very readily hydrolyzed by acids, therein differing markedly from other disaccharides, and it yields on hydrolysis, one molecule of d-Glucose and one of d-Fructose (levulose). It will be recollected that d-fructose is levorotatory, and the levorotatory power of d-fructose being greater than the dextrorotatory power of d-glucose, the mixed products of cane-sugar hydrolysis are levorotatory. Cane- sugar, on the contrary, is dextrorotatory, so that hydrolysis of cane- sugar in solution leads to a change of optical rotation from right to left. Hence the process of the hydrolysis of cane-sugar is frequently termed Inversion. Cane-sugar is built up by the union of a molecule of d-glucose with one of d-fructose. The question arises, however, from which of the two d-glucoses is cane-sugar derived; the a-d-glucose or the /3-d -glucose? This question is answerable in a very simple way. It is possible to hydrolyse cane-sugar very much more rapidly than a-d-glucose can undergo transformation into /3-d-glucose or vice versa. It will be recollected that a-d-glucose possesses a much higher dextrorotatory power than /3-d-glucose. Now we find that the glucose produced in the hydrolysis of cane-sugar possesses, initially, a high rotatory power. On adding ammonia, which accelerates the transformation of a- into 0-glucose, the rotation due to glucose falls. Hence the glucose set free in the hydrolysis of cane-sugar is a-glucose, and cane-sugar is therefore to be regarded as a derivation of a-d-glucose. Cane-sugar does not react with phenylhydrazine. It contains eight hydroxyl groups, for it forms an octa-acetate, in which these groups have been replaced by acetyl groups. Apart from this it has not proved possible to ascribe any satisfactory constitutional formula to cane-sugar. The synthesis of cane-sugar has, however, been accom- 78 DISACCHARIDES, POLYSACCHARIDES AND GLUCOSIDES plished, by the interaction of potassium fructosate and acetochlor- glucose. 1 Cane-sugar is not attacked by any ferments excepting Invertase, an enzyme found in many yeasts, moulds, and in some of the higher plants. Invertase, as its name implies; effects the hydrolysis of cane-sugar into its constituent parts, glucose and fructose; that is, it brings about "inversion." Cane-sugar, or, rather, its product, glucose, does not undergo alcoholic fermentation in the presence of yeasts until it is broken down into glucose and fructose. Hence yeasts which do not contain invertase are not able to cause alcoholic fer- mentation in solutions of cane-sugar. Maltose is a disaccharide which results from the hydrolysis of starch or of glycogen by acids or by ferments. Acids, however, continue the process of hydrolysis by splitting the maltose itself, so that maltose is only a transient stage in the hydrolysis of starch or glycogen by acids. On the other hand the ferments which split starch or glycogen do not hydrolyze maltose, so that if maltose-splitting ferments be absent the process of hydrolysis ceases at this stage. Maltose is highly dextrorotatory, exhibits, mutarotation, reduces Fehling's solution and forms a phenylosazone. When hydrolyzed by acids it yields two molecules of glucose, but it is much less readily hydrolyzed by acids than cane-sugar. The ferment Diastase, which hydrolyzes starch and glycogen, the ferment Invertase which hydrolyzes cane-sugar, the ferment Lactase which hydrolyzes milk-sugar, and the ferment Emulsion which hydrolyzes amygdalin and isomaltose, are all without action upon maltose, which is hydrolyzed only by a ferment known as Maltase, found in many animal tissues and in the majority of yeasts. Maltose itself does not undergo alcoholic fermentation, and must first be split by maltase or by acids into glucose, but as the majority of yeasts contain maltose, these yeasts can accomplish the reduction of alcohol from maltose. The glucose which maltose yields upon hydrolysis is initially highly rotatory; on adding ammonia the rotation falls. Hence maltose is a derivative of a glucose. It is, in fact, glucose-/* glucoside. It can, of course, exist in two forms, according to whether the glucose moiety which still contains a potential aldehyde group is in the a or )8 form. The one maltose, a-maltose, is therefore a-glucose-a-glucoside; the other is /3-glucose-a-glucoside. Maltose can be synthesized from glucose by the condensing action of strong acids. But in addition to maltose another disaccharide is obtained by this process. This disaccharide is isomeric with maltose and yields, like maltose, two molecules of glucose on hydrolysis, differs from maltose in the characteristics of its phenylosazone, and also in the fact that it is not fermentable by yeasts. The ferment maltase, in fact, has no action upon it, while the ferment emulsion, 1 Marchlcwski in 1899. DISACCHARIDES 79 which is found in certain plant-tissues and which has no action upon maltose, hydrolyzes this sugar with the production of .two molecules of glucose. This glucose, unlike the glucose which is produced in the hydrolysis of maltose, is of low initial rotatory power. On adding a drop of ammonia to its solution the rotatory power increases. Hence, this sugar, which is called Isomaltose, is a derivative of 0-glucose. It is a mixture of a-glucose-/3-glucoside, and /3-glucose-/3-glucoside. Milk-sugar, also called Lactose, has not been encountered in the vegetable kingdom. It does not occur preformed in any item of our diet excepting in milk, nor does it appear likely that one of its con- stituent hexoses, galactose, is commonly obtainable from any other dietary source than milk. Of course it might be obtained from brain- tissue, but this cannot be regarded as a customary item of our dietary. Lactose yields, on hydrolysis, one molecule of d-glucose and one of d-galactose. Lactose exhibits mutarotation, reduces Fehling's solution, and forms a phenylosazone. Lactose is not hydrolyzed by maltase, invertase, diastase or emulsin, but it is hydrolyzed by a specific fer- ment designated Lactase, and found in the gastric mucous membrane and in a few yeasts such as Kephir yeast. This yeast is employed by the Arabs to make a sparkling alcoholic beverage, "Kephir," from the milk of mares. Milk-sugar is found in varying amounts in the milk of all mammals. During pregnancy it is often found in small quantities in the urine; and after weaning it also tends to escape for a few days through the kidneys. Extirpation of the mammary glands in milch-goats and cows gives rise to a notable increase in the amount of sugar in the blood (Glucohemia) and also to the appearance of glucose in the urine (Glycosuria) . It thus appears probable that in the mammary glands milk-sugar is formed from glucose alone, and not from glucose and galactose in the diet. On comparing the formulae of glucose and galactose: HCOH HCOH - ;: HCOH\ \ HCOH > < HOCH / \ HOCH CH CH HCOH HCOH CH 2 OH CH 2 OH d-glucose. d-galactose. it will be seen that the transformation of galactose into glucose involves the rupture of the oxide-ring and its closure again on the opposite side. 80 DISACCHARIDES, POLYSACCHARIDES AND GLUCOSIDES No enzyme has yet been isolated which is capable of bringing about this transformation. It is for this reason and possibly also for others connected with the metabolism of the intestinal bacteria, that maltose or cane-sugar can- not be regarded as satisfactory substitutes for milk-sugar in the diet of young infants. On the other hand, the assimilation-limit or quantity which may be ingested at once without leading to alimentary glycosuria, is lower for lactose than for the other sugars, so that if large quantities of sugar have to be given to make up the requisite calorific value of the diet, as in the case of fat-intolerant infants, maltose may be used as an accessory to lactose in the food. Cane-sugar being the disaccharide which is most foreign to animal tissues, and also the sweetest in taste, is much less suitable than milk-sugar or maltose for the dietary of young infants. The galactose which milk-sugar yields on hydrolysis is of low rotatory power, and its rotation increases on adding ammonia. Hence lactose is glucose-/3-galactoside, since it can be shown by forming the osazone of the sugar and hydrolyzing, when the phenylhydrazine remains attached to the sugar with the free (potential) aldehyde group, that it is the glucose radical which contains the potential aldehyde-group, the aldehyde-group of the galactose offering the point of union for the glucose molecule. The potential aldehyde-group of the glucose radical can exist either in the a- or the /3-form. The a-lactose (rotatory power = -f- 86) is therefore a-glucose-jft-galactoside, while ,8-lactose (rotatory power = + 35) is /3-glucose = 0-galactoside. No derivative of a- galactose is certainly known to occur in nature. If, however, kephir lactase be allowed to act upon a concentrated mixture of equal parts of glucose and galactose, two isomeric lactoses are produced, both exhibiting mutarotation, and both yielding d-glucose and d-galactose on hydrolysis. One of these is ordinary lactose, the other has been termed Isolactose and is possibly a mixture of a- and 0-glucose-a:- galactosides. Each of the disaccharides which contains a potentially active aldehyde-group can, therefore, exist in four different forms. Thus for maltose we have: a glucose-a-glucoside \ a- glucose-a-glucoside / ft- maltose a glucose-/3-glucoside \ a- . ., ft glucose-0-glucoside / ft- ^omaltose and for lactose we have: a glucose-a-galactoside \ a- . , ft glucose-a-galactoside / ft- lsolactose a glucose-/3-galactoside \ a- . ft glucose-0-galactoside / ft- lact< POL YSACCHA RIDES 81 These relationships are very important, and we shall have occasion to refer to them again in later chapters. Melibiose is a galactoside of glucose. It is derived from the trisac- charide raffinose by hydrolysis. POLYSACCHARIDES. We must now take up the consideration of the Polysaccharides, or carbohydrates formed by the union of more than two molecules of the simple sugars, with the elimination of a corresponding number of molecules of water. A few tri- and tetra-saccharides are tolerably well known and defined; of these the most important is Raffinose, CigH^Oie, a trisac- charide which is found abundantly in many plant-tissues and products, particularly molasses, eucalyptus-manna, wheat, barley, fungi, bacteria and yeast. It may be distinguished from cane-sugar by its greater solubility in methyl alcohol, and by the fact that it is split by emulsin, yielding d-fructose and melibiose, while cane-sugar is not attacked by this ferment. Hydrolysis by acids yields first d-fructose and melibiose, then the melibiose is hydrolyzed more slowly, yielding d-galactose and d-glucose. Raffinose does not reduce Fehling's solution. Raffinose is not split by animal tissue-extracts nor by any of the digestive juices with the exception of gastric juice which slowly inverts it owing simply to the fact of its acidity and not to any ferment con- tained in the juice. As the gastric contents are only distinctly acid for a brief period during digestion we may infer that this mode of splitting raffinose is of no nutritive significance since it must be of very trivial extent. A portion of the raffinose contained in the food is probably absorbed unaltered and excreted as such in the urine, the remainder with the exception of the very small proportion inverted in the stomach, remains unaltered until it reaches the large intestine (cecum) where it is rapidly inverted by the bacteria which inhabit this portion of the alimentary canal and is thus rendered available for nutritive purposes. We here meet with a phenomenon which is yearly growing of greater significance in our eyes, namely the Symbiotic Relationship between the mammals and the bacterial parasites which inhabit their intestines. While the bacterial flora of the intestines constitute a parasitic growth, yet their tenure of the intestine is not wholly to the disadvantage of the host, and through the multifarious enzymes which they produce these organisms render available to mammals foodstuffs which would other- wise be indigestible and excreted unaltered. It is probably for this reason that chickens and rats fed upon a strictly aseptic diet do not grow normally. While in the instance chosen, that of raffinose, the products thus rendered available may not be of indispensible impor- tance to the animal economy, yet in many cases, as for example in the splitting of chlorophyll by the intestinal bacteria, the products which 6 82 DISACCHARIDES, POLYSACCHARIDES AND GLUCOSIDES result (containing methyl-pyrrole groupings), may very possibly be unobtainable by mammals in any other way. The higher polysaccharides are very imperfectly defined. We have no reliable methods which are available for determining their molec- ular weights, and we do not know, therefore, how many molecules of sugar take part in their formation. The group is a very large one, and the general formula (C 6 H 10 O 5 )n may be ascribed to the majority of its best-known members, indicating that they are formed by the union of an indefinite number, n, of hexose anhydrides. The following sub- stances are important and typical members of the group: Starch, Glycogen, Dextrins, Inulin, Pectin, Humin, Cellulose, Gums, and Vege- table Mucilages. It is very important to recollect, however, that these are merely arbitrary terms used to describe very ill-defined members of the series. Thus we cannot be certain that there is only one chemical individual Starch ; on the contrary, it appears probable that there may be many starches, and starch is certainly known in two widely different forms, to wit : a form insoluble in water and a form which is soluble in water. On the other hand it should be recollected that the differences which are observed between these forms of starch may possibly be purely physical, and not chemical differences at all. We here encounter, in fact, a problem which is presented generally by the colloids, and which we shall meet with again in connection with the proteins. Starch, inulin, gums, mucilages and glycogen do not reduce metallic oxides in alkaline solutions. They do not, therefore, contain potentially active aldehyde-groups. Dextrins, on the contrary, do contain alde- hyde-groups, for they reduce Fehling's solution. With the possible exceptions of glycogen and inulin, the polysaccharides do not form crystals, or at least, they have not as yet been prepared in crystalline form. Water dissolves some of them, others only swell in cold water and dissolve in hot water, others are unaffected by water. Solutions of the polysaccharides do not taste sweet unless held in the mouth for a sufficient period to enable the diastase (Ptyalin) in the saliva to bring about hydrolysis. Solutions of the polysaccharides are optically active. The higher polysaccharides do not diffuse through parchment paper, thus behaving typically as colloids. They do not form com- pounds with phenylhydrazine. The polysaccharides play a wide variety of parts in the vegetable kingdom. In the first place, they serve as reserve materials, or stores of sugar, laid up against a future time of need; such a part is that played by starch (or vegetable glycogen, as it may be called in analogy to animal glycogen, which plays a similar part in the animal economy) and also by inulin. The gums and mucilages, on the contrary, serve, in part at least, to close up injuries and protect them while healing. The celluloses, again, have yet another function to perform. They, or their derivatives, constitute the supporting tissues of plants, just as bones or exoskeletons constitute the supporting tissues of animals. The Celluloses (the plural is employed because it appears highly POLYSACCHARIDES 83 probable that there are many modifications of cellulose), are insoluble in all ordinary solvents, such as water, alcohol, ether, and so forth. Cellulose dissolves, however, in solutions of many metallic salts in the presence of excess of strong acid, for example in zinc chloride in acid solution, and in the hydrochloric acid solutions of antimony, mercuric, or bismuth chlorides. The requisite condition for solution appears to be, the presence of a salt of a weak metallic base in acid solution. Another solvent for cellulose is an ammoniacal solution of cupric oxide, known as " Schweitzers' Reagent." In the presence of con- centrated sulphuric acid, sulphuric-acid esters of cellulose are formed and pass into solution. If this solution be diluted and boiled, glucose is formed and glucose only, hence cellulose is an anhydride of glucose. A preliminary stage in this hydrolysis is the formation of Amyloid, a soluble colloidal substance which resembles starch in yielding a blue color with iodine. Cellulose is indigestible by any of the ferments contained in or produced by mammalian tissues. It is, however, digestible by bacteria, and as much as seventy per cent, of unlignified cellulose may be dis- solved in vitro by the juices from the lower intestine of the horse. The products of this form of digestion are not sugars, but carbon dioxide, methane and fatty acids. Human beings have been found to utilize as much as forty per cent, of young and tender cellulose, doubtless through the agency of the intestinal bacteria. Hence the nutritive value of cellulose, especially in animals such as the cow and horse which possess very long intestines, is by no means negligible. But the celluloses are of significance to the animal economy from yet another point of view. By virtue of their incomplete digestibility they communicate bulk and substance to the f eces and thus facilitate their passage through the intestines, in the first place by bringing about a favorable distention of the intestinal muscular walls, and in the second place by furnishing these muscles with material upon which to exert leverage. Prior to the introduction of "War-breads" the ten- dency of our times was to eliminate indigestible carbohydrates more and more thoroughly from the diet and the prevalence of intestinal stasis and chronic constipation in modern communities is doubtless attri- butable, in part at least, to this "refinement" of our foodstuffs. A crude endeavor to correct this deficiency in our diet is frequently made by mixing bran or other coarsely ground cellulose-rich materials with the flour from which bread is made. This remedy may in many instances, however, be worse than the disease, for the ingestion of large, horny and sharp-edged indigestible fragments with the food may lead to lacerations of the intestine, and consequent inflammatory reactions or enteritis. What is required is finely ground cellulose-rich material, such as our ancestors enjoyed when they ground up their grains by hand between two hard stones. Agar is frequently employed to com- municate indigestible bulk to the diet or, in recent years, heavy taste- less petroleum oils, but in administering these substances we are merely 84 DISACCHARIDES, POLYSACCHARIDES AND GLUCOSIDES striving to remedy the consequences of a totally unnecessary dietary habit which arises from a threefold origin of public ignorance, a fancied superiority of things which are white, and therefore "pure," white bread, white eggs, white (i. e., sulphured) dried fruits, white sugar (made to appear white by the addition of litmus) and so forth, and in the irresponsible self-interest of millers and bakers. The War, through the introduction of more thorough utilization of grains to make "War flour" and "War bread" has thus no doubt proved a veritable blessing in disguise to many chronic sufferers and it is not improbable that the reinstatement of our former foolish and wasteful habits of milling will be prevented or delayed by a more general public recog- nition of the beneficial role of indigestible residues in the food. Cellulose occurs almost exclusively in the vegetable kingdom. It is found, however, in the shells of Tunicata. Otherwise it is unknown in the animal kingdom. In the cell- walls of plants, not only true cellu- lose is found but other cellulose-like substances, some of which yield not only glucose, but other sugars, even pentoses such as arabinose, xylose, etc. Polysaccharides which yield only pentoses on hydrolysis are also found, and are known as Pentosans. As the cell-walls of plants advance in age they undergo a peculiar change resulting in the acquirement of greater rigidity. This process is known as Lignification. The exact nature of the change which occurs is not known, but it has been suggested that Lignin is formed from cellulose by the formation of compounds with aromatic derivatives. The vegetable gums and mucilages are a very heterogeneous group of polysaccharides. The gums are insoluble, the mucilages soluble in water. The majority of them yield galactose and arabinose when hydro- lyzed by dilute acids. Agar-agar, so widely used in culture-media for bacteria, is a representative of this class of carbohydrates; it is derived from certain marine algae. From marine algae of the Fucus type is also obtained a polysaccharide yielding pentoses on hydrolysis which is designated Algin. It is a colloidal substance which behaves like a weak acid, forms insoluble salts of aluminium and lime, and is employed as a waterproofing material and a substitute for size. Closely related to the gums and mucilages is a group of substances, the Pectins, which are of very great industrial importance inasmuch as they are responsible for the gelation of fruit-jellies. The pectins are white amorphous gelatinous substances, which form colloidal solu- tions in water, do not reduce Fehling's solution, and yield galactose, glucose and pentoses on hydrolysis by acids. They are believed to be derived by partial hydrolysis, due to the organic acids present in fruit-extracts, from a series of parent-substances, the Pectoses, which are present in plant-tissues in the form of insoluble calcium salts. The pectins are converted by dilute alkalies or by the ferment Pectase into Pectic Acid, the calcium salt of which is insoluble in water and forms jellies. Since pectase is destroyed by heat, the formation of fruit-jellies by extracting fruits with hot sugar-solutions is not to be POLYSACCHARIDES 85 attributed to the action of pectase, but rather to the production of insoluble jelly-forming substances from pectose or pectin by the hydrolyzing action of the fruit-acids. The pectins are not hydrolyzed by diastases, they are, however, hydrolyzed by special enzymes, the Pectinases, found in malt and in certain moulds which liquify pectin jellies with the production of reducing sugars. Coming now to those polysaccharides which are primarily of nutri- tive importance, Starch is the form in which sugar is chiefly stored up by plants for future consumption, although cane-sugar, inulin and other carbohydrates frequently play a similar part. Starch is found in the greatest amounts in those portions of plants which are subsequently to be drawn upon for the materials of growth. Thus seeds, roots, bulbs, tubers and the pith of deciduous trees in winter are particularly rich in starch, this carbohydrate frequently comprising as much as eighty per cent, of the dry weight of the material. The starch is stored up in these tissues in the form of stratified granules, which differ characteristically in form and size in different plants. It is by means of these characteristics of form, size and stratification of the granules that we can tell very readily whether a starch alleged to have been derived from one specified source has or has not been adulterated in the pursuance of "legitimate business enterprise" with starch derived from some other and cheaper source. The concentric rings, or stratifications of starch-grains represent their gradual growth, and intimate that the growth of starch-grains takes place rhythmically, periods of desposition alternating with periods of rest. Starch is only slightly and very slowly changed by cold water, but in hot water the grains swell up and finally burst, forming what is known as "starch-paste." Neither starch nor starch- paste reduces metallic oxides in alkaline solution. A very familiar test for starch is the formation of a very deep indigo- blue coloration when it is acted upon by iodine solutions in the presence of hydriodic acid or of an iodide. The color disappears on boiling and reappears on cooling. In applying this test it is necessary to remember that it is not given in the presence of excess of reagents which are oxidized by iodine, such reagents, for example, as hydroxides of the alkalies, or sulphurous or arsenous acids. It is in connection with this test that we meet with very clear indications that starch is not a homogeneous chemical unit, for varieties of starch are known which do not give a blue, but a reddish-brown or a "port-wine" color with iodine. We do not know to what these colorations are due, or whether they are specific, i. e., yielded by one chemical individual alone, or generic, i. e., yielded by a group of similar chemical individuals. On boiling starch with dilute mineral acids, glucose and only glucose is obtained. Starch is therefore an anhydride of glucose. If the acid is allowed only to act upon the starch in the cold, or with very gentle heating, a modification of starch, known as "soluble starch" is obtained. If we act upon starch for several weeks with cold dilute mineral acids, 86 DISACCHARIDES, POLYSACCHARIDES AND GLUCOSIDES or for an hour with four per cent, sulphuric acid at 80 (/., we obtain "Amylodextrin," which yields a port-wine coloration with iodine. Further hydrolysis of the amylodextrin yields a mixture of simple dextrins which give no color with iodine (" Achroodextrin") ; still further hydrolysis yields Glucose, an intermediate product of hydrolysis being Maltose which, however, in the acid-hydrolysis of starch, is immediately broken down into glucose, so that in the hydrolysis of starch by acids maltose is only transiently present in the system. In the hydrolysis of starch by diastatic ferments, however, unless Maltase be also present, the final product of hydrolysis is the disaccharide maltose, the inter- mediate stage of hydrolysis being so far as we know, similar to those observed in the hydrolysis of starch by acids. The hydrolysis of starch takes place, therefore, step by step, with the production of intermediate stages of hydrolysis before the final product, glucose, is obtained. We shall meet with analogous phenomena among the proteins, and if we draw a parallel, which is of course only justifiable in a formal, not in a chemical sense, between the hydrolysis of starch and the hydrolysis of proteins, then we would have the following table of analogues: Starch analogous to Proteins "Soluble starch' Amylodextrin Achroodextrins Maltose Glucose Albumoses Peptones Polypeptids Dipeptids Amino-acids Inulin, a polysaccharide found in the tubers of dahlias, and in other situations, bears the same relationship to fructose that starch does to glucose. On hydrolysis by acids it yields only fructose; it is not hydrolyzed by any of the diastatic ferments which hydrolyze starch or glycogen. It is> however, hydrolyzed by a special ferment Inulinase. Inulin differs very markedly from starch, in that it dissolves readily in warm water with the formation of a solution instead of a paste, and it yields a yellow color with iodine. In various other situations in the vegetable kingdom other poly- saccharides resembling starch and inulin are found, differing from these, however, in certain characteristics. Thus we have Amylin, Lavosin, Cerosin, and Secalin, etc., found in grain-seeds, some of which yield glucose on hydrolysis, others fructose. In Lupinus luteus is found Galactin, a polysaccharide which yields only galactose on hydrolysis. In Lichens is found a polysaccharide, Lichenin, which yields only glucose on hydrolysis by acid, but which, curiously enough, is not hydrolyzed by diastatic ferments. It yields a yellow color on treat- ment with iodine. Glycogen is to the animal economy what starch is to that of the plant. It was observed by the distinguished French investigator, Claude Bernard, in 1848, that the sugar-content of the liver, excepting after starvation, is very high. He further found that the sugar which the POLYSACCHARIDES 87 liver yields on standing is not present as such, but in a form resembling starch, which is rapidly hydrolyzed by enzymes contained in the tissues, or by acids, yielding glucose. Glycogen may be prepared from fresh liver by extracting the tissues with strong potassium hydroxide solu- tion, which decomposes the proteins but does not hydrolyze the glycogen, and then precipitating with alcohol. If the liver be allowed to stand before extraction, a much smaller quantity of glycogen will be obtained, and simultaneously it will be found that sugar has appeared in the liver. If the liver be heated to boiling before being allowed to stand, the glycogen does not disappear and no increase in the sugar content of the liver is observed. Evidently, therefore the disappearance of glycogen in the liver on standing is due to the action of a hydrolyzing ferment which is destroyed or inactivated by heating. Glycogen, although like starch, an anhydride of glucose is never- theless readily and sharply distinguishable from starch. It forms when pure a fine white amorphous powder. Its molecular weight is unknown. It dissolves in cold water, forming opalescent solutions, but it is a typical colloid and does not diffuse through parchment. With iodine glycogen yields a reddish-brown or port-wine coloration which dis- appears on heating and reappears on cooling. The hydrolysis of glycogen, like that of starch, takes place in step- like stages. Intermediate products of hydrolysis are dextrins and maltose. In the absence of maltase the diastatic ferments hydrolyze it as far as the maltose-stage and then their action stops. It is not by any means certain that there is only one glycogen or that there are not a variety of different reserve-carbohydrates in animal tissues, but if this is the case no means has yet been found of positively separating and identifying them. Glycogen is found in a variety of tissues, but the chief storehouses in the vertebrates are the liver and the muscles. In invertebrata glycogen occurs in organs which correspond in function to the liver. It also occurs in the protoplasm of unicellular animals and is abundant in yeast. It appears never to occur in the nucleus. The glycogen which is stored up in the striated and smooth muscles of the vertebrata is of peculiar significance, in that it stands quantita- tively in direct relation to the work which the muscles perform. As the muscles do work, glycogen disappears from them. As might be expected, therefore, the percentage of glycogen in muscle varies very much in different animals and under different conditions. The follow- ing figures, given by Cramer, show this very clearly: Glycogen, Animal. Muscle. per cent. Dog Number 1 Dog Number 2 Dog Number 3 Dog Number 4 Biceps brachii 0.17 Quadriceps femoris 0.53 Biceps brachii 0.25 Quadriceps femoris 0.32 Dorsal muscul ature 0.135 Posterior adductors . 077 Dorsal musculature 0.417 Posterior adductors . 444 88 DISACCHARIDES, POLYSACCHARIDE8 AND GLUCOSIDES Glycogen is also found in glandular, epithelial and connective tissues and in the brain. The distribution of glycogen in the body is very variable; the following figures were obtained by Schondorff, employing dogs which had been well fed with carbohydrates and meat shortly before death : One hundred grammes of glycogen were distributed in different parts of the body in the following proportion in seven dogs employed : Minimum Minimum observed. observed. Average. Blood 0.04 0.001 0.015 Liver 56.74 20.09 37.97 Muscle 62.55 31.22 44.23 Bone . 12.88 5.36 9.25 Skin 11.38 1.42 4.49 Viscera 7.30 0.38 3.81 Heart 0.28 0.08 0.17 Brain 0.23 0.04 0.09 It will be observed that the heart-muscle, which is in continual activity, contains very little reserve-stock of carbohydrates. It is evidently unable to accumulate a reserve or capital of carbohydrate and maintains its activity upon its current income. With this may be correlated the fact that after each beat of the heart a definite and relatively lengthy period occurs, the "refractory period" during which even the application of stimuli fails to elicit a contraction from the heart-muscle, whereas ordinary striated muscle, containing abundant stores of reserve carbohydrate, may be stimulated repeatedly at exceed- ingly brief intervals until relaxation between the contractions becomes a mechanical impossibility, and the contractions fuse into one "tetanic" contraction which relaxes only when the muscle becomes exhausted and its stores of glycogen depleted. It will be noted, also, that the percentage of glycogen in the blood is extraordinarily low. In fact it appears that the only form in which carbohydrate material circulates in the Vertebrata is that of glucose, and that this is also the only form in which carbohydrate food is utilized by the tissues for the production of energy, or the manufacture of reserve-materials. Now the carbohydrates of the food are usually ingested in the form of starch, glycogen, and other polysaccharides, or in the form of disaccharides, such as cane-sugar or lactose, and these carbohydrates are readily utilized by the organism. Preparatory to utilization therefore, these carbohydrates must undergo elaborations and transformations resulting in the formation of glucose. AMINO -POLYSACCHARIDES. The hydrolysis of proteins which contain a glucosamin radical yields in some instances an amino-disaccharide, presumably diglucosamin. The most important amino-polysaccharide in the animal economy is, however, Chitin, which forms the exoskeleton of the Jnsecta and the GLUCOSIDES 89 Crustacea. It may be obtained in colorless semi-transparent lamellae which are stained reddish-brown by iodine; on addition of sulphuric acid or zinc chloride the color changes to blue or violet. Hydrolysis with strong acids yields about seventy-five per cent, of d-glucosamin. Chitin also contains acetyl radicals which are liberated as acetic acid on fusion with alkali. Prolonged treatment with alkali in the cold leads to the formation of " soluble chitin" which is diffusible through parchment, but has an extraordinary affinity for water, carrying the water in the dialyzer with it as it traverses the parchment, and withholding it from the cavity of the dialyzer against hydrostatic pressure. Other products of the partial hydrolysis of chitin are crystallizable (chitosans). GLUCOSIDES. The glucosides are a large and important class of substances, occur- ring in great variety in certain vegetable tissues, and also in exceedingly important tissues and localities in the animal body. They yield monosaccharides on hydrolysis and other radicals which differ widely in different glucosides. Reference has already been made to the glucoside Amygdalin which occurs in the kernels of cherries and almonds and is hydrolyzed by the ferment Emulsin, yielding glucose, hydrocyanic acid and benzaldehyde. Various species of the Cruciferce contain irritant glucosides, notable among which are Sinigrin or Potassium Myronate in the oil of black mustard, obtained from the seeds of Sinapis nigra, and Sinalbin, in the oil of white mustard, obtained from the seeds of Sinapis alba. Both sinigrin and sinalbin are hydrolyzed in the presence of water by a ferment, Myrosin, which occurs in the tissues of the plants from which they are obtained. The products yielded by the two glucosides are, however, very different, sinigrin yielding dextrose, potassium bisul- phate, and allyl isosulphocyanate, while sinalbin yields dextrose, sinapin sulphate (a sulphate of an alkaloid) and methyl phenyl isosulpho- cyanate. Both glucosides are intensely irritant when applied to the skin, and are utilized for this purpose in therapeutics. Glucosides of great therapeutic importance are also found in the leaves and seeds of Digitalis purpurea, Strophanthus and Scilla, and comprise the most important active constituents of the pharmacopceial preparations made from these plants. They have a characteristic action upon heart-muscle of which advantage is taken in the medical treatment of cardiac affections. The same plants also contain gluco- sides which are either without effect upon the heart, or else have an effect which is of secondary importance. Some of these glucosides are members of the saponin series and contribute to the effectiveness of aqueous extracts of the plants by holding in solution substances which would otherwise be insoluble in water. 90 DISACCHARIDES, POLYSACCHARIDES AND GLUCOSIDES From a biochemical point of view, and in our present state of knowl- edge, perhaps the most noteworthy glucosides which occur in plant- tissues are the various members of the Saponin and Sapotoxin group of glucosides These substances are found in a very great variety of plant- tissues, but especially in Quillaja, (soapbark), Saponaria (soapwort), Cyclamen (cyclamin), Solatium (nightshade and potato) and Smilax (sarsaparilla). These glucosides behave like weak acids and are split on hydrolysis with acids into sugars and other substances which are for the most part, as yet undefined. They possess to a very remarkable degree the property of reducing the surface-tension at surfaces in con- tact with water in which they are dissolved and coating these surfaces with an insoluble film, with the result that the forces tending to cause coalescence of bubbles are very much reduced, so that the water con- taining saponins form froths like soap-solutions, when it is shaken up with air. Hence the names "soapbark," " soapwort," etc. For the same reason they have the property of holding otherwise insoluble substances in solution or suspension, since the suspended particles have less tendency than usual to clump together and thus form masses large enough to fall out of the solution. The saponins and solanins readily dissolve or form colloidal solutions of a variety of fatty substances, particularly the Lecithins, an important group of phosphorus-containing fatty substances which will fall under discussion repeatedly in future chapters. They also form, in many cases, insoluble compounds with Cholesterol, an aromatic alcohol, which is found associated with lecithins in all living tissues. The power of the saponins to dissolve fatty substances is undoubtedly the origin of their remarkable action upon red-blood corpuscles, the stroma of erythrocytes being very rich in lecithins and other fatty substances. As little as one part of cyclamin added to 100,000 parts of blood causes complete liquefaction or Hemolysis of the stroma of the corpuscles with resultant setting free of the enclosed hemoglobin, while liquefaction of a proportion of the corpuscles is brought about by even smaller amounts. Cholesterol tends to prevent this action of the saponins by combining with them to form insoluble compounds, and hence blood serum or plasma, since it contains a small amount of cholesterol, to some extent inhibits the hemolytic action of the saponins. A saponin, digitonin, which occurs in Digitalis but is devoid of action upon the heart, is employed in the quantitative estimation of cholesterol. In animal tissues glucosides are found especially among the decom- position-products of Nucleic Acids and in the tissues of the brain. The nucleic acids will fall under special consideration in a later chapter and it need merely be stated here, in passing, that they are phosphoric acid compounds of glucosides, the Nucleosides, which yield either d-glucose or d-ribose and nitrogenous bases on hydrolysis. A nucleo- side is also found in minute traces in the blood and exerts an action upon the egg-cells of the Sea-urchin (Strongylocentrotus) similar to that CARBOHYDRATE ESTERS 91 of a saponin; it is, however, devoid of lytic action upon the red blood- cells themselves. The glucosides in the brain, the Cerebrosides, occur in complex fatty compounds which yield the free glucosides, Phrenosin and Kerasin oil partial hydrolysis. They also exist in part preformed in brain-tissue, or at any rate can be directly extracted therefrom by solvents such as pyridine or hot alcohol containing benzole or chloroform. These substances are not confined to nervous tissue but are also present in small amounts in the kidney and liver and probably in other organs as well. They also occur in the yolks of eggs. The cerebrosides are nitrogen-containing substances which are hydrolyzed by acids, yielding fatty acids, galactose, and a nitrogenous base, Sphingosine, which is a diatomic amino-alcohol containing unsatu- rated linkages: : CH.CHOH.CHOH.CH 2 NH 2 The fatty acid which is yielded by Phrenosin is a hydroxy-acid, Cerebronic Acid, C25H 5 oO 3 , while Kerasin yields Lignoceric Acid, C^JH^sC^. The two cerebrosides differ furthermore in their solubilities, phrenosin being almost insoluble in boiling acetone, while kerasin is readily soluble. Both cerebrosides are insoluble in water or in ether, but they dissolve in hot alcohol, from which they crystallize in needles or plates on cooling. Solutions of phrenosin are dextrorotatory, those of kerasin being levorotatory. With sulphuric acids the cerebrosides yield, first a yellow, and later a purple-red coloration. In the presence of cane-sugar and sulphuric acid, they yield a purple coloration immediately. This reaction is attributable to the sphingosine radical. Cerebrosides are absent in the brains of fetal animals, but with the advance of medullation they appear in abundance. It is therefore assumed that the cerebrosides are constituents originating in medullary sheaths rather than in the axons or nerve-cells. THE CARBOHYDRATE ESTERS. Phosphoric acid esters of d-glucose and d-ribose occur among the products of the partial hydrolysis of nucleic acids. They will fall under more extended consideration in a later chapter. Sulphuric acid esters, the Glucothionic Acids have been found by Levene and Mandel in a variety of animal tissues, the nature of the carbohydrate radical is not yet established. A sulphuric acid ester of an amino-polysaccharide, Chondroitin- Sulphuric Acid, CigH^NOisHSCX occurs in important amounts in bones and other sclerous tissues and also in the walls of the great arteries and in certain pathological tissues. It is a normal constituent of urine in very small amounts. It is soluble in water, yielding levorotatory 92 DISACCHARIDES, POLYSACCHARIDES AND GLUCOSIDES solutions, and is precipitable from aqueous solutions by alcohol. Hydrolysis by dilute hydrochloric acid yields sulphuric acid and Chondroitin: + H 2 Ci 8 H 27 NOi4 + H 2 SO 4 chondroitin sulphuric acid + water = ohondroitin -f- sulphuric acid. Chondroitin reduces Fehling's solution. On further hydrolysis it yields d-galactose and d-glucuronic acid; it appears to be a compound of glucuronic acid and an amino-hexose, Chondrosamin, or amino- galactose. REFERENCES. GENERAL: Armstrong: Simple Carbohydrates and the Glucosides. London. 2d edition. SYMBIOSIS: Schottelius: Arch. f. Hygiene, 1898, 34, p. 210; 1902, 42, p. 48; 1908, 67, p. 177. Nuttal and TUerf elder: Zeit. f. physiol. Chem., 1895, 21, p. 109; 1896, 22, p. 62. Armsby: U. S. Bureau of Animal Industry, Bull. 139, 1911. McCollum and Davis: Jour. Biol. Chem., 1915, 20, p. 641. Kuriyama and Mendel: Ibid., 1917, 31, p. 125. AMINOPOLYSACCHARIDES AND CARBOHYDRATE ESTERS: Alsberg and Hedblom: Jour. Biol. Chem., 1909, 6, p. 483. Morgulis: Am. Jour. Physiol., 1917, 43, p. 328 (chitin). Mandel and Levene: Zeit. f. physiol. Chem., 1905, 45, p. 386; Biochem. Zeit., 1907, 4, p. 78. Levene and La Forge: Jour. Biol. Chem., 1913, 15, p. 155; 1914, 18, pp. 123 and 237; 1915, 20, p. 433. Levene: Ibid., 1916, 26, p. 143. Levene and Lopez-Sudrez: Ibid., 1916, 25, p. 511; 1916, 26, p. 373. Levene: Ibid., 1917, 31, p. 609. CHAPTER V. THE HYDROAROMATIC DERIVATIVES: THE CYCLOSES, CHOLESTEROL AND CHOLIC ACID. GENERAL CHARACTERISTICS. A class of bodies here claims our consideration, the members of which, while chemically distinct, are, in their physical behavior and physiological properties intermediate in character between the carbo- hydrates and the fats. At the one extremity we have the cy closes, which although polyatomic alcohols, nevertheless resemble sugars in their solubility in water, their percentage-composition which is repre- sented by the formula CeH^Oe, and their decidedly sweet taste. At the other we have cholesterol and the cholesterol esters or waxes which resemble the fats in their insolubility in water and solubility in organic solvents, and which are constantly associated with fats and fatty substances in the tissues in which they occur. They all contain a reduced benzole-ring and are thus related to the Terpenes; they are furthermore hydroxy-derivatives and thus yield a variety of color- reactions which depend upon the presence of a hydroxyl radical in the benzole -ring. The extreme importance of these substances in the life of tissues has only very recently come to be suspected, but the variety of parts they are now known to play in essential activities of the living cell is so extensive that we have come to regard them as constituting a very significant factor indeed in the life-economy. Thus Inosite in combina- tion with phosphoric acid is an important constituent of seeds and the rapidly growing parts of plants, while in animal tissues inosite is found in a variety of situations and forms an integral part of the molecule of the active principle of the anterior lobe of the Pituitary Gland. Cholesterol is found wherever fats occur in animal tissues, and the remarkable effects which it exercises upon the growth of epithelial tissues, 1 show that it plays an important physiological role. Choles- terol esters or Waxes occur in abundance in vegetable tissues, while in mammals they occur in noteworthy amounts in the fatty sheaths of medullated nerves, and in the cortex of the Suprarenal Gland. Cholic Acid, which is probably a derivative of cholesterol, occurs combined with amino-acids (amino-acetic acid or ethyl amino-sulphonic acid) in the bile, and the salts which these acids form with sodium, play an essential part in accomplishing the digestion and assimilation of fats. 1 Cf. Chapter xx. 94 CYCLOSES, CHOLESTEROL AND CHOLIC ACID It is questionable whether animal tissues are able to accomplish the synthesis of any of these substances; in fact all the evidence at present available contributes to show that they cannot, and that we are absolutely dependent upon vegetable tissues for our supplies of these very essential materials. The investigations of Gardner, Denis Chalatov and Anistchakov have shown that addition of cholesterol to the dietary in abnormal amounts, increases the cholesterol-content qf the tissues, while a diet extremely deficient in cholesterol-results in a like deficiency of cholesterol in the blood and tissues. On the other hand, in vegetable tissues terpenes and terpene-derivatives abound so that the ultimate source of cholesterol in the diet w^ould appear to reside in these products of the synthetic activity of plants. The power of the animal organism to destroy cholesterol is very limited, and if a considerable excess be administered in the diet, the unutilized cholesterol is stored away in various tissues, particularly in the liver, spleen and suprarenal bodies. In certain animals, for example rabbits, but not in others, the excess of cholesterol is in part deposited in the interior of the arterial walls, leading to the formation of lesions, which simulate arteriosclerotic lesions of the arteries in human beings. The normal channel of excretion of cholesterol would appear to be the bile, in which it is present in part in the form of unal- tered cholesterol, and in part in the form of cholic acid, combined with amino-acetic acid or amino-ethyl-sulphonic acid to form the "bile- acids." Both of these substances are in part reabsorbed from the intestine, so that there is a tendency for cholesterol and its products to circulate in the body, and accumulate in the tissues. Of course this process cannot go on unchecked, otherwise the accumulations of cholesterol in the tissues would soon extinguish their functional activities. It appears possible from the abundance of cholesterol esters in the suprarenal cortex, particularly during cholesterol over- feeding, that the suprarenal glands may play a part in assisting to eliminate or destroy cholesterol, but regarding the nature of the ultimate products which may be formed in this process we are entirely in the dark. Inosite, on the other hand, which contains within itself a much higher proportion of oxygen than cholesterol, is partially oxidized by animal tissues and the products of its oxidation appear to be indistinguishable from those of carbohydrate-metabolism. Not even inosite, however, and still less cholesterol are of importance from the purely nutritive aspect, i. e., as sources of energy. The calorific value of the hydro-aromatic fraction of the diet is so small as to be negligible in comparison with the total. Their significance lies elsewhere, and if we revert to the analogy of inanimate machines we must class them with the lubricants and other accessory substances which are essential to the smooth running of the machine, rather than with the fuel which supplies the energy of the machine. Indirectly, indeed, they must contribute to the available energy-value of the diet by permitting its more efficient consumption, just as the judicious CYC LOSES 95 employment of lubricants will diminish the necessary consumption of gasoline in an automobile-engine. Their influence upon the nutrition of animals is indirect, however, and not direct, and the hydro-aromatic derivatives must for this reason be classified as Accessory Foodstuffs or foodstuffs which are primarily utilized for other purposes than the production of work and heat, or the building up of the structural elements of tissues. THE CYCLOSES. The hydro-aromatic compounds which lie nearest to the carbo- hydrates in their physical properties and physiological behavior are the Cy closes, or hexa-hydroxy-benzoles, which are represented by the formula : CHOH HOHC CHOH HOHC CHOH \/ CHOH A number of isomeric compounds are represented by this formula, differing from one another in the arrangement of hydrogen and hydroxyl groups about the carbons. The form which occurs in animal tissues is optically inactive, the levo- and dextrorotatory carbons being balanced and equalized within the molecule. This cyclose is designated Inosite. In vegetable tissues it is found widely distributed, occasionally in the form of ester-like compounds (dambonite, bornesite), but chiefly in the form of the hexaphosphate, the calcium-magnesium salt of which is known commercially as Phytin. This substance occurs particularly abundantly in seeds and grains, the husks of which also contain a fer- ment, Phytase, which is capable of splitting the compound, in aqueous solution, into inosite and phosphoric acid, a hydrolysis which other- wise can only be accomplished completely by exposing the substance in acid solution to a temperature very considerably above that of boil- ing water. Intermediate steps in the hydrolysis of inosite hexaphos- phate by phytase are the tri- and mono-phosphates which do not, however, occur preformed in the tissues of grains. In mammals i-inosite is found in small amounts in muscular tissue, from which it was first obtained and recognized as a distinct chemical entity. It is also found in combination with a complex fatty substance, containing phosphorus and nitrogen, in the tissue of the anterior lobe of the pituitary body. This compound, to which the name Tethelin has been applied, is probably the physiologically active principle of the gland. On somewhat prolonged hydrolysis by alkalies and acids the substance breaks up and yields free i-inosite. Inosite is readilv soluble in water and alcohol and is obtained in the 96 CYCLOSES, CHOLESTEROL AND CHOLIC ACID form of fine white acicular crystals by the addition of ether to an alcoholic solution. It has a sweet taste, but being neither actually nor potentially an aldehyde or ketone, it does not reduce metallic oxides in alkaline solution, and hence, of course, does not reduce Fehling's solution. It is precipitated from aqueous solutions by lead acetate containing an excess of lead oxide ("basic lead acetate"). Inosite may be recognized by the above peculiarities, by its melting- point (225), and by the following characteristic reactions: Gallois' Reaction. A drop of inosite solution is mixed with a drop of mercuric nitrate solution and heat gently applied until the water has evaporated. A yellow color at first appears which changes on further heating to a deep red. This color disappears on cooling, and reappears on reheating. Scherer's Reaction. A few crystals of inosite are dissolved in a drop or two of nitric acid of specific gravity 1.2, and an equal volume of ten per cent, calcium chloride solution is added and the same volume of a one per cent, solution of platinic chloride. This mixture is evaporated to dryness and the residue heated, when a rose-red color appears, which disappears on cooling, and reappears with a bluish nuance on reheating. Inosite is found in small amounts in normal urine, and the amount increases in certain pathological conditions, particularly in diabetes insipidus and in Bright's disease. The administration of inosite in unusual amounts by mouth gives rise to transient diarrhoea and to an increase in the Creatinine output in the urine, a fact which, in the light of considerations which will be detailed in subsequent chapters, may possibly indicate increased destruction of tissue-substances. Only a very small proportion of the inosite administered by mouth is excreted in the urine, the remainder being oxidized and eliminated in the form of products which are apparently indistinguishable from those of ordinary carbohydrate-combustion. In phloridzinized dogs the excre- tion of d-glucose in the urine, already a maximum, is increased by administration of inosite, and if the ^additional output of glucose be added to the inosite which is excreted unchanged in the urine, the sum is approximately equal to the inosite administered. Under these circumstances, therefore, the ring-formation appears to undergo a simple splitting with the partial transformation of inosite, molecule for molecule, into glucose. Cycloses other than i-inosite occur in vegetable tissues but with one exception have not as yet been identified among the constituents of animal tissues. The exception is Scyllite which is found in the tissues of the bony (Teleost) fishes. It gives Scherer's reaction and is optically inactive, but it may be distinguished from i-inosite by its very high melting-point; 380 as contrasted with 225. In vegetable tissues occur 1-inosite in the form of the methyl ester in quebracho bark, d-1-inosite or racemic inosite (a mixture of the d- and 1-varieties) in the leaves of mistletoe, and d-inosite in the rosin and needles of conifers, in senna leaves and in India-rubber. CHOLESTEROL AND THE PHYTOSTEROLS 97 CHOLESTEROL AND THE PHYTOSTEROLS. Cholesterol, C27H 45 OH, may be represented so far as our knowledge at present extends, by the formula of von Fiirth: CH 3 CH 3 HOHC it is found in all animal fats or oils, in small quantities, in bile, blood, milk, yolk of egg, the medullated sheaths of nerve-fibers, the liver, kidneys and suprarenal bodies. It is contained in considerable amount in cod-liver oil. Under pathological conditions it is found to constitute a very large proportion of the most frequently occurring type of gall- stones, the conditions which ordinarily hold cholesterol in solution in bile, being in these cases, it appears, deficient. It occurs also in atheromata of the arteries, in tubercular cysts and in carcinomatous tissue. When precipitated from alcoholic solution by the addition of water, or when deposited in the body, as in gall-stones, cholesterol forms characteristic crystals with one re-entrant angle, resembling flat rectangular plates with one corner knocked out (Fig. 2). These crystals contain one molecule of water and are white, of a waxy con- sistency, insoluble in water, soluble in alcohol, ether, benzol, etc., and in fatty oils. When crystallized from anhydrous alcohol-ether mixtures cholesterol forms acicular crystals without any water of crystallization. Cholesterol may be held in solution or suspended in emulsified form in water by the addition of soaps, saponins, bile-salts, or lecithin, and it is by this means that it is held suspended in the bile and other tissue- fluids. As has been stated above, there is reason to suppose that cholesterol may possibly be decomposed in the suprarenal glands, and a portion is possibly converted into cholic acid in the liver, but for the rest, so far as we know at present, the main channel of excretion for cholesterol is the bile. The cholesterol which thus finds its way into the upper part of the small intestine, along with the cholesterol of the food, is in part reabsorbed and in part retained in the intestine until it is voided 7 98 CYCLOSES, CHOLESTEROL AND CHOLIC ACID with the feces. This latter portion of the cholesterol becomes subject in the lower intestine to the putrefactive action of bacteria, which results in its reduction to a derivative of cholesterol designated Coprosterol, containing two additional hydrogen atoms, and represented by the formula C 2 7H 4 7OH. This inefficient method of excretion would lead undoubtedly to a continual accumulation of cholesterol within the tissues, if it were not assisted by some means of destruction of the accumulated excess. The power of the body to destroy cholesterol is, however, very limited, and if cholesterol be administered in the dietary in unusual quantities, it forms deposits in various organs, notably the liver and suprarenal glands, and may ultimately lead to the formation of serious lesions. There is therefore, under ordinary circumstances, rather a delicate balance between the intake of cholesterol in the food on the one hand, FIG. 2. Cholesterol crystals. (After Hawk.) and its output in the feces, and destruction in the tissues on the other. If the power of the tissues to destroy or alter cholesterol is diminished for any reason we may anticipate that the excretory apparatus will be found inadequate, and that cholesterol will accumulate in the body. It is to this that we must probably attribute the accumulation of cholesterol which has been observed by Wacker in the subcutaneous fatty tissues of aged people, the decline in the activity of the tissues which accompanies age probably resulting in a deficient power of destroying cholesterol. It has been observed by Luden that the cholesterol-content of the blood in carcinomatous patients is usually high and that oxidation-products of cholesterol which are present in normal blood are frequently absent in these cases. The administration of unusual amounts of cholesterol to young animals results in marked effects upon their Growth, which will be fully discussed in a later chapter. If cholesterol be administered to animals CHOLESTEROL AND THE PHYTOSTEROLS 99 (rats) inoculated with carcinomatous tissue, the cancer grows much more rapidly than in normal animals and "metastases" or fresh growths in localities distant from the site of the primary growth, are formed much more numerously and in a much higher proportion of animals. In this connection it is significant to observe that Carcinoma is primarily a disease of old age so far as manifest growth or accretion of the parasitic tissue is concerned. It very rarely manifests itself in man before thirty and increases in frequency very decidedly with advancing age, the incidence between the ages of sixty-five and seventy-five being no less than ten times as great as between thirty-five and forty-five. It is, however, impossible to initiate carcinomatous growths in animals by administration of cholesterol, unless carcinoma-tissue is already present as a result of inoculation or spontaneous development, so that cholesterol cannot be looked upon as a cause, but rather as a favoring condition of cancer-growth. It must be remembered that our estimate of the age of incidence of carcinoma is founded upon the date at which the growth obtrudes itself upon the attention of the patient or physician. For how long prior to this its beginnings have been actually resident in the body, we have no means of estimating, but judging by the analogy afforded by other growth-phenomena (cf . Chapter XX) we may infer that the date of origin of the growth probably precedes by a consider- able interval the date of its obvious manifestation, so that despite the fact that cholesterol cannot initiate cancer, the date of its diagnosis, and therefore its "apparent" or "statistical" date of incidence may very possibly be determined by the acceleration of its growth due to an accumulation of cholesterol in the tissues. Cholesterol yields the following series of color reactions together with others, for description of which the student is referred to special monographs : Salkowski's Eeaction. Cholesterol is dissolved in chloroform and an equal volume of concentrated sulphuric acid is added. The solution is colored blood-red which changes gradually to purple. If the mixture is poured out in a shallow layer and exposed to the air, the purple changes to blue, then green and ultimately yellow. Liebermann-Burchard Reaction. Cholesterol is dissolved in a small amount of chloroform in a dry test-tube, a few drops of acetic anhydride are added and then concentrated sulphuric acid is added drop by drop. The mixture becomes red, then blue and finally, if not too much cholesterol and sulphuric acid have been added, a permanent green. Obermuller's Reaction. Dry cholesterol is heated in a glass tube with two or three drops of propionic anhydride until it melts. On cooling the mass turns first violet, then blue, green, orange, and finally red. Schiff' s Reaction. To dry cholesterol in an evaporating dish add a trace of ferric chloride, strong hydrochloric acid and chloroform, and evaporate the mixture nearly to dryness, when the edge of the residue begins to turn violet. Then add more chloroform, evaporate to dry- ness and heat. The whole mass turns violet first with a reddish and later with a bluish nuance, and finally a dirty green. 100 CYCLOSES, CHOLESTEROL AND CHOLIC ACID Neuberg-Rauchwerger's Reaction. This reaction is of exceptional interest because it is also given by the bile-acids and certain other derivatives of the terpenes. A common origin of the bile-acids (cholic acid) and cholesterol is thus indicated. With rhamnose or better still, with d-methyl-furfurol and concentrated sulphuric acid, an alcoholic solution of cholesterol gives a pink ring, or after mixing the two liquids and cooling, a pink solution. Lifschutz's Reaction. Dissolve a few milligrammes of cholesterol in two c.c. of glacial acetic acid, add a few drops of benzoyl superoxide, and boil. On adding four drops of concentrated sulphuric acid to the solution a green coloration is obtained, which rapidly changes to violet, then to blue. Oxidation-products of cholesterol yield this reaction without preliminary treatment with benzoyl superoxide, and in this way oxidation-products of cholesterol have been detected in the blood and tissues, and especially in cholesterol-concretions (gall-stones) in the gall-bladder. In plant-tissues there are found a variety of substances, the Phyto- sterols, which are more or less closely allied to cholesterol. The best, known of these is Sitosterol, an isomer of cholesterol, which occurs in wheat, rye, linseed-oil and the calabar bean. It differs from choles- terol in crystalline form, melting-point (137 contrasted with 148.5 for cholesterol) and optical rotatory power. Its solubilities in various organic solvents, and the color reactions which it yields are similar to those of cholesterol. It is absorbed together with cholesterol from the intestine. In fungi a series of phytosterols are found which contain a smaller proportion of hydrogen than cholesterol, and furthermore, differ from cholesterol in not yielding Salkowski's reaction. BILE-CONCRETIONS; AMBERGRIS. The concretions which occasionally form in the gall-bladder are of three types, formed respectively of Calcium Carbonate, Bile-pigments and Cholesterol, Each of these types of gall-stones is usually con- taminated with a larger or smaller proportion of the constituents of the other types. The cholesterol-stones have a waxy glistening and cry- stalline fracture, and are frequently deposited in concentric layers. They are often facetted by the pressure of adjacent stones, while their color is sometimes white, but more frequently tinged with bile-pigments. The cholesterol-stones are the type which most frequently occur in man. The conditions leading to their formation are unknown but it is parhaps a significant fact, in view of the accumulation of cholesterol in the tissues with advancing age, that the incidence of cholelithiasis increases progressively with the advance of years, over 75 per cent, of cases occurring in persons over forty years of age. It is furthermore stated that cholelithiasis is more frequent in carcinomatous than in non-carcinomatous subjects. CHOLESTEROL ESTERS : On the other hand the deposition of cholesterol may frequently originate, not so much in the abundance of this substance in the bile, as in its diminished solubility therein. An increase in the albumin- content of bile, as in inflammatory conditions, or by the addition of egg-albumin to bile in vitro may lead to the deposition of cholesterol and it is stated that certain bacteria, particularly the typhoid bacillus, diminish the solubility of cholesterol in bile which they inhabit. The proportion of cholesterol in cholesterol-stones varies from sixty-four to ninety-eight per cent. In addition there occur derivatives of cholesterol which yield Lifschiitz's reaction without preliminary oxidation, and are probably, therefore, derivatives originating from cholesterol by oxidation. Similar substances are found in the blood of normal persons, but are deficient in or absent from the blood of persons afflicted with carcinoma (Luden). The biliary concretions of the sperm whale (Physeter macrocephalw) are occasionally found floating upon the sea, or cast up upon the shores of oceans inhabited by these mammals. They are found in dull gray or black masses, having a peculiar sweet earthy odor, and form the Ambergris of commerce. When taken directly from the intestinal canal of whales it is of a deep gray color, soft consistence and disagree- able odor, but on exposure to air, it hardens and acquires the charac- teristic odor just described. Ambergris formerly enjoyed a high reputation as a therapeutic agent but its therapeutic virtues probably resided in its scarcity and expensiveness. At the present time ambergris is of importance solely in the manufacture of perfume in which its utility depends upon the rather extraordinary property it possesses, when added to perfumes in minute amounts, of very markedly enhancing their "floral" fragrance. Ambergris consists in the main, frequently to the extent of eighty- five per cent., of a substance, Ambrine, which very closely resembles cholesterol in its solubilities, general appearance and composition. It is insoluble hi water, highly soluble in alcohol, ether and oils, and crystallizes in white shining plates. CHOLESTEROL ESTERS. Cholesterol esters of the fatty acids are very widely distributed in the vegetable kingdom. In the animal kingdom they are found in the blood and lymph, in the medullated sheaths of nerves, in the cortical tissues of the suprarenal- gland and in the secretions of the sebaceous glands. The so-called fat or grease of sheeps' wool, which, when refined is commercially known as "Lanoline," consists almost entirely of a mixture of the palmitate, oleate and stearate of cholesterol together with a variable proportion of water. The fatty acid esters of cholesterol resemble the true fats, or fatty acid esters of glycerol, in their solubility in organic solvents, and insolubility in water. They differ, however, from the fats in the ,162 -: :*: : cy$LO&E& CHOLESTEROL AND CHOLIC ACID comparative difficulty with which they are hydrolyzed or "saponified" by alkalies, in their resistance to the action of bacteria, so that they do not become "rancid," and in the property they possess of absorbing or mechanically imbibing a large proportion of water to form a mass which still retains a fatty consistency. For this reason lanoline has of late come to be employed very widely in therapeutics as a vehicle for aqueous solutions of drugs which, through this agency, may be applied as salves. The cholesterol esters differ from cholesterol is not being emulsi- fiable in water containing soaps. Acetyl cholesterol C^yELs.OOC.CHs is also devoid of the characteristic action of cholesterol upon the growth of carcinomata. It would seem unlikely that this is due merely to the replacement of a hydroxyl-group by an acetyl-group, more especially since a variety of soluble and insoluble hydroxyl-derivatives of hydroaromatic substances have been found to be devoid of action upon the growth of carcinoma. It appears more likely that the loss of emulsifiability consequent upon the replacement of the hydroxyl- group prevents the distribution of acetyl cholesterol by the blood and tissue-fluids to the cells of the carcinomatous tissue. The cholesterol esters are of exceptional interest to the physical chemist because they are the substances which were first observed by Lehmann to display the remarkable phenomenon of "fluid crystals" or drops which, while spherical and retaining the characteristics of fluids, nevertheless display evidence, afforded by the unequal refrac- tion of light in different axes, of the possession of an internal crystalline structure. While other bodies are now known which display this peculiarity, the cholesterol esters still constitute a group which is pre- eminently suitable for the investigation of fluid crystals. They may be very readily obtained by gently heating the esters on a microscope slide until somewhat above the melting-point, and allowing to cool to a little above the melting-point. According to Adami fluid crystals, presumably containing choles- terol esters, may be observed in the myelin droplets which form during the degeneration of the fatty sheaths of medullated nerves. THE BILE-SALTS AND CHOLIC ACID. The mixed bile-salts, sodium glycocholate and sodium taurocholate may readily be obtained from ox-bile by mixing the bile with animal charcoal, evaporating to dryness, extracting with hot alcohol and add- ing ether to the cooled extract. If the process has been properly per- formed, a snow-white precipitate of fine acicular crystals ("Plattner's crystallized bile") is obtained which, in one or two crystallizations, may be almost freed from contamination. The two salts may be separated by adding lead acetate to their aqueous solution, by which means the glycocholate is precipitated, while the ta irocholate remains in solution. Sodium glycocholate is the most abundant BILE-SALTS AND CHOLIC ACID 103 constituent of the bile-salts in herbivorous animals and in man, but is absent from the bile of carnivorous animals. Sodium taurocholate, on the contrary, only occurs in small amounts in the bile of herbivora and man, while it is abundant in the bile of carnivora. The bile-salts are readily soluble in water, yielding solutions which, like solutions of the saponins, have a very low surface-tension, foam readily, and hold otherwise insoluble substances in solution or suspen- sion. This is especially true of the lecithins, which are very readily emulsified by bile-salts. The low surface-tension of these solutions is utilized very frequently in manometers for measuring very minute changes of gas-pressure. The solution of bile-salts does not "stick" or form drops on the sides of the containing tube as water frequently does, and the meniscus or surface of the fluid is flatter than that of water, enabling a reading of the height of a column to be made with greater ease and accuracy. The bile-salts and the free acids are further characterized by the peculiar taste of their solutions, at once bitter and sweet. The dry salts form a very fine light powder which is irritating when it comes into contact with the nasal mucous membranes. Hydrolysis of glycocholic acid by boiling with barium hydroxide yields Glycocoll and Cholic Acid: C23H 39 O 3 .CO.HN.CH 2 .COOH + H 2 O = C^H^Os.COOH + CH 2 NH 2 COOH glycocholic acid + water = cholic acid + ammo-acetic acid. while hydrolysis of taurocholic acid yields Cholic Acid and amino-ethyl sulphonic acid (Taurin) : C 23 H39O3.CO.HN.CH 2 .CH 2 SO 2 .OH + H 2 O = C 23 H39O3COOH + H 2 N.CH 2 .CH 2 .SO 2 OH taurocholic acid + water = cholic acid + amino-ethyl-sulphonic acid. The characteristic peculiarities of the bile-acids are determined by their common radical, the cholic-acid fraction. Free cholic acid, is almost insoluble in water, but its salts readily dissolve, forming bitter- sweet solutions which are dextrorotatory. The alkali salts of cholic acid, on the other hand, are only sparingly soluble in alcohol, while the free acid dissolves readily in this solvent. Cholic acid may also be recognized by the following characteristic reactions: Hammarsten's Reaction. If finely powdered cholic acid be added to a twenty-five per cent, solution of hydrochloric acid, a violet-blue colora- tion slowly appears which gradually changes through green to yellow. Mylius' Reaction. If an alcoholic (about five per cent.) solution of cholic acid in alcohol be mixed with two volumes of y^ iodine solution in alcohol, and the mixture slowly diluted with water, microscopic needles of an iodine addition-product are formed which are blue by- transmitted light. This reaction is characteristic for cholic acid and is not given by the conjugated bile-acids (glycocholic or taurocholic acids). Pettenkofer's Reaction. With a little cane-sugar, on careful addition of strong sulphuric acid, it yields a red coloration. This reaction, which is probably due to furfurol formed from cane-sugar by the action 104 CYCLOSES, CHOLESTEROL AND CHOLIC ACID of the sulphuric acid, is not absolutely to be relied upon, since similar reactions (differing from one another, however, in the absorption- spectra of the fluids produced) are yielded by a variety of substances, for example proteins, oleic acid, phospholipins, amyl alcohol and morphine. Neuberg-Rauchwerger's Reaction. Also given by cholesterol, which see. The structure of cholic acid has not yet been fully elucidated, but it appears to be definitely established that it is a derivative of the hydro- aromatic series. The decomposition-products resulting from the variety of procedures contain fractions which are closely related to products which are similarly obtained from other hydro-aromatic derivatives, such as cholesterol, turpentine and camphor, and the identification by Panzer of hydroxy-hexahydro-phthalic acid: OH H 2 C H 2 C \/ c /\ COOH / \ CH 2 CH S / CH COOH among the oxidation-products of cholic acid leaves very little room for doubt that hydro-aromatic nuclei exist preformed in the undecomposed molecule. Apart from these inferences, however, it is known that cholic acid contains one carboxyl-group and two primary and one secondary alcohol-groups united to a hydrocarbon-complex which contains cyclic linkages. The formula may therefore be written: C 2 oH fCHOH CH 2 OH 31 CH 2 OH I COOH A related acid which is found in small amounts in ox-bile and also in gall-stones is Choleic Acid, which differs from cholic acid in its per- centage composition ^JLoCX) and in its relative insolubility in alcohol. It yields a blue compound with iodine and also gives Ham- marsten's reaction with hydrochloric acid. Desoxycholic Acid, how- ever, which also occurs in bile and in gall-stones yields neither of these reactions although it is isomeric with choleic acid. BILE-SALTS AND CHOLIC ACID 105 In the bile of animals other than man or the ox are found a variety of acids, which have as yet been very imperfectly studied but which differ in composition and physical characteristics from one another and from cholic acid. A common origin of these substances is probably to be sought in hydro-aromatic radicals contained in the diet and derived ultimately in all probability from vegetable tissues. The bile-salts are, in part at least, reabsorbed from the intestine, and bile-salts administered by mouth cause a remarkable increase in the secretion of bile, in fact, with the possible exception of salicylic acid, the bile -salts appear to be the only true Cholagogues or stimulants of the secretion of bile. 1 When they are injected into the blood or forced into the blood owing to an obstruction of the bile-ducts, leading to icterus or "jaundice," they have a markedly depressant action upon the heart-muscle, slowing the beat very decidedly, and in large amounts they dissolve the red blood-corpuscles just as the saponins do. Under these circumstances bile -salts are probably excreted in part in the urine, but no reliable method of confirming their presence in the urine has yet been devised. For clinical purposes however, this is not an incon- venience since the presence of bile in the circulating blood is always evidenced by the appearance of Bile-pigments in the urine which are readily detected in a variety of ways. REFERENCES. INOSITE: Starkenstein-' Zeit exp. Path. u. Therp., 1908-9, 5, p. 378. Rose: Biochem. Bull., 1912-13, 2, p. 21. Anderson: Jour. Biol. Chem., 1912, 11, p. 471; 1912, 12, pp. 97 and 447; 1912- 1913, 13, p. 311; 1914, 17, pp. 141, 151, 165, 171; 1914, 18, pp. 425 and 441; 1915, 20, pp. 463, 475, 483, 493; 1916, 25, p. 391. Anderson and Bosworth: Ibid., 1916, 25, p. 399. CHOLESTEROL: von Filrth: Biochem. Zeitschr., 1915, 49, p. 416. Bang: Chemie und Biochemie der Lipoide. Wiesbaden, 1911. Glikin: Chemie der Fette, Lipoide und Wachsarten. Berlin, 1913. Lifschutz: Zeit. f. physiol. Chem., 1909, 58, p. 175; 1909, 63, p. 222. Biochem. Zeit., 1913, 52, p. 206. Zeit. f. physiol. Chem., 1914, 91, p. 309; 1914, 92, p. 383; 1914, 93, p. 209. Doree and Gardner: Proc. Roy Soc. B M 1908, 80, pp. 212 and 227; 1909, 81, p. 109. Ellis and Gardner: Ibid., 1909, 81, pp. 129 and 505; 1912, 84, p. 461; 1912, 85, p. 385; 1913, 86, p. 13. Fraser and Gardner: Ibid., 1909, 81, p. 230; 1910, 82, p. 559. Doree: Biochem. Jour., 1909, 4, p. 72. Gardner and Lander: .Biochem. Jour., 1913, 7, p. 576. Proc. Roy. Soc. B., 1914, 87, p. 229. Chalatow: Virchows Arch. Path. u. Anat., 1912, 207, p. 452. Beitr. Path. Anat. u. Allg. Path., 1914, 57, p. 85. Anitschkow: Beitr. Path. Anat. u. Allg. Path., 1913, 56, p. 379; 1914, 57, p. 201. Deutsch. med. Wchnschr., 1913, 39, p. 741. Weltmann and Biach: Zeit. exp. Path. u. Therap., 1913, 14, p. 367. Bailey: Proc. Soc. Exp. Biol. and Med., 1914, 12, p. 68; 1915, 13, p. 60. 1 In the opinion of some investigators, however, the increase in the secretion of bile which results from the administration of bile-salts is no greater than that which would be equivalent to the amount of bile-salts administered. 106 CYCLOSES, CHOLESTEROL AND CHOLIC ACID RELATIONSHIP OF CHOLESTEROL TO CARCINOMA: Robertson and Burnett: Jour. Exp. Med., 1913, 17, p. 344. Proc. Soc. Exp. Biol. and Med., 1913, 10, pp. 140 and 143; 1913, 11, p. 42; 1914, 12, p. 33. Jour, of Cancer Research, 1918, 3, p. 75. Robertson and Ray: Jour. Biol. Chem., 1919, 37, p. 443. Wacker: Zeit. f. physiol. Chem., 1912, 80, p. 383. Luden: Jour. Lab. and Clin. Med., 1916, 1, p. 662; 1917, 3, pp. 93 and 141; 1918, 4, p. 1. Jour. Biol. Chem., 1916, 27, p. 273; 1917, 29, p. 463. Sweet, Corson, White and Saxon: Jour. Biol. Chem., 1915, 21, p. 309. CHAPTER VI. THE FATS. THE TRUE FATS. The true fats are compounds, or Esters of the fatty acids with the triatornic alcohol glycerol. 1 Thus tripalmitin is formed by the union of three molecules of palmitic acid with one molecule of glycerol and the elimination of a corresponding number of molecules of water. CH 2 OH HOOC.Ci 5 H 31 CH 2 OOC.C 15 H 3 i CHOH + HOOC.C 15 H 3 i = CH.OOC.CuHn + 3H 2 O CH 2 OH HOOC.C 15 H 3 i CH 2 OOC.Ci 5 H3i By the action of alkalies this process is reversed, and the fatty acids which are thus set free combine with the excess of alkali to form soaps. The process of the hydrolysis of fats by alkali is therefore known as Saponification. Monoglycerides, i. e., glycerides containing only one fatty acid molecule, and Diglycerides are readily procurable in the laboratory, but they do not usually occur in natural fats unless they have been exposed to the action of fat-splitting enzymes (Lipases) or other saponi- fying agencies. In the Triglycerides the fatty-acid radicals need not all be identical and two or even three different fatty acids may be combined with one and the same molecule of glycerol to form neutral fat. The specific gravity of the fats is less than that of water, arid when liquid, or liquefied by heat, those which are insoluble in water float upon the top of it. The fats which are formed from the higher fatty acids are insoluble in water, while the solubility of the lower members in water decreases as the number of carbon atoms in the fatty acid molecule increases. They are soluble in a variety of organic solvents, and form very stable suspensions or emulsions in water in the presence of emulsifying (surface-tension reducing) agents such as soaps, bile- salts, saponins and so forth. 1 The separation of glycerol from fats was first accomplished by the Swedish chemist Scheele, in 1779. News of this discovery had, however, not yet reached the legislative assembly of one of the allied nations in 1914, with the result that in 1915 a responsible official of the executive, in reply to the inquiry of a legislator stated that it had only recently been discovered that nitroglycerin could be made from fats. It is perhaps time that a civilization which is based on mechanics, physics and chemistry should insist on a rudimentary knowledge of the practical import of these sciences on the part of its legislators and executives. 108 FATS The fatty acids which are found in the naturally occurring fats belong to two series, the saturated series, represented by the general formula C n H 2n 02 and the unsaturated or oleic acid series represented by the general formula C n H 2n . 2 O2. In this latter series of acids two of the carbon atoms are united by a double bond or unsaturated link- age which enables them to react very readily with hydrogen, oxygen or the halogens, the double bond being converted into a single one, and the remaining valencies of the carbons saturated by combination with the reacting atoms. The higher fatty acids which occur in nature usually have even values of n and the chain of carbon atoms is not branched. The lower acids, having small values of n, are formed in the secre- tions of the sebaceous glands, and in butter, while the tissue-fats and vegetable oils are in the main composed of fats derived from higher fatty acids. Thus in sweat we find: Formic acid, H.COOH Acetic acid, CH 3 COOH Propionic acid, C 2 H 5 COOH Butyric acid, CsHyCOOH Isovalerianic acid, C4H 9 COOH Caprylic acid, C 7 H 15 COOH These acids are probably secreted in combination with glycerol, but if the sweat be allowed to remain in contact with the skin, the glycerides are attacked by bacteria which hydrolyze them, liberating the free acids, to which the characteristic odor of the "unwashed" is attribut- able. The odor .of the lowest members of the series, Formic and Acetic Acids, is sharp, reminiscent in the former instance of ants, in the latter of vinegar. Propionic Acid has an intermediate odor, while Butyric Acid has the odor of rancid butter and Valerianic Acid the most intensely disagreeable odor of decomposing perspiration. The highest members of the fatty-acid series are non-volatile and have only very faint odors. In butter the lowest member of the series is butyric acid, while caproic and caprylic acids also occur together with higher fatty acids particularly Palmitic and Oleic Acids. The majority of the tissue -fats are, however, mixtures of the glycer- ides of Palmitic Acid, Ci 5 H 3 iCOOH and Stearic Acid Ci 7 H 35 COOH, of the saturated series, with glycerides of Oleic Acid, Ci 7 H 33 COOH, of the unsaturated series. The separation of the saturated from the unsaturated acids of the higher series may be accomplished by convert- ing them after hydrolysis of the fat, into the lead-salts, and extracting them with ether. The lead-salts of the higher fatty acids of the satur- ated series are almost insoluble in ether, while those of the unsaturated series readily dissolve in this solvent. The ether extract therefore contains all of the unsaturated fatty acids in combination with lead. The lead may be removed by extraction of the ether with dilute hydro- chloric acid, leaving the free fatty acids dissolved in the ether. CHARACTERISTICS OF THE NATURAL FATS 109 THE CHARACTERISTICS OF THE NATURAL FATS. The various animal fats and vegetable oils differ from one another very strikingly in their physical characteristics and chemical behavior. These differences are in the main determined by the relative propor- tions in which the glycerides of the three fatty acids above mentioned occur in the fat. The glycerides of oleic acid have the lowest melting- point, those of stearic acid the highest, and hence olive' oil, which con- sists very largely of glycerol trioleate is fluid at ordinary tempera- tures, while mutton-fat, which contains a high proportion of glycerol tristearate, is solid or semi-solid at ordinary temperatures. The melting-points of the pure fats are as follow: Triolein. . . . . . -. ; . t. . .- . y .... -6.0C. Tripalmitin 65.0,C. Tristearin ............'.'....'. 71. 5 C. a small admixture of triolein, however, reduces the melting-point of a fat to a very considerable degree. The chemical reactivity of the fats is also strongly influenced by their content of oleates. The unsaturated bond in oleic acid renders it capable, under appropriate conditions, of directly absorbing hydrogen, being thereby converted into the corresponding saturated acid. The artificial hydrogenation of vegetable oil is now being very largely practised and results in the production of a solid fat, utilizable for a variety of household purposes for which the fluid oil would be unsuited. The significance of the process, however, goes far beyond this. The addition of two atoms of hydrogen to the oleic acid molecule adds considerably to its calorific value, since the heat of combustion of the hydrogen to water is thus rendered available for nutritive purposes. In the aggregate the hydrogenation of vegetable oils adds to the nutritive value of these fats an amount 1 which would otherwise require a very great deal of space and labor to produce. From an economic point of view therefore, and as a means of food con- servation, the hydrogenation of vegetable oils is a very desirable thing to encourage. It is true that the vegetable oils fail in important respects to furnish the nutritive equivalent of animal fats, for, as we shall see in later chapters, the animal fats contain accessory foodstuffs which are essential for growth, and even for the maintenance of health, while the vegetable oils are lacking in these. To the extent, however, to which fats are employed in the diet for their mere fuel-value, the vegetable oils are fully equivalent substitutes for the animal fats, and only a small proportion of the total fat-consumption, at any rate in adults, is requisite to furnish the accessory foodstuffs which we acquire from the animal fats. It is probable that there would be no danger of shortage of the accessory foods being caused by the utilization of vege- 1 Roughly 7 per cent. 110 FATS table fats, unless meat and dairy products were at the same time very deficient in the dietary. The unsaturated bonds in the oleates also confer upon them the property of absorbing halogens, and the power of various natural fats and oils to absorb iodine is used as a means of characterizing and identifying them. The "Iodine Number" is the number of grammes of iodine which is absorbed by a hundred grammes of fat dissolved in chloroform and treated with a solution of iodine in alcohol or acetic acid. Other characteristics which are employed to differentiate the natural fats are: The "Hehner Number" or weight of water-insoluble fatty acids yielded by 100 grammes of fat; the "Acid Number" or proportion of free fatty acid in the fat, estimated by titration in alcoholic solution; the "Reichert-Meissl Number," or proportion of volatile fatty acids yielded by distilling the hydrolyzed fats with steam; the Saponification-value or milligrammes of potassium hydroxide neutralized by the saponification of one gramme of the fat; and the Acetyl Number, or amount of acetic acid yielded by 1 gramme of fat after treatment with hot acetic anhydride. In the following table the melting-points, iodine numbers and saponification-values of some of the fats most commonly employed are enumerated: Saponification Fat. Melting-point. Iodine number. value. Butter-fat 28-33 C. 26- 38 220-233 Pork-fat 36-46 C. 50- 70 195-197 Beef-fat . . . . . 40-48 C. 36-48 193-200 Sheep-tallow 44-49 C. 33- 46 192-195 Human fat Cod-liver oil Cotton-seed oil Olive oil . . Linseed-oil 17.5 C. 57- 66 193-199 0-10 C. 144-168 175-193 3- 4 C. 105-117 191-196 2-10 C. 78- 91 185-194 -27 C. 173-202 190-195 Cod-liver Oil is of especial interest to the physician because of its widespread employment as a food and therapeutic agent in chronic wast- ing diseases such as tuberculosis, and rickets. It is obtained from the livers of codfish by extraction with steam and water. It consists of a mixture of the glycerides of a great variety of saturated and unsatur- ated fatty acids together with a considerable proportion of phos- pholipins, a small amount of cholesterol, numerous nitrogenous bases and traces of iron, manganese, bromine and iodine. The therapeutic value of the oil has been variously attributed to each of these con- stituents in turn, and on the other hand to the readily digestible char- acter of the oil itself. Modern opinion inclines to the view that the efficacy of cod-liver oil resides mainly in its high calorific value, and the fact that it is usually added in considerable dosage to the pre- established dietary. On the other hand it is very rich in accessory foodstuffs and the possible significance of some of these must not be overlooked. From this point of view it is not impossible that the therapeutic applications of cod-liver oil may be destined to increase rather than to diminish, as our growing knowledge of the exact require- WAXES 111 ments of the tissues enables us to use it with more judgment and less empirically than heretofore. Cotton-seed Oil consists of a mixture of the glycerides of oleic and linoleic and palmitic acids, while Olive Oil consists almost entirely (89 per cent, to 98 per cent.) of the triglyceride of oleic acid. Linseed-oil is of very great importance in the industries on account of its peculiar property of hardening when it dries in thin films exposed to the air, forming a transparent waterproof surface and accelerating the drying of other substances (pigments, etc.), with which it is mixed. This process of hardening takes place at first slowly, and then more rapidly, the products of oxidation which are formed accelerating the further stages of the process. The oxidation of linseed-oil which results in hardening is, in fact, an "autocatalytic," that is, a self- accelerated reaction, producing its own catalyzers. These substances are believed to be unstable peroxides which readily break down, liber- ating oxygen or possibly ozone, which oxidizes adjacent molecules of the oil. Other substances which accelerate the hardening are powdered lead, zinc, copper, platinum or their oxides. This phenomenon depends upon the very large proportion of unsaturated linkages which linseed oil contains; it consists of a mixture of the glycerides of linoleic, linoleinic and isolinoleinic acids (fatty acids of the unsaturated series Cnltn-eQj) with a small proportion of oleic, palmitic and myristic acids, and a trace of unsaponifiable material. Castor-oil is obtained by expression from the seeds or "beans" of the castor-oil plant (Ricinus communis). It consists in the main of the glycerides of ricinoleic acid Ci7H 32 (OH).COOH, a hydroxy-acid of the unsaturated series. It is without aperient action until saponified by the bile and pancreatic juice in the upper part of the sinall intestine and is therefore devoid of irritant action upon the walls of the stomach. WAXES. In addition to the glycerides of fatty acids there are found in a variety of living tissues and tissue-products, fatty acid esters of monatomic alcohols which are collectively and somewhat loosely designated Waxes. This term is not infrequently extended to include the cholesterol esters of the fatty acids, for no better reason than that cholesterol is a monatomic alcohol, and that the cholesterol esters somewhat resemble the waxes in certain of their properties, more particularly in the difficulty with which they are saponified. It would be preferable, however, to restrict the term "wax," as we are doing here, to the fatty acid esters of the higher monatomic alcohols of the paraffin series. In this way we will include in the class all of the most typical waxes of commerce, and we will exclude the entirely atypical esters of cholesterol. The waxes are characterized by their high melting-point and the difficulty with which they are saponified. They are not hydrolyzed 112 FATS by the fat-splitting ferments (lipases), and it is only with comparative difficulty that they are split into their components .by alkalies. They are hydrolyzed by bacteria, and hence do not turn sour or "rancid" on standing. While their high melting-points prevent them from being " sticky" or exhibiting any of the characteristic properties of fluids or oils at ordinary temperatures, yet they retain the " greasy" or slippery qualities of the fats (more accurately expressed as the possession of a low coefficient of friction), and their insolubility in water, a com- bination of qualities which renders them ideal and indeed indispens- able agents for polishing and waterproofing the surfaces of rough or porous materials. In the skull of the white whale or cachelot (Physeter macrocephalus) , there is found a large cavity which during life, is filled with an oily liquid. This liquid partially solidifies after the death of the animal, and consequent fall in temperature, and separates into two portions, a solid crystalline part ordinarily called Spermaceti and a liquid known as Spermaceti-oil. Spermaceti is also found in some other whales and in certain species of dolphins. Purified spermaceti is a mixture of fatty acid esters of monatomic alcohols. The chief constituent is the palmitic acid ester of Cetyl Alcohol, Ci e H 33 OH, mixed with small quantities of the lauric, myristic and stearic acid esters of the twelve, fourteen and eighteen carbon atom alcohols of the paraffin series (general formula C n H 2n +iOH). Spermaceti is used for making "wax-candles," as a finishing material or waterproof polish, and in pharmacy as a means of stiffening emol- lients and salves, and raising their melting-point, particularly in hot climates. Spermaceti-oil is a very valuable lubricant for small and delicate machinery or apparatus. The Beeswax of commerce is a digestion-product of the honey-bee, Apis mellifica. It is elaborated by special glands and the production of honey and wax stand in inverse proportion to one another, the pro- duction of one gramme of wax diminishing the yield of honey by from ten to fourteen grammes. Regarding the mode of origin of the wax from the foodstuffs of the bee, we are wholly in the dark. The chief constituent of beeswax is the palmitic acid ester of Myricyl Alcohol C 30 H 6 iOH with an admixture of other acids and esters. Bees- wax is employed in a variety of industries too numerous to mention here, it is an important constituent of a variety of commercial waxes, which are prepared by the admixture of paraffin and other substances with the beeswax to obtain the combination of physical qualities which is desired for the purposes for which the wax is to be employed. Adulter- ation with paraffin and other non-saponifiable materials may be detected by the low saponification-value of the mixture, the sapomfica- tion-value of pure beeswax lying between 90 and 97. Waxes are produced by a variety of insects, notably the Hymenoi)- tera (wasps and bees) and Homoptem (cicadas and scale insects). Japan Wax (or Chinese Wax) is obtained from a scale-insect which THE PHOSPHOLIPINS OR PHOSPHATIDS 113 infests the Chinese Ash. The most widely employed vegetable wax is carnaiiba wax, obtained from the leaves of the Wax-palm or Car- naiiba-palm which grows in tropical South America. It consists of a complex mixture of esters of higher monatomic alcohols. THE PHOSPHOLIPINS OR PHOSPHATIDS. In all living tissues and without exception, we find a variety of complex substances resembling the fats in their solubility in organic solvents, and yielding fatty acids, alcohols (usually glycerol), phos- phoric acid and nitrogenous bases when hydrolyzed. These sub- stances constitute the group of Phospholipins, and on account of their constant association in the tissues with cholesterol and cholesterol derivatives they are sometimes included with these in the larger group of Lipoids or fat-resembling substances, the common characteristic of the group consisting in their high solubility in fats and oils, and in the various fat-solvents. The term lipoid, however, is merely a con- venient brief designation of a heterogeneous group of substances which may be chemically unrelated to one another. The phospho- lipins, on the contrary, are a rather well-defined and homogeneous group of chemically related substances. The best known and most abundant representatives of the phos- pholipin group are the Lecithins. These substances, which are found in every living cell, yield fatty acids, glycerol, phosphoric acid and choline (=oxyethyl trimethyl ammonium hydroxide) on hydrolysis. One molecule of phosphoric acid is yielded for every molecule of choline, and the phosphorus and nitrogen-contents of these substances stand therefore in the proportion to one another of 1 : 1. The struc- ture of the lecithins is believed to be represented by the formula: CHz.O (fatty acid radical) CH.OL (fatty acid radical) CH 2 .0 HO - P = O C 2 H 4 O ' / N = (CH 3 ) 3 \ OH. The fatty acid radicals consist, as a rule, of palmitic, stearic or oleic acid, but at least one oleic acid radical would appear to be invariably present, since the lecithins exhibit to a very high degree the character- 8 114 FATS istic instability of the unsaturated fatty acids. This instability is in fact enhanced in the phospholipins generally to a remarkable degree, and the difficulties attending their preparation and purification are rendered exceptionally great by their extreme susceptibility to oxi- dation. It is a fact which is doubtless of very great significance that the tissues of the Brain are notably rich in phospholipins, while the activities of the brain are exceptionally dependent upon an abundant and continuous supply of oxygen, the first bodily activities to dis- appear in asphyxia being those of the higher cerebral centers. The various members of the phospholipin group resemble one another very closely in physical and chemical behavior. They differ among themselves mostly markedly, first in the proportion of phos- phorus to nitrogen which they contain, and secondly in their solubilities in certain organic solvents. Those phospholipins which, like lecithin, contain one atom of phosphorus (i. e., one molecule of phosphoric acid) for every atom of nitrogen, are termed Monoamino-monophosphatids ; those which con- tain two molecules of phosphoric acid for every atom of nitrogen (P : N = 2 : 1), are termed Monoamino-diphosphatids ; those which contain two atoms of nitrogen for every atom of phosphorus are termed Diamino-monophosphatids (P : N = 1 : 2), and so forth. The highest proportion of nitrogen to phosphorus which has been found to occur in a phosphatid is that of four atoms of nitrogen for every atom of phosphorus. The majority of the phospholipins are soluble in alcohol and in ether, but some of them are insoluble in ether, and others, while soluble in alcohol or in ether alone, are insoluble, or but sparingly soluble in certain mixtures of the two. The great majority of the phospholipins, but not all of them, are precipitated from ether solu- tions by the addition of acetone, a fact which is utilized very frequently in their preparation. They are also precipitated by a variety of metallic salts, and platinum chloride, and particularly cadmium chloride are frequently employed for their separation and purification. The phospholipins are amorphous substances which are generally white or cream-colored when pure, but darken rapidly on exposure to the air. The iodine-number simultaneously diminishes, indicating that the unsaturated linkages have been partially neutralized by com- bination with oxygen. This oxidation is particularly accelerated by heat and by traces of moisture, and the dried or partially dried phos- pholipins are unfortunately extremely hygroscopic, rapidly attracting and condensing moisture when exposed to the air. The drying of phospholipins without decomposition can therefore only be achieved at low temperatures, and in vacuo or in an atmosphere composed of some indifferent gas. There seems to be some reason for supposing that the lability of the phospholipins may be greatly enhanced by impurities which are commonly associated with them, and that in the absence of these, they may be comparatively stable. THE PHOSPHOLIPINS OR PHOSPHATIDS 115 The majority of the phospholipins are rapidly hydrolyzed by the fat-splitting ferments or Lipases, yielding fatty acids, glycero-phosphoric acid and nitrogenous bases. Glycero-phosphoric acid is not split by lipase, but is readily decomposed by dilute acids yielding phosphoric acid and glycerol. Since glycero-phosphoric acid is not liberated from phospholipins until they reach the small intestine, where the reaction is alkaline, it would appear unlikely that it is split before absorption. According to some authors, an enzyme exists in tissue extracts from the liver, kidneys and intestinal mucosa, which is capable of bringing about the decomposition of glycero-phosphoric acid, but the constant presence of undecomposed glycero-phosphoric acid in small amounts in normal urine would appear to render this doubtful. Optically in- active glycero-phosphoric acid is readily prepared synthetically from glycerol, and phosphoric acid; the glycero-phosphoric acid yielded by hydrolysis of phospholipins is, however, levorotatory. It is soluble in water and insoluble in alcohol. The calcium salt is readily soluble in cold, but almost insoluble in boiling water. The Lecithins, the composition and probable structure of which, have already been discussed, occur in all plant and animal cells. A lecithin or a mixture of lecithins, is particularly abundant in the yolks of eggs, and may be obtained in impure condition by extracting the broken yolks with ether, and adding acetone to the extract. Lecithins are particularly abundant in young and rapidly growing or embryonic tissues. They progressively diminish as development proceeds and, in the embryos of sea-urchins at all events, they are probably the source from which the phosphoric acid is obtained which is required to build up the nucleic acids in the nuclei of the new cells. The lecithins are soluble in alcohol, ether, chloroform, carbon bisulphide, benzol, and fats or fatty oils; they are precipitated from ether by the addition of acetone. In water they swell up and form pasty masses which throw out oily drops and threads, the so-called "myelin forms," into the body of the liquid. This probably represents the beginning of an imperfect emulsification, and the addition of soaps, saponins or bile-salts acceler- ates and completes the process with the formation of relatively stable milky emulsions which are coagulated by the addition of small quanti- ties of salts of the alkaline earths (calcium, barium or strontium). The lecithins have a peculiar greasy odor which is rather sharp and reminiscent of dried brain-tissue. They are tasteless. It was formerly believed that lecithins stood in a peculiar relation- ship to certain types of snake-venom and other hemolyzing poisons. If a pure hemolyzing snake-venom be allowed to act upon thoroughly washed blood-corpuscles, no hemolysis occurs. Upon the addition of blood-serum or of impure lecithin, which by themselves are of course without action, hemolysis ensues at once. It has been ascertained by Bang, however, that pure lecithin prepared from yolk of eggs is devoid of activating influence upon cobra -venom. It would thus appear probable that the activating action of other preparations of 116 FATS lecithin is attributable to impurities which may nevertheless be phospholipins. Similarly the alleged action of lecithin in "activating" fibrin-ferment (the blood-coagulating ferment), has recently been shown to be due, in fact, not to lecithin but to kephalin. In egg-yolk and in a variety of tissues we find another mono-amino- monophosphatid, designated Kephalin which differs from lecithin in being insoluble in alcohol. It is particularly abundant in brain-tissue and may be extracted by dehydrating the tissue with acetone, extract- ing with ether and adding alcohol to the concentrated extract. It may be purified by precipitation with cadmium chloride. It used to be alleged that kephalin differed from lecithin only in the addition of a methyl group, but recent investigations have shown the difference to be much more profound, inasmuch as the nitrogenous base in keph- alin is not cholin, but amino-ethyl alcohol. Besides its great abun- dance in brain-tissues, where it doubtless plays an important role. Kephalin is of very great physiological importance in consequence of the part which it plays in the coagulation of the blood. The recent investigations of Howell and McLean have shown that kephalin possesses in high degree the power of neutralizing the anti-thrombin in the blood and thus permitting the thrombin, or fibrin-ferment to transform fibrinogen into fibrin and bring about coagulation of the blood. Very small amounts of kephalin, therefore, decisively acceler- ate the coagulation of the blood and kephalin is now being utilized extensively for this purpose in surgery. Cuorin which is found in heart-muscle is a mono-amino-diphosphatid (P : N = 2 : 1). It is insoluble in alcohol and in acetone, but very soluble in ether, chloroform and benzol. It readily emulsifies in water and on the addition of alkalies the emulsions form clear solutions. The nature of the nitrogenous base in cuorin is not known. It con- tains three fatty acid radicals for every two molecules of phosphoric acid. Sphingomyelin, which is found in the brain, is a diamino-monophos- phatid. It is soluble in hot alcohol but sparingly soluble in cold alcohol, and insoluble in ether. It yields on hydrolysis an alcohol of unknown structure, Sphingol instead of glycerol, and it yields' two different nitrogenous bases, namely Neurine and a base of which the structure is not yet completely defined, which is designated Sphingo- sine (see p. 197). GLUCOSIDES OF THE PHOSPHOLIPINS. If glucose be added to an ether solution of lecithin, the sugar, which is ordinarily insoluble in ether, dissolves and becomes so closely associated with the lecithin that repeated precipitation and resolution do not remove it. We infer that lecithin forms a compound with glucose, which, however, would appear to be a very loose one, since the analyses of various preparations indicate a very inconstant composition. GLUCOSIDES OF THE PHOSPHOLIPINS 117 In the tissues of the liver we find considerable quantities of a water- soluble phospholipin, Jecorin, which yields glucose, fatty acids, glycero- phosphoric acid, choline and hydrogen sulphide on hydrolysis by alka- lies. Early observers were inclined to regard jecorin simply as lecithin glucoside, but the sulphur-content of jecorin precludes the adoption of this simple interpretation. Jecorin, like other phospholipins, is exceedingly hygroscopic and susceptible to oxidation. It is soluble in alcohol containing w^ater and in ether, but is insoluble or but spar- ingly soluble in absolute alcohol. With silver nitrate an aqueous solution of jecorin yields a precipitate which dissolves in excess of the jecorin solution; on the addition of ammonia this mixture turns dark red. It appears highly probable from the variability of the analytical data obtained with jecorins from different sources that a variety of substances of this general type exist in the tissues; more especially is this indicated by the variable ratio of phosphorus to nitrogen which in some preparations approximates to the value P : N = 1 : 2, while in others P : N = 1 : 4. In any case it is probable that the phospho- lipin portion of the molecule is not simply lecithin. All preparations contain sodium, which appears to be present in chemical combination. In the anterior lobe of the pituitary gland is found another water- soluble phospholipin which, however, instead of yielding glucose on hydrolysis, yields the cyclose Inosite. This substance, Tethelin, is soluble in water, alcohol or ether, but is insoluble in a mixture of Alcohol and ether in the proportion of one part by volume of alcohol to one and one-half parts by volume of ether. It contains phosphorus and nitrogen in the proportion of 1 : 4 and one-half of the nitrogen is present in the molecule in the form of amino-groups. On hydrolysis the yield of amino-nitrogen increases to three -fourths of the total nitrogen, indicating, probably, that one of the nitrogen atoms is present in the form of an imino-group (= NH). Tethelin is exceedingly hygroscopic and susceptible to oxidation which is accelerated by traces of moisture. Oxidation is accompanied by progressive darkening of the originally cream-colored powder, so that it ultimately becomes dark brown or almost black; at the same time the iodine absorption number decreases. With a 2 per cent, solution of p-dimethylamino-benzaldehyde in hydrochloric acid cf sp. gr. 1.09, aqueous solutions of tethelin yield a pink coloration (Ehrlich's Reaction, indicating an acetylated oxyamino-acid radical). The nitrogenous base has not as yet been identified, but it probably contains an Iminazolyl radical: This is of great significance in view of the fact that the active prin- ciple of the Posterior Lobe of the Pituitary Body is also an iminazolyl derivative and the possibility is indicated, 'which the anatomical 118 FATS relations of the two glands tend to confirm, that the active principle of the posterior lobe is derived by partial decomposition from that of the anterior lobe. Tethelin, in very small dosages administered by mouth, has a remarkable effect upon the growth of animals, consisting in the main of an initial retardation followed by a notable acceleration of growth. These phenomena will fall under fuller consideration in a later chapter. When administered locally in aqueous solution or incorporated with lanoline and applied as a salve it very markedly accelerates the repair of slowly healing lesions of the skin, such for example as various ulcers. The effects of tethelin upon growth almost exactly reproduce those of the whole anterior lobe tissue, and it is inferred that tethelin is the active principle, the absence or superabundance of which is responsible for the remarkable clinical manifestations of hyperactivity (gigantism and acromegaly) of the pituitary gland. Tethelin in aqueous solution has no effect upon the uterus, and only causes in very large dosages a slight transient fall in blood pressure when injected intravenously in animals. After acidifying the solution (or rendering alkaline), heating for a brief period to nearly boiling- point and then cooling and neutralizing, however, the solution has a powerful effect in causing tonic contractions of the uterus similar to those caused by extracts of the posterior lobe, and when injected intravenously causes the characteristic rise in blood pressure which is brought about by small doses of posterior lobe extract (Pituitrin). In the tissues of the brain we find a complex substance or series of substances which arise by combination of phospholipins with the cerebrosides which are glucosides yielding galactose on hydrolysis. This substance, Protagon, may be obtained by extracting dehydrated brain-tissue (dehydrated by acetone) with 85 per cent, alcohol, and cooling to zero when the protagon in impure form is precipitated. On partial hydrolysis it yields sphingomyelin (see p. 116) and cerebrosides (see p. 91). Protagon when dry forms a fine white powder, it dissolves in 85 per cent, alcohol at 45 C., but on cooling is precipitated in groups of small acicular crystals. It is difficultly soluble in cold alcohol or ether, but dissolves in warm ether. It dissolves in methyl alcohol contain- ing chloroform but, on standing, this solution decomposes and a cerebroside, Phrenosin is deposited. In water protagon swells up and forms a pasty mass which dissolves in excess of water, forming an opalescent solution. Solutions of protagon in pyridin are dextro- rotatory, but on standing they decompose, depositing sphingomyelin, and become levorotatory. We are not certain whether protagon is a chemical individual or not. The composition as reported by different observers, varies very considerably, and yet preparations have been obtained which failed to alter in composition or optical rotation of their solutions after repeated resolution and recrystallization. GLUCOSIDES OF THE PHOSPHOLIPINS 119 REFERENCES. CHARACTERISTICS OF THE NATURAL FATS: Lewkowitsch: Chemical Technology and Analysis of Oils, Fats and Waxes. London, 1913. THE PHOSPHOLIPINS: Maclean: Lecithin and Allied Substances. London, 1918. Levene and West: Jour. Biol. Chem., 1913-14, 16, p. 419; 1916, 24, pp. 41, 47, 50 and 111; 1916, 25, p. 517; 1918, 33, p. Ill; 1918, 34, p. 175. McLean: Am. Jour. Physiol., 1916, 41, p. 250; 1917, 43, p. 586. Waksman: Ibid., 1918, 45, p. 375. GLUCOSIDES or THE PHOSPHOLIPINS , Rosenheim: Biochem. Jour., 1914, 8, p. 110; 1916, 10, p. 142. Levene and West: Jour. Biol. Chem., 1917, 31, p. 635. Koch and Koch: Ibid., 1917, 31, p. 395. Robertson: Ibid., 1916, 24, p. 409. Schmidt and May: Jour, of Lab. and Clin. Med., 1916-1917, 2, p. 708. CHAPTER VII. THE PROTEINS AND THE AMINO-ACIDS. GENERAL CHARACTERISTICS OF THE PROTEINS. THE greater and most characteristic part of the organic matter in protoplasm consists of colloidal substances containing nitrogen which are designated Proteins. As examples of the proteins we may recall white of egg, which is practically a solution of protein in dilute sodium chloride solution, or casein, which is flocculated out of milk by the addition of acids, and gelatin, which is derived from the connective tissues by extraction with hot water. The proteins all contain carbon, hydrogen, nitrogen and oxygen, while the great majority of them also contain sulphur and a very great many of them contain phosphorus. Other constituents, for example, iron, copper and iodine are found in certain exceptional proteins or in. compounds of the proteins with non-protein radicals containing these elements. The average composition of the more typical proteins is represented in the following table: Element. Per cent. C . . . . . .... . . . . 50. 6 to 54.5 H 6. 5 to 7.3 N 15.0to 17.6 S 0.3 to 2.2 P 0.4to 0.9 O 21. 5 to 23. 5 When perfectly free from water, the proteins form loose white powders, but when imperfectly dry, and especially if exposed to heat, they tend to form horny semi-transparent flakes or plates, so that in most of the older literature, before the modern methods of dehydration at low temperature by absolute alcohol and ether were employed, the proteins are usually described as horny substances when in the dry condition. While drying, and in the presence of traces of moisture the proteins show a marked tendency to discoloration, with the production of heavily pigmented insoluble substances which are probably related to the " humin-substances" which are produced in the presence of carbohydrates by boiling the tryptophane radical of proteins with acids. Many proteins have curious and characteristic faint odors, but they are generally tasteless and amorphous. Notwithstanding their colloidal character and very slight diffusi- bility in solutions, many proteins may, nevertheless, under suitable GENERAL CHARACTERISTICS OF THE PROTEINS 121 conditions, be obtained in crystalline condition. This is particularly true of hemoglobin, of egg-albumin, the serum-albumin of the horse, and a variety of vegetable proteins. The solutions of the proteins are always optically active and with the exception of the solutions of hemoglobin and the nucleo-proteins, are levorotatory. The great majority of the proteins are soluble in water or in very dilute acids or alkalies. Some exceptional proteins, however, such as Elastin from elastic fibers of connective tissues, Keratin from horn, Fibroin from silk and Spongin from sponges, are insoluble in water or in dilute acids and alkalies and require strong acids or alkalies to bring them into solution. The action of strong soda upon a sponge may be cited in illustration. The proteins are usually insoluble in organic reagents, although some of the vegetable proteins, particularly those obtained from a variety of grains, are soluble in 80 per cent, alcohol. Many of the proteins not commonly regarded as alcohol-soluble are, however, soluble in faintly alkaline alcohol, if they are first dissolved in alkaline water, and alcohol added up to 80 or 90 per cent. Casein is soluble in warm anhydrous formic acid, but the protein undergoes decomposition if the solution is allowed to stand. The proteins combine with both acids and bases, neutralizing them wholly or in part, and causing a diminution of hydrogen ions in the case of combination with acids, or of hydroxyl ions in the case of combi- nation with bases. They therefore belong to the class of substances designated Amphoteric Acids, or acids which are simultaneously capable of acting as bases. Under certain conditions the proteins are also capable of combining with neutral salts. When dissolved in water, especially in faintly acid solutions, the proteins are usually modified by heat in such a way as to render them less soluble. This generally leads to flocculation or Coagulation of the protein, or if the solution be very concentrated, to the formation of a firm jelly, such as, for example, the white of a hard-boiled egg. The true characterization of the proteins depends upon the presence among their hydrolytic cleavage products of a preponderating pro- portion of Amino-acids. No other single "test" can be relied upon to demonstrate the presence of a protein in a solution containing unknown substances, nor can the individual proteins be accurately characterized, as a general rule, in any other terms than the propor- tions of various cleavage-products which they yield on hydrolysis. By employing a multiplicity of tests, however, the presence of protein in a solution may be established by the fact that the unknown sub- stance yields several positive reactions. For the identification of any particular protein we depend upon slight peculiarities of solubility in various salt solutions, dilute acids and alkalies, etc., and upon physical peculiarities and the nature of the tissue or fluid in which the protein occurs. The various reactions which the majority of the proteins yield may be subdivided into Coagulation-reactions which involve or depend 122 THE PROTEINS AND THE AMINO-ACIDS upon dehydration of the protein and the formation of complex insoluble anhydrides, Precipitation-reactions, which depend upon the formation of insoluble compounds with the precipitating-agents employed, and Color-reactions which depend upon chemical interaction with the reagents employed, resulting in the production of distinctive colors. The most important of these reactions are the following: COAGULATION-REACTIONS. 1. Heat. Heat applied to solutions acidified with ace_tic acid. If mineral acids are employed, compounds of the protein with the acid may be formed which are incoagulable by heat. 2. Alcohol. Alcohol added to neutral or acid solutions. 3. Concentrated Neutral Salts. Concentrated neutral salts, particu- larly ammonium sulphate or magnesium sulphate. In ajcidified solutions concentrated sodium chloride or sodium sulphate are also coagulants of protein. 4. Strong Mineral Acids. Upon the ability of the strong_mineral acids to coagulate proteins depends Heller's Test for protein in urine. The suspected sample of urine is placed in a test tube and concen- trated nitric acid is allowed to flow into the bottom of the tube from a pipette. At the junction of the two fluids a white ring of coagulated protein is formed. Precipitation Reactions. 1. The Salts of Heavy Metals, such as cupric sulphate, lead acetate, mercuric chloride, silver nitrate, etc., form insoluble compounds with proteins. In the presence of excess of the reagent, the precipitate which at first forms not infrequently redissolves. . 2. The so-called Alkaloidal Reagents, such as phosphotungstic or phosphomolybdic acids, tannic acid, potassium mercuric iodide, picric acid, trichloracetic acid, phenol and salicyl-sulphonic acid. Other reagents which similarly precipitate proteins are metaphosphoric acid, nucleic acids, chondroitin-sulphuric acid and taurocholic acid. Potassium ferrocyanide and acetic acid yield an insoluble compound of the protein with hydroferrocyanic acid. Color -reactions. 1. Millon's Reaction. A solution of mercury in strong nitric acid yields a mixture of mercuric and mercurous nitrates dissolved in a mixture of nitric and nitrous acids. If this reagent be added to a protein solution, a precipitate is produced which turns brick-red on heating. 2. The Xanthoproteic Reaction. On adding strong nitric acid to protein solutions and heating to boiling, a pale yellow solution or coagulum results. On rendering the mixture alkaline with ammonia it becomes orange-yellow. 3. The Hopkins-Cole Reaction. A solution of glyoxylic acid, formed by acting upon a concentrated solution of oxalic acid with sodium amalgam, is added to the protein solution in a test tube, and sulphuric COAGULATION-REACTIONS 123 acid introduced at the bottom of the tube by means of a pipette. A reddish- violet ring is formed at the junction of the two liquids. 4. Acree's Reaction. To the solution is added an equal volume of a 0.02 per cent, solution of formaldehyde containing a trace of ferric chloride. Concentrated sulphuric acid is then introduced below the mixture and at the junction of the two fluids a violet ring is formed. 5. The Biuret-reaction. The protein solution is rendered strongly alkaline with concentrated sodium or potassium hydroxide, and j, dilute solution of cupria^ulphate ls_added, one^drop at a time. A reddish or bluish violet results in solutions of proteins, and a pink color in solutions of their digestion-products, the peptones. Excep- tions are afforded by the protamiiie group of proteins, which yield a pink biuret-reaction without preliminary hydrolysis. 6. The Ninhydrin Reaction. One-tenth of a gram of Triketohydrin- denehydrate ("Ninhydrin"): CO is dissolved in from thirty to forty c.c. of water, one or tw.o drops of this solution are added to one c.c. of the protein solution, and the mixture is heated for a short time to boiling. On cooling, an intense blue or bluish violet color develops. This reaction is given not only by proteins, but also by their cleavage-products, proteoses, peptones and even amino-acids, with the exception of proline and oxyproline. This reaction is exceedingly delicate and is given by substances con- taining at least one free carboxyl-group, and one amino-group; it will detect glycine (amino-acetic acid) in solutions containing only one part in ten thousand. This extreme delicacy, in fact, renders the reaction rather unserviceable as a practical test for proteins or their decom- position-products, since such extraordinary precautions have to be taken to ensure that a positive test may not have been attributable to accidental contamination of reagents or apparatus with traces of the many substances that will yield a positive reaction. All of these color reactions, with the exception of the biuret reaction and the ninhydrin reaction, depend" upon specific atomic groupings which are usually, but not invariably present in the protein molecule. Thus, Millon's reaction is attributable to a hydroxy-benzene radical which is usually present in proteins in the form of the amino-acid Tyrosine, but is absent from certain proteins, for example gelatin. The xanthoproteic reaction is attributable to aromatic groups which are provided by the Tyrosine, Phenylalanine and Tryptophane radicals in the protein molecule. The xanthoproteic reaction is therefore not given by proteins such as the members of the protamine group in which 124 THE PROTEINS AND THE AMINO-ACIDS these radicals are lacking. The Hopkins-Cole reaction is attributable to the indole linkage: c C 6 H 4 CH \ / NH which is present in the tryptophane (indole aminopropionic acid) radical of protein. It is therefore not given by proteins from which this radical is absent, such as, for example, zein, a protein obtained from corn (maize). The biuret-reaction, on the contrary, is yielded by a variety of sub- stances such as oxamide, biuret, etc., which are not proteins. The Deaminized Proteins which are laboratory-products formed by acting upon proteins with nitrous acid, and in which the NH 2 groups have been replaced by hydroxyl-groups, no longer give the biuret-reaction, although they otherwise resemble the natural proteins in physical and chemical behavior. THE CLASSIFICATION OF THE PROTEINS. American and English biochemists have unfortunately adopted slightly different systems of classification of the proteins. The Ameri- can classification, adopted by the American Physiological Society and the American Society of Biological Chemists, is as follows: I. SIMPLE PROTEINS: Albumins. Globulins. Glutelins. Prolamins (alcohol-soluble proteins). Albuminoids. Histones. Prot amines. H. CONJUGATED PROTEINS: Nucleoproteins. Glycoproteins. Phosphoproteins. Hemoglobins. Lecithoproteins. HI. DERIVED PROTEINS: 1. Primary Protein Derivatives: Proteans. Met a proteins. Coagulated Proteins. 2. Secondary Protein Derivatives: Proteoses. Peptones. Peptides. THE CLASSIFICATION OF THE PROTEINS 125 The classification adopted by the British Medical Association is the following : I. SIMPLE PROTEINS: Protamines. Histones. Albumins. Globulins. Glutelins. Alcohol-soluble Proteins. Scleroproteins. +- Phosphoproteins. V H. CONJUGATED PROTEINS: Glucoproteinsr Nucleoproteins M Chromoproteins. * m. PRODUCTS OF PROTEIN HYDROLYSIS: Infraproteins. Proteoses. Peptones. Polypeptides. . Neither of these systems of classification is free from objection. To them both the general objection applies, that the distinctions drawn are largely based upon variations in physical behavior which do not necessarily correspond to fundamental differences of chemical architecture while, on the other hand, many protein or protein-like substances are known which display intermediate characteristics, or individual peculiarities which render their inclusion in any of the classes enumerated, a matter of more or less arbitrary opinion. In particular the defect of the American system lies in the rather intangible dis- tinctions which are made between various classes of primary protein derivatives, and the inclusion of coagulated proteins which are almost certainly derived from native proteins by abstraction of water from the molecule, in the same class with proteans and metaproteins which are derived from native proteins by partial hydrolysis, is unfortunate. The English classification has the merit of simplicity, but it would be more advisable to include the phosphoproteins among the conjugated proteins, as in the American classification, and to add a fourth group to accommodate the coagulated proteins. The term prolamine is also preferable to the designation a alcohol-soluble proteins," because it draws attention to the high content of the amino-acid proline which characterizes these proteins more fundamentally than the physical property of solubility in eighty per cent, alcohol. The salient characteristics of these various classes of protein sub- stances are as follows: 126 THE PROTEINS AND THE AMINO-ACIDS I. THE SIMPLE PROTEINS. Protamines. The protamines are the simplest proteins which are as yet definitely known to occur in nature. They are found in sperma- tozoa, and especially in the spermatozoa of fishes in combination with nucleic acid, forming a simple type of nucleoprotein. They are pre- dominantly basic substances, indeed so strongly basic that a solution of salmine (the protamine from salmon spermatozoa) reacts alkaline to litmus and absorbs carbon dioxide from the air, forming carbonates of the protamine. The acid function of these substances is correspond- ingly weak, although they are, like all proteins, amphoteric acids, and in the presence of excess of strong bases will partially combine with them. The protamines are soluble in water and form definite salts with acids which are coagulated by alcohol and thrown out of solution without decomposition, the combined acid being carried down quanti- tatively with the protein. They yield a pink biuret-reaction resembling in this respect the derivatives of the partial hydrolysis of other native proteins. They yield, when completely hydrolyzed, a preponderating proportion of diamino-acids. Histones. The histones are somewhat more complex and colloidal in character than the protamines, and their basic function is less marked. They are still predominantly basic, however, and occur, in cellular tissues, combined with nucleic acid, and in the chromoprotein, hemo- globin, combined with a colored acid radical, Hematin. They are soluble in dilute acids or dilute solutions of the strong bases, but are precipitated from acid solutions by the addition of ammonia. Albumins. The albumins are markedly colloidal substances which are soluble in distilled water and in salt solutions. The basic function is almost equal to the acid function. Representative examples are egg- albumin and the albumin which is found in blood-serum. They are coagulated by saturation of their solutions with ammonium sulphate. Globulins. The globulins are very decidedly colloidal substances passing, for example, with difficulty, or not at all through clay filters. They are insoluble in distilled water, but are soluble in dilute solutions of strong acids or bases, or of inorganic salts. The acid function pre- dominates slightly over the basic, so that they neutralize bases more readily and completely than acids. Typical examples are afforded by serum-globulin, the globulin which is precipitated from egg-white by dilution with distilled water, and a variety of vegetable proteins such as edestin, obtained from seeds -of hemp (Cannabis Sativa). They are coagulated by half-saturation of their solutions with ammonium sulphate or complete saturation with magnesium sulphate. Glutelins. The glutelins are a group of vegetable proteins of which only two, the Glutenin of wheat and the Oryzenin of rice have as yet been prepared in sufficient quantity, and purity to render analysis and characterization possible. They are insoluble in water or dilute THE SIMPLE PROTEINS 127 salt solutions but they are soluble in dilute solutions of strong bases or acids. Prolamins. The prolamins are soluble in 70 per cent, to 90 per cent, alcohol. They are insoluble, or nearly so, in distilled water, but dis- solve readily in dilute solutions of strong acids or bases. They occur in a variety of grains, typical members of the group being Gliadin, found in the seeds of wheat and rye, Hordein found in the seeds of barley and Zein found in the seeds of maize. They are characterized by the high proportion of Proline which they yield when hydrolyzed. Scleroproteins. The scleroproteins, termed albuminoids in Ameri- can and Continental European publications, form a very heterogeneous group of substances. The various proteins which we have hitherto been considering are either constituents of cellular tissues, concerned in the life and maintenance of the protoplasm, or else they form reserve-materials which are sooner or later to be called upon to supply the requirements of protoplasm. Quite other is the function of the scleroproteins, for these are proteins of a primarily structural or architectural rather than nutritional significance. They are binding, cementing and supporting substances which contribute in a mechanical rather than in a chemical fashion to the furtherance and maintenance of life. They occur especially in the various connective tissues, and corresponding with their peculiar function, we find that they display a variety of physical characteristics, distinguishing them from the proteins of cellular origin, and also distinguishing the individual members of the group very sharply from one another. Typical members of this class are Gelatin and its parent-substance Collagen which forms the chief constituent of white fibrous connective tissue, and also the main organic constituent of bones. On boiling, especially in the presence of dilute acid, Collagen yields the cleavage-product Gelatin. Collagen itself is insoluble in water, salt solutions and dilute acids or alkalies, but gelatin swells in cold water and dissolves in warm water, forming jellies on cooling if the solutions are sufficiently concentrated. Reticulin, occurring in the reticular fibrous tissues of glands differs from collagen in several respects, notably in containing phosphorus. Keratin is another scleroprotein and forms the chief constituent of the horny epidermal structures, hair, wool, nails, hoofs, horns, feather, tortoise-shell, etc. A form of keratin, Neurokeratin, also occurs in nervous tissues. Keratin is insoluble in water, dilute acids or alkalies and salt solutions; it is soluble with difficulty in strongly alkaline solutions. It is also characterized by the high percentage of sulphur w r hich it contains and which is attributable to the amino-acid radical Cystine. Elastin forms the chief constituent of the elastic fibers of connective tissue. It is distinguished by its elasticity and tensile strength and also by its extreme insolubility, being soluble only in strong caustic alkalies or concentrated mineral acids. Fibroin, the substance forming 128 THE PROTEINS AND THE AMINO-ACIDS the core of silk fibers, is characterized by possibly even greater tensile strength, while it is somewhat more readily dissolved by concentrated acids and alkalies than elastin. Sericin or silk gelatin forms the outer coating of the silk fiber, and is sticky when freshly secreted, so that it enables intersecting and adjacent fibers to adhere. It is soluble in hot water, and the solution resembles a solution of ordinary gelatin in that, if concentrated, it gelatinizes on cooling. Finally, Spongin forms the chief part of the ordinary sponge from which the originally living protoplasm has been extracted. It is insoluble in acids but soluble in concentrated alkalies. Some of the spongins contain iodine as an integral part of the molecule. The scleroproteins are for the most part incomplete proteins in the sense that they do not yield when completely hydrolyzed, all of the amino-acids that we are accustomed to obtain from the more typical proteins of cellular tissues. Thus gelatin yields neither tyrosine nor trypto;phane, elastin and fibroin yield neither aspartic nor glutamic acids, and spongin yields neither tyrosine nor phenylalanine. The extraordinary variety of physical properties and peculiarities displayed by the various scleroproteins reveals the possibility of sub- stances of very unique physical characteristics being derived from proteins, and would point to the ultimate possibility of very important industrial applications of such derivatives. At the present time, horny derivatives of the protein of milk, casein, are extensively used in the manufacture of substitutes for ivory, celluloid and bone. The animal proteins, being among the most expensive foodstuffs we require, can never be employed very extensively in the industries, except ing when they form by-products of the foodstuffs-industry, as in the manufacture of glue from slaughter-house or fish- wastes, and of casein products from skimmed milk. Certain vegetable proteins might, however, be rendered relatively cheap and abundant and offer an interesting field for the investigation of the special physical characteristics of their derivatives. H. THE CONJUGATED PROTEINS. Nucleoproteins. The conjugated proteins are complex substances formed by the union of a protein with a non-protein radical, which may be termed the Prosthetic Group. The Nucleoproteins, for example, are compounds of Nucleic Acids, which are substituted phosphoric acids containing carbohydrate and nitrogenous radicals, with a protein which plays the part of a base in the compound. These compounds are the most characteristic constituents of the nuclei of cells. When the protein constituent is a histone, the compound is termed a Nucleo- histone. The nucleoproteins are insoluble in distilled water, but soluble in dilute alkalies from which solutions they are precipitated by weak acids, such as acetic acid or carbon dioxide. They are as a rule incom- pletely digestible by the pepsin of gastric juice, leaving an indigestible THE CONJUGATED PROTEINS 129 residue which still contains protein and is termed Nuclein. All of the nucleoproteins appear to be very closely associated, or possibly com- bined with Iron. Glucoproteins. In the glucoproteins the prosthetic group is either, an ammo-carbohydrate, a polysaccharide derived from glucosamin or acetylated derivatives of glucosamin, or else chondroitin-sulphuric acid (see p. 91). The glucoproteins are subdivided into Mucins, Mucoids and Chondroproteins. The true mucins yield extraordinarily glutinous or mucilaginous solutions from which the mucin is precipi- tated by acetic acid. The mucoids are not precipi table by acetic acid and do not, as a rule, yield such highly viscous solutions as the mucins. The chondroproteins are insoluble in water, but are soluble in dilute alkalies, from which solutions the protein is precipitated by neutral- ization with strong acids or by an excess of acetic acid. They yield Chondroitin-sulphuric Acid on hydrolysis, a substituted sulphuric acid formed by the union of a molecule of Chondroitin with a molecule of sulphuric acid. Chondroitin resembles gum-arabic in physical char- acteristics, and is a compound of Glucuronic Acid and Glucosamin. The mucins occur in mucous secretions, as for example the secretions from the skin-gland of snails or slugs. Mucoids are found in connec- tive tissues, in the vitreous humor of the eye and in the white of egg (ovomucoid) . The chondroproteins occur especially in cartilaginous tissues, and in the interstitial substance of connective tissue. Chondro- proteins are also found in the accumulations of colloidal material which characterize the "amyloid degeneration" of certain organs under pathological conditions. Phosphoproteins. The phosphoproteins are proteins which yield phosphoric acid when hydrolyzed. The most typical example of this group is Casein, the chief protein constituent of milk, but phospho- proteins also occur in a variety of vegetable tissues, and in the yolk of egg (ovovitellin) . They are predominantly acid in character, as might be expected, not only from their content of phosphoric acid, but also from the fact that they yield a high proportion of dicarboxylic amino-acids on hydrolysis. Chromoproteins. The chromoproteins are compound proteins in which the prosthetic group is colored. The most typical examples of the group are Hemoglobin, the red coloring-matter and oxygen-carrier of blood, in which the prosthetic group is a complex iron-containing organic acid Hematin, and Hemocyanin, a blue pigment containing copper which plays a role corresponding to that of hemoglobin in the Arachnidce and Crustacea. The chromoproteins, hemoglobin and hemo- cyanin, are exceptional among proteins in the relative ease with which they are obtainable in crystalline condition. The protein radical in hemoglobin is a predominantly basic protein, known as Globin and related to the histones. Lecithoproteins. The lecithoproteins are compound proteins in which the prosthetic group is a phospholipin. This is rather a con- 9 130 THE PROTEINS AND THE AMINO-ACIDS jectural group of substances, for although proteins associated with phospholipins have been prepared from yolk of egg, and from vegetable tissues, it is not yet certain whether the phospholipin is an integral part of the protein molecule, or merely a contamination which "is physically adherent to it. Evidence of an electrochemical character has demonstrated, however, that compounds between lecithin and proteins are formed when the two substances are mixed in aqueous solution, and we may infer that similar compounds may not improbably exist in nature. HI. THE PRODUCTS OF PROTEIN HYDROLYSIS. Infraproteins. The infraproteins are substances produced in the initial stages of protein hydrolysis which still retain the characteristic properties of the proteins. Examples are the Acid- and Alkali-albumin- ates, formed from albumins by gentle heating in acid or alkaline solu- tions, and which differ from albumins in being insoluble in neutral distilled water. Other examples are Paracasein, formed by the action of rennet or weak pepsin upon casein, and the Paranucleins which are formed by the partial digestion of a variety of phosphoproteins. Proteoses. The proteoses are hydrolytic cleavage-products of the proteins which have lost the characteristic protein property of being coagulable by heat, but they retain the coagulability by ammonium sulphate. They are usually subdivided into Primary Proteoses which are coagulable by half-saturation of their solutions with ammonium sulphate, and Secondary or Deuteroproteoses which are coagulated by complete saturation of their solutions with ammonium sulphate. The majority of the proteoses are coagulable by alcohol, but certain of them are soluble in alcohol. They yield a reddish violet or pink biuret- reaction. A considerable number of the proteoses are toxic when injected into the circulation, while the native proteins with a few marked exceptions, such as the Ricin in castor-oil beans (Ricinus Communis) are non-toxic. On the other hand the native proteins are Antigenic that is, they give rise, on repeated injection into the circulation of animals, to sub- stances which circulate in the blood serum and have the property of precipitating the particular protein against which the animal has been immunized. The proteoses on the contrary are as a rule non- ant igenic. Peptones. The peptones are still simpler products of the hydrolytic cleavage of proteins. They are slightly diffusible, and they are inco- agulable either by heat or by ammonium sulphate. They are, however, precipitable by tannic acid, phosphotungstic acid or lead acetate. They are usually coagulable by alcohol, although certain peptones, especially when combined with acid, are not coagulable by alcohol. They yield a clear pink biuret-reaction, and are non-antigenic and, as a rule, non-toxic. THE END-PRODUCTS OF PROTEIN HYDROLYSIS 131 IV. THE COAGULATED PROTEINS. The coagulated proteins may be subdivided into two classes, namely, those in which the coagulation-process has gone so far as to be irrevers- ible, so that the coagulum cannot be brought back into solution again without preliminary decomposition into simpler substances, and those in which the coagulum remains soluble after removal of the coagulating- agent and in which the coagulation-process has therefore remained reversible. The majority of the heat-coagulated proteins belong to the first class, although the incipient stages of heat-coagulation are some- times reversible. On the other hand the coagula produced by alcohol or by ammonium sulphate belong to the second class, although in some instances after more or less prolonged contact with alcohol the coagula produced by alcohol become irreversible. The polypeptides or chains of amino-acids out of which proteins are built up, form anhydrides with exceptional ease, either by internal neutralization of carboxyl- and amino-groups, or by the condensation of several molecules, and this tendency increases with increasing length of the amino-acid chain. We can hardly suppose, therefore, that this property has been lost in the much more bulky amino-acid complexes which constitute the proteins. On the other hand the agencies which bring about coagulation are all of such a character (heat, alcohol and concentrated salts) as to suggest that the with- drawal of water from the protein is the chemical basis of the coagulation- process. It appears very probable, therefore, that coagulation is due to the formation of protein anhydrides, and that the irreversible coagula are those in which the anhydride-formation has proceeded furthest. THE END-PRODUCTS OF PROTEIN HYDROLYSIS: THE AMINO-ACIDS. Decomposition of proteins into simple crystal! izable substances may very readily be brought about by a variety of agencies, but the only methods of decomposition which yield easily interpretable results are those which bring about Hydrolysis, or decomposition of the mole- cule by successive splittings with the addition of the elements of water. Hydrolysis of the proteins (autohydrolysis) will take place spontaneously in neutral protein solutions or even if precipitated proteins be left in long-continued contact with neutral and sterile water. The process is, however, greatly accelerated by the application of heat, especially by temperatures considerably exceeding the tem- perature of boiling water, or by catalyzers, of which the most efficient are acids, alkalies and the protein-digesting (proteolytic) enzymes. Whatever the means of hydrolysis employed, however, the end-result, provided the hydrolysis has been complete, is the same, namely, the production of a mixture of amino-acids. 132 THE PROTEINS AND THE AMINO-ACIDS Incomplete hydrolysis, however, results in the production of a number of intermediate substances, variously designated, in the order of decreasing complexity, Proteoses (Albumoses) , Peptones, Polypep- tides and Dipeptides. The hydrolysis of the proteins, therefore, occurs in stages, just as, in the hydrolysis of starch, intermediary stages (the dextrins and maltose) are passed through before the attainment of the last stage of hydrolysis and the quantitative conversion of the starch into glucose. It is not certain, however, whether the various intermediate pro- ducts of protein hydrolysis represent successive stages of hydrolysis or whether in some instances comparatively simple products may not be split off from the proteins or proteoses, leaving complex residues, so that complex and simple intermediate substances are produced simultaneously. Probably both types of cleavage occur at different points in the protein molecule. Those linkages which are most acces- sible to the action of the particular catalyzer employed will be dis- rupted firstj and if some of them chance to lie near the extremities of the molecule, simple products and a complex residue will result, while disruption of more internal linkages will break the molecule into parts of more equal weight and complexity. It was early recognized that amino-acids form the chief part of the decomposition-products which result from the hydrolysis of protein. The separation of the individual amino-acids from one another, and their quantitative estimation, was a much more difficult matter. The first attempts to isolate individual amino-acids from the mixture which the complete hydrolysis of a protein yields, depended upon the frac- tional crystallization, either of the free amino-acids or of their salts. Except in the case of the very slightly soluble amino-acids, such as tyrosine, these methods were not even approximately quantitative, and even the isolation and identification of a given amino-acid could only be effected with certainty when that acid was present in relatively large amounts. The attainment of our present relatively extensive knowledge of the nature and quantities of the amino-acids which result from protein hydrolysis, is an achievement of the past twenty years, and we owe it in the first place to the investigations of Kossel and of Emil Fischer and their pupils. The various amino-acids which are yielded by the proteins are limited in number, and probably do not exceed eighteen or nineteen. These however, fall into several very distinct classes, namely: Monoamino-monocarboxylic acids, general formula: H 2 N.R.COOH Monoamino-dicarboxylic acids, general formula: /coon H 2 N.R THE END-PRODUCTS OF PROTEIN HYDROLYSIS 133 Diamino-monocarboxylic acids, general formula: , \ R.COOH Diaminohydroxy-monocarboxylic acids. Heterocyclic compounds, i. e., amino-acids containing a ring of atoms, one or more nitrogen atoms being included in the ring. The first necessary step in the analysis of the amino-acids produced by the hydrolysis of protein, consists in the separation of the diamino- acids from the monoamino-acids. This may be accomplished by means of phosphotungstic acid, which precipitates the diamino-acids while the monoamino-acids are left in solution. The diamino-acids Arginine and Histidine may be separated in the form of their silver salts, while the remainder of the precipitate produced by phosphotungstic acid consists of the diamino-acid Lysine. The monoamino-acids may be estimated by evaporating the whole mixture to dryness in vacuo and then dissolving the mixture of acids in alcohol, and passing into the solution dry hydrochloric acid gas which catalyzes the formation of alcohol esters of the amino-acids. These esters are volatile and may be separated into fractions each containing only a small number of acids, by means of fractional dis- tillation in vacuo. The esters in each fraction are then reconverted into free acids and alcohol by hydrolysis, and the individual acids separated and estimated by special methods adapted to the peculiar properties of each of the acids present in the particular fraction con- cerned. The difficultly soluble acids, Tyrosine, Cysline and Diamino- trioxydodecanic Acids are separated from the digest before ester ification. This method, due to Emil Fischer, is laborious and inaccurate, but it greatly surpasses the methods which were formerly in use, and which did not even permit a partial separation of the various mono- amino-acids in a protein digest. The method permits the accurate quantitive determination of only five acids, namely, the diamino-acids histidine, arginine and lysine, and the monoamino-acids tyrosine and cystine. The estimates of the other acids are only approximate, and must be regarded as minimum values, since it has been found that in a known mixture of amino-acids it is only possible to account for about two-thirds of the nitrogen, by Fischer's method. For many purposes, in which a knowledge of the total proportion of nitrogen present in the form of monoamino-acids suffices, Fischer's method has now been largely superseded by the method of Van Slyke which is described below (p. 144), but if we desire to ascertain approximately the quantity of the individual monoamino-acids contained in a protein digest, Fischer's method, or modifications of it, still affords the only available procedure. 134 THE PROTEINS AND THE AMINO-ACIDS if By these various methods the following ammo-acids have been isolated from among the products of hydrolysis of various proteins: A. Monoamino-monocarboxylic acids. 1. Glycine, or ammo-acetic acid: 2. Alanine, or a-amino-propionic acid: CH 3 .CH(NH 2 ).COOH 3. Valine, or a-amino-iso-valerianic acid: CHs CH.CH(NH 2 ).COOH CH 3 4. Leucine, or a-amino-isocaproic acid: CH 3 CH.CH 2 .CH(NH 2 ) .COOH CH 3 5. Isoleucine, or a-amino-/3-methyl-/3-ethylpropionic acid : CH 3 \ CH.CH(NH 2 ).COOH C 2 H 6 6. Caprine, or Glycoleucine, or a-amino-normal-caproic acid: CH 3 .CH 2 :CH 2 .CH 2 CH(NH 2 ) COOH 7. Phenylalanine, or 0-phenyl-a-amino-propionic acid : C 6 H 5 CH 2 .CH(NH 2 )COOH 8. Tyrosine, or jS-parahydroxyphenyl-a-amino-propionic acid: HO.C 6 H 4 .CH 2 .ciNH 2 ).COOH 9. Serine, or jS-hydroxy-a-amino-propionic acid: CH 2 (OH) .CH(NH 2 ) .COOH 10. Cystine, or dicysteine or di- (/3-thio-a-aniino-propionic acid) : HOOC.CH.(NH 2 ).CH 2 S SCH 2 .CH(NH 2 ).COOH V THE END-PRODUCTS OF PROTEIN HYDROLYSIS 135 B. Monoaminodicarboxylic acids. 11. Aspartic acid, or amino-succinic acid: HOOC.CH 2 CH(NH 2 ) .COOH . 12. Glutamic acid, or a-aminoglutaric acid: HOOC.CaHjCHaCHCNHiO.COOH C. Diaminomonocarboxylic acids. 13. Arginine, or a-amino-3-guanidine-valerianic acid: x NH 2 HN = C NH, .CH 2 CH 2 .CH(NH 2 ) .COOH 14. Lysine, or a-oj-diaminocaproic-acid : C WvH 2 N.CH 2 .CH 2 .CH 2 .CH(NH 2 ) .COOH D. Heterocyclic amino-acids. .15. Histidine, or /3-immazolyl-a-amino-propionic acid: N NH CH= =C.CH 2 .CHCNH 2 ).COOH * 16. Proline, or pyrrolidine carboxylic acid: CH 2 - - CH 2 CH 2 CH.COOH 17. Oxyproline, or hydroxy-a-pyrrolidine-carboxylic acid Either:' HOCH CH 2 CH 2 CH.COOH or: CH 2 - CHOH CH 2 CH.COOH 18. Tryptophane or /3-indole-o:-aminopropionic acid C.CH 2 .CH. (NH 2 ) .COOH /\ C 6 H 4 CH / \/ NH 136 THE PROTEINS AND THE AMINO-ACIDS In addition to these acids two hydroxy-diamino-acids have been iso- lated from among the cleavage-products of one protein, namely, casein. The very great variety of proteins and protein derivatives which exist in nature are therefore constructed out of a relatively small and limited number of amino-acid building-stones, differing proportions and arrangements of these components being responsible for the wide variety of characteristics displayed by the native and derived proteins. In many instances a definite parallelism can be traced between the chemical and physical behavior of the proteins and their amino-acid content. Thus, the Albumins, which are soluble in distilled water and are not coagulated by half-saturation of their solutions with am- monium sulphate, contain no glycine, while the Globulins, which (when uncombined "with acids or bases) are insoluble in distilled water and are coagulated by half-saturation of their solutions with ammonium sulphate, do contain this amino-acid. The alleged transformation of serum albumin into globulin by warming in alkaline solutions observed by Moll, and not infrequently quoted, is therefore, an impossibility, since it would involve the synthesis of amino-acetic acid and its union with the albumin molecule which could not be brought about by any such simple procedure. The product actually obtained by Moll was an infraprotein, alkali-albuminate, which mimics globulin in being insoluble in neutral water, but differs from it in fundamental consti- tution. The alcohol-soluble vegetable proteins (Prolamines) contain a trace (probably attributable to associated impurities) of glycine, and some of them contain no glycine, their content of diamino-acids is very small, while their content of glutamic acid and of proline is very high. The phosphoproteins, Casein and Vitellin, are also rather high in glutamic- acid content. Gelatin is characterized by its high glycine content and Keratin by its high content of cystine. The Histones, which are pre- dominantly basic substances, contain about thirty per cent, of diamino- acids, while the Protamines, which are still more predominantly basic, contain only small amounts of monoamino-acids, Salmine (from salmon sperm) containing over eighty per cent, of arginine, while Sturine (from sturgeon sperm) contains sixty-seven per cent, of its nitrogen as arginine, ten per cent, in the form of histidine, and from six to seven per cent, in the form of lysine. /" The amino-acids are white, crystalline, readily diffusible substances / and the crystal form is characteristic for each amino-acid. The \ crystal forms of glycine, leucine and histidine are shown in the V accompanying figures (3-5). The amino-acids are usually readily soluble in water, cystine and tyrosine affording exceptions to this rule. They are, with the exceptions of proline and oxyproline, insoluble in alcohol and ether. They have high melting-points and melt with decomposition, splitting off carbon dioxide. With the exception of glycine the amino-acids are optically active, some of them being dextrorotatory and others levorotatory. TH'E END-PRODUCTS OF PROTEIN HYDROLYSIS 137 FIG. 3. Glycine crystals. (After Hawk.) FIG. 4. Leucine crystals. (After Funke.) k FTG. 5. Histidine monochloride crystals. 138 THE PROTEINS AND THE AMINO-ACIDS The amino-acids, since they contain trivalent nitrogen and a car- boxyl-group, are simultaneously bases and acids, in other terminology are Amphoteric Acids. They form crystalline salts with metallic bases and with mineral acids. On treatment with nitrous acid the amino-acids lose their amino- group, which is replaced by a hydroxyl-group as follows : CH 2 .NH 2 CH 2 OH + HNO 2 =| + N 2 + H 2 O COOH COOH It will be observed that all of the amino-acids obtained in the hydrolysis of proteins are a-amino-acids, that is, an ami no-group is attached to the carbon atom immediately adjacent to the carboxyl- group. This is probably a fact of great significance, since, as we shall see, the proteins are formed by the union of long chains of amino-acids, linked together by means of their amino- and carboxyl -groups. These groups being closely adjacent, the resultant chains are shorter, and the weight of the other radicals in the molecule more evenly distributed than would be the case if the carboxyl- and amino-groups were separated by a long chain of carbon linkages, and the possibility of such heavy compounds as the proteins possessing sufficient stability to permit their formation probably resides in this device for shortening the chain of serial linkages. Corresponding to this view we find that the oo-amino- group which is also present in lysine, is not united in proteins to any carboxyl-group but remains free and reacts with nitrous acid just as the amino-acid does. THE SYNTHESIS OF PROTEINS. The marked predominance of amino-acids among the products of protein hydrolysis long ago led biological chemists to surmise that the amino-acid structure, or some derivative of that structure, must be represented in a high degree in the protein molecule, and it was in following up this clue that Schiitzenberger in 1888 carried out one of the earliest and most successful attempts to synthesize bodies of a protein character. Recognizing that the decomposition of proteins into amino-acids is essentially a phenomenon of hydrolysis, he regarded .dehydration as an essential feature of any attempt at protein syn- thesis, while the abundance of amino-acids among the products of protein hydrolysis, and the presence therein, as he thought, of bodies related to urea, led him to believe that protein synthesis must consist in the linkage of amino-acids with molecules of urea and the elimination of water. Accordingly amino-acids were mixed with urea and phos- phorus p'entoxide and heated to 125 C. The product was a pasty solid, soluble in water and readily coagulated by alcohol. It was furthermore precipitated from aqueous solutions by the usual protein precipitants and gave the biuret- and xanthoproteic reactions. THE SYNTHESIS OF PROTEINS 139 This experiment of Schiitzenberger's left us, however, very much where we were, so far as real knowledge of the structure of the protein molecules is concerned. The knowledge of the fact that a mixture of amino-acids and urea yields, under certain treatment, a body or bodies more or less closely resembling the proteins, furnished us with little or no information regarding the structure of the protein molecule which we did not already possess in the fact that the disintegration- products of the proteins are predominantly amino-acids. Prior to Schiitzenberger, Grimaux, in 1881, had shown that condensatioa- products of aminobenzoic acid and aspartic acid resemble the proteins in many of their properties; but these experiments also threw no light upon the structure of the protein molecule beyond emphasizing the already sufficiently evident probability that the amino-acid grouping plays an important part in the building up of the protein molecule. The clue which led, through a series of remarkable researches, to our present comparatively extensive knowledge of the groupings within the protein molecule, was obtained in 1883 by Curtius when he discovered that ethyl glycocollate (the ethyl ester of glycine) in watery solution tends to form Glycine Anhydride: (In the absence of water) H 2 N.CH 2 .COOH + C 2 H 6 OH = H 2 N.CH 2 COOC 2 H 6 + H 2 O (Glycine) + (Ethyl alcohol) = (Ethyl glycocollate). (In the presence of water). H 2 N.CH 2 COOC 2 H 6 + Hj-N.CH^COOCzHs = (Ethyl glycocollate) + (Ethyl glycocollate) . CH 2 N HL O = C C = O + 2C 2 H 6 OH ^NH CH/ (Glycine anhydride) + (Ethyl alcohol) . Obviously, if the closed ring representing the glycine anhydride molecule could be opened up without destroying the stability of the molecule, a new amino-acid would be formed, one degree more complex than the original amino-acid (glycine) . This possibility was realized by Emil Fischer, who found that if the glycine anhydride which is thus prepared be boiled for a short time with concentrated hydro- chloric acid, the following change occurs: /CH 2 .NHx /CH ? .NH 2 .HC1 O = C C - O + HC1 + H 2 O = O = \ / (Glycine anhydride.) (Glycyl-glycine chloride.) 140 THE PROTEINS AND THE AMINO-ACIDS On now treating the glycyl-glycine chloride with silver oxide, silver chloride is precipitated and free Glycyl-glycine is obtained. If, how- ever, the glycine anhydride be originally treated with Alcoholic instead of with an aqueous solution of hydrochloric acid, the ethyl ester of glycyl-glycine is obtained: xCH 2 .NHv /CH 2 NH 2 C C = O + C*H 6 OH = O=C \ / \ (Glycine anhydride.) (Glycyl-glycine ester.) It would appear, therefore, as if we had only to repeat this cycle of operations indefinitely in order to secure the most complex poly- amino-acids; but this is not so easy as it might appear at first sight; the instability of polyamino-acids consequent upon the high reactivity of the NH 2 group, and the consequent difficulty of obtaining simple anhydrides renders this procedure impossible. Moreover the anhy- dride-ring is in many cases (e. g., leucine anhydride) very difficult to break up when it has once been formed. In the search for methods of overcoming these difficulties Fischer found that the instability of the amino-acids could be eliminated by the introduction of radicals into the NH 2 group, and he and Fourneau synthesized phenylcyanate-glycyl-glycine (C 6 H5.NH.CO-NHCH 2 CO -NHCH 2 COOH) and carboxethyl-glycyl-glycine ester (C 2 H 5 O.OC. NH.CH^CO-NH.CH^COOC,!^ which are both chemically stable bodies. In subsequent investigations Fischer found that, by gentle heating, combination between the esters of the carboxethyl-amino- acids and other amino-acids could be directly brought about, and in this way carboxethyl-diglycyl-leucine ester was formed: C 2 H 6 OOC.NH.CH 2 CO.NH.CH 2 .CO.NH.CH.(C4H 9 )CO.OC2H 5 . The difficulty was here encountered, however, that the carboxethyl- group having been once introduced, cannot be eliminated again. The method which was devised to overcome this difficulty was extremely ingenious. The introduction of a radical into the NH 2 group appeared to be a necessity, forced upon us by the impossibility of otherwise securing simple anhydrides of the acids. It occurred to Fischer, however, that the radical thus introduced into the NH 2 group might itself be made a carrier of amino-acid groups into the molecule. This anticipation proved to be correct. The radical which was first utilized was the chloracetyl group (C1CH 2 .CO ). When chloracetyl chloride is allowed to act upon glycyl-glycine ester (ob- tained by the methods described above), chloracetyl-glycyl-glycine- ester is obtained: C1CH 2 COC1 + H 2 N.CH 2 CO.NH.CH 2 COOC 2 H 6 = Chloracetyl chloride) + (Glycyl-glycine ester) = C1CH 2 .CO.NH.CH 2 .CO.NH.CH 2 .COOC 2 H 5 + HC1 (Chloracetyl glycyl-glycine ester) . THE SYNTHESIS OF PROTEINS . 141 By saponification of this ester, free chloracetyl-glycyl-glycine is obtained. On now treating this with a concentrated aqueous solution of ammonia, the chlorine atom in the chloracetyl group becomes, by a usual reaction, replaced by an amino-group, and thus Diglycyl-glycine is obtained: C1CH 2 .CO.NH.CH 2 .CONH,CH2COOC 2 H 5 + 2NH 3 = (Chloracetyl glycyl-glycine ester) H 2 N.CH 2 .CONH.CH 2 CONH.CH 2 COOC 2 H 5 + NEUC1 (Diglycyl-glycine ester). In other words, the chloracetyl-group, introduced to protect the NH 2 -group of the amino-acid is, after it has performed its protective function, itself transformed into an amino-acid-group, through the replacement of the halogen atom by NH 2 . Obviously, other halogen- containing acid groups may be used in place of chloracetyl, and in this way a great variety of amino-acid-groups can be introduced into the NH2-group. Among others the following are employed: Chloracetyl chloride for the introduction of glycyl. a-Bromopropionyl chloride for the introduction of alanyl. a-Bromisocapronyl chloride for the introduction of leucyl. a-Phenylbromopropionyl chloride for the introduction of phenyl- alanyl. a-S-Dibromovaleryl chloride for the introduction of prolyl. By this method the chain of amino-acids is lengthened at the amino- group end. Theoretically it appeared possible to also lengthen the chain at the carboxyl-end of the molecule, by acting upon the esters of the amino-acids with the acid chlorides of other amino-acids. Until 1904, however, the acid chlorides of amino-acids were unknown, and all attempts to prepare them had failed, owing to the same reason which limits the use of the first method of synthesizing poly-amino- acids described above, namely the reactivity of the NH 2 -group. It will be recollected that Fischer found that the NH 2 -group could be protected by the introduction of radicals, and, utilizing this fact, in 1904 he succeeded in devising a method of preparing the acid chlorides of the amino-acids. The acid chlorides thus prepared, react with the esters of other amino- or polyamino-acids to form polyamino-acid chains of greater length. Thus: C4H 9 CHBr.CO.NH.CH 2 .COCl + 2NH 2 .CH 2 .COOC 2 H 5 = (Bromisocapronyl-glycyl chloride) Glycine ester) + C4H9CHBrCONH.CH 2 CO.NH.CH 2 COOC2H 5 (Glycine ester hydrochloride) (Bromisocapronyl-glycyl-glycine ester) Subsequent saponification of the bromisocapronyl-glycyl-glycine ester and treatment with ammonia yields the polyamino-acid or (tripeptide) , Leucyl-glycyl-glycine : aH 9 CH(NH 2 )CO.NH.CH 2 CONH.CH2.COOH 142 THE PROTEINS AND THE AMINO-ACIDS If the bromisocapronyl-glycyl chloride be made to act upon glycyl- glycine ester, and the product treated with ammonia, the tetrapeptide Leucyl-diglycyl-glycine results: C4H9.CH(NH 2 )CO.NHCH 2 .CO.NHCH 2 CO.NHCH 2 COOH By these methods, and modifications of these methods, Fischer and others have succeeded in building up long chains of amino-acid-groups, these chains being collectively termed by Fischer, Peptides. Chains consisting of two links, i. e., combinations of two amino-acids are designated Dipeptides; such, for example, are glycyl-glycine, alanyl- alanine and leucyl-leucine, chains consisting of three links are termed Tripeptides, such being, for example, diglycyl-glycine and leucyl-glycyl- glycine. Chains consisting of four links are termed Tetrapeptides, and so on, the higher members of the series being collectively termed Polypeptides. The surpassing interest of these investigations lies in the fact that many of the polypeptides are considered to be, in all probability, identical with certain of the natural peptones derived from proteins by partial hydrolysis, while others probably merit inclusion among the proteins themselves. Thus the Octadecapeptide, 1-leucyl-triglycyl- 1-leucyl-triglycyl-l-leucyl-octaglycyl-glycine, and the Tetradecapeptide, 1-leucyl-triglycyl-l-leucyl-octaglycyl-glycine so closely resemble, in general properties, the ordinary proteins, that they would undoubtedly have been classed among the proteins had they been first met with in nature. Thus, they give the biuret-reaction, and form opalescent watery solutions, and the tetradecapeptid is coagulated by ammonium sulphate and precipitated by tannic acid and by phosphotungstic acid. As they do not contain tyrosine, tryptophane, phenylalanine or cystine, they fail to give such protein color reactions as depend upon the presence of these groups. The molecular weight of the octadecapeptide is 1213, and the sub- stitution of phenylalanine, tyrosine and cystine in the place of glycine groups would increase this weight two or three times, giving a value which is of the same order of magnitude as the more modern esti- mations of the (minimal) molecular or combining-weights of many of the natural proteins. A whole series of the polypeptides give the typical peptone biuret reaction (pink), and such as contain tyrosine also give Millon's reac- tion. The biuret-reaction is, with the glycine compounds, first encoun- tered in the tetrapeptide, but it is given by tripeptides built up to include other amino-acids. The biuret-reaction is, generally speaking, more intense the greater the length of the polypeptide chain. The majority of the polypeptides are readily soluble in water, and such as are soluble in water with difficulty, are readily soluble in dilute mineral acids and alkalies, with which they combine; they are less soluble in solutions of acetic acid. As a rule they are insoluble in absolute alcohol, but in alcohol containing a little watery ammonia PEPTIDES AMONG PROTEIN HYDROLYSIS 143 they may be soluble, in which case they are precipitated on boiling off the ammonia. Under conditions involving dehydration, as for example heating, or treatment of the esters with alcoholic ammonia, the dipeptides are converted into anhydrides which are ring-compounds, designated Dikelopiperazines, and having the general formula: /CH.R.CCK NH I > Br > NO 3 > Cl > CH 3 COO while the cations inhibit coagulation in the order: Li > Na > K > NH 4 > Mg We have seen that, in order that Precipitation of a protein by salts may occur, the protein must be ionized, but for Coagulation this condi- tion is not requisite. In determining the rate of precipitation the valency of the precipitating ion is of prime importance; in determining the rate of coagulation it is of comparatively subordinate importance. For precipitation very low concentrations of the precipitating salt suffice, intermediate concentrations frequently, and indeed usually redissolve the precipitate, and for coagulation high concentrations of the salt are required. This latter fact, and the fact that the presence of coagulating-salts aids coagulation by alcohol and by heat, suggests, as it did to Hofmeister, that coagulation is dependent upon Dehydration of the protein. Starting from the observation of Jones and Ota, that certain salts when dissolved in water, produce an abnormal depression of the. freezing-point, H. C. Jones and his pupils have built up a very large body of evidence for the existence of hydrates (or " solvates") of sub- stances in solution. These investigators find that both ions and undis- 166 COMPOUNDS, OF THE PROTEINS sociated molecules can form "solvates," and that these hydrates or "solvates" are readily decomposed at temperatures which approach the boiling-point of the solvent, and by the presence of other agents in the solution which compete for the solvent. The determination of the quantity of water bound in this way by substances in aqueous solution, is frequently a matter of difficulty and uncertainty, but the existence of such "solvate" compounds may be demonstrated in a variety of ways, although their quantitative composition remains, in general, unknown. A very striking experiment which illustrates the formation of " solvates" is that cited by Pickering. If a mixture of Propyl Alcohol and water be placed in a semipermeable vessel and surrounded with water, it is found that water enters the cell, but that no propyl alcohol escapes. If, however, the same semipermeable vessel, containing the same mixture of propyl alcohol and water, be immersed in propyl alcohol, propyl alcohol enters the cell and water does not leave it. In other words, the vessel is permeable to either propyl alcohol or water when these are pure, but it is impermeable to mixtures of the two, the inference being that large molecular complexes are formed on mixing these reagents which cannot pass through the pores of the vessel. From these and similar experiments Poynting concludes that osmotic pressure is an expression of the diminution in the active mass of the solvent due to the formation of compounds with the dissolved substance. It is a familiar fact to chemists that anhydrous Cobalt Chloride is blue, but that on taking up water it becomes violet or red. Ostwald believed that the undissociated cobalt chloride molecule is blue, while the cobalt ion is red. Since, however, the color of a concentrated solution of cobalt chloride can be changed from purplish-red to blue by the addition of relatively small amounts of calcium salts, or still smaller amounts of aluminium chloride, or by the addition of a few drops of alcohol, it is more probable that this change in color is due to dehy- dration of the cobalt chloride molecule in solution, by the abstraction of water from it by the added substance. Similarly the progressive change in color of Cupric Chloride solutions, from blue to greenish-brown, on concentration or dehydration is attributed to the loss of water on the part of cupric chloride water-complexes. G. N. Lewis finds that if various bromides be added to concentrated solutions of Cupric Bromide the copper salt is dehydrated (turned brown) by the salts of mono- valent metals in the order: Li > Na > NEU > K. Divalent metals dehydrate more strongly, the order being: Mg > Ca > Sr > Ba while trivalent metals (Al) act still more energetically. The resemblance between the order of effectiveness of the monovalent metals in dehy- drating cupric bromide and their order of effectiveness in coagulating "electronegative" protein is very evident. The peculiar interest to the biological chemist of the possibility thus indicated, that substances dissolved in water form loose combinations with the solvent, lies in the especial significance of water in relation to PRECIPITATION OF PROTEINS BY INORGANIC SALTS 16? the protein and polypeptide structure. Dehydration of a protein may result in one or more of the following series of reactions: NHs.OOC.R.NHsOH + H 2 O HOHsN 3OOH NHs.OOC.R.NHsOH /NHOC.R.NH 3 OH + H 2 O NHOC.R.NHaOH /NHOC.R.NH 2 = R \ + H2 NHOC.R.NH 2 /NHOC.R.NH = R + H2 and hydration, of course, may result in the reversion of this series of changes. That proteins may be thrown out of solution in two very different conditions of hydration is very clearly shown by the following experi- ments : Anhydrous Casein dissolves readily in cold anhydrous Formic Acid; still more readily in hot formic acid. If, to a two per cent, solution of casein in formic acid, we add a fairly concentrated solution of Cupric Chloride, the mixture is at first green, indicating the presence of lower hydrates of cupric chloride, but on adding more of the solution it becomes blue, and simultaneously with the appearance of a pure blue color, but not before, precipitation of cupric caseinate occurs. If, to five c.c. of a two per cent, solution of casein in formic acid, we add 1J, 2 or 2J c.c. of a saturated solution of cupric chloride, no precipita- tion of the caseinate occurs, but on diluting this mixture with water a precipitate results, and the appearance of this precipitate coincides with the attainment of a clear blue color on the part of the mixture. About six cubic centimeters of water are required to produce a permanent precipitate. This precipitate redissolves on heating, and the mixture simultaneously becomes green; on cooling the blue color reappears and with it the precipitate. If formic acid be added to the mixture the precipitate redissolves as soon as the mixture becomes green. If the precipitate be very slight it will redissolve on adding alcohol. It cannot be urged that the formation of cupric caseinate requires the presence of more cupric ions than are present in green solutions, because green solutions of cupric chloride contain an abun- dance of ions, and casein will react with very small amounts of metal 168 COMPOUNDS OF THE PROTEINS ions, for although it is itself insoluble it will drive carbonic acid out of the sparingly soluble calcium carbonate to form a freely soluble casein- ate of calcium. If instead of adding water to a mixture of five c.c. of two per cent, casein in formic acid, and two c.c. of saturated cupric chloride, we add alcohol; no coagulation occurs until the mixture changes in color from green to brown, w r hen a Coagulum of cupric caseinate is produced which redissolves on adding water. Similar results are obtained when a 2-molecular solution of Cobalt Chloride is employed instead of a saturated solution of cupric chloride. If to five c.c. of a two per cent, solution of casein in formic acid we add two to three c.c. of this cobalt chloride solution, we obtain a blue- purple mixture. On adding water to this mixture it changes in color from blue-purple, through red-purple to clear pink. Not until a pure pink color is obtained does a precipitate result. If, instead of adding water, we add a considerable volume of alcohol (ten volumes) the mix- ture rather abruptly changes to a clear pale blue, and then, but not before, we obtain a coagulum of cobalt caseinate. Electronegative casein (i. e., casein dissolved in alkalies) is not precipitated by the salts of the alkalies, although it is readily precipi- tated by salts of the alkaline earths. Electropositive casein (i. e., casein dissolved in acids) is, however, very readily precipitated by salts, and these precipitates are not soluble upon dilution. Thus if two c.c. of tenth normal hydrochloric acid be added to five cubic centimeters of a one per cent, solution of casein in 0.008 N. potassium hydroxide, a clear, acid solution of casein results. The casein is precipitated from this solution by the addition of four drops of a saturated solution of sodium chloride, or by one drop of a saturated solution of ammonium sulphate. This latter precipitate does not dissolve on diluting the mixture to one-sixteenth. Casein Formate affords no exception to the rule that salts of casein with acids are precipitable by relatively small concentrations of neutral salts, but the precipitation will only occur 'in the presence of a sufficiency of water. If to five cubic centimeters of a two per cent, solution of casein in formic acid we add a saturated solution of ammonium sulphate, three cubic centimeters of this solution just suffice to produce a coagu- lum, this becomes more abundant on adding water, and redissolves on adding formic acid. If, however, instead of adding three we add two cubic centimeters of the saturated ammonium sulphate solution, a clear solution is obtained. On adding water to this a precipitate results which redissolves on heating and reappears on cooling. Analogous results may be obtained with Ovomucoid. It is clear, therefore, that protein may be thrown out of solution by electrolytes in two grades of hydration, the one of high, the other of very low hydration. The former process is what we have termed Precipitation, the latter we have defined as Coagulation. At grades of PRECIPITATION OF PROTEINS BY INORGANIC SALTS 169 hydration intermediate between these extremes the protein may be soluble. Dehydration, partial or complete, leading to resolution or to coagulation may be induced by heat, by non-electrolytes possessing an affinity for water, or by concentrated electrolytes. The importance of a high degree of dehydration in the production of Coagula, irresistibly suggests that this phenomenon is dependent upon the formation of anhydrides, analogous to the Diketopiperazines which may be formed from the amino-acids and polypeptides by dehy- drating-agents, and of the general formula: NH xlNXl.V^V^V R< | or R<^ ?O.HN' Such bodies may exist either in the keto-form, illustrated by the above formulae, or in the enol-form, such as: ,N.(HO)Cx Coagulation by mineral salts appears invariably to be accompanied or preceded by chemical interaction of the coagulating-salt and the protein salt of an acid or base which preexisted in solution before the coagulant was added. The coagulated protein in these instances, therefore, does not represent the unaltered protein salt as it existed in solution before the coagulant was added. When Alcohol is used as the coagulant however, it is found, at least in the case of the Caseinates of the Alkaline Earths, that the protein salt as such is coagulated, carrying down with it the amount of mineral base with which it was combined before the coagulant was employed, so that after washing out the alcohol with ether, and absorbing the ether by desiccation over sulphuric acid, calcium caseinate is obtained in the form of a dry powder which is soluble in water, whereas free casein is insoluble in water. If coagulation by alcohol is attributable to dehydration of the protein, the elements of water must be contributed chiefly by the interaction of free amino- and carboxyl-groups with the formation of ring-anhydrides, and that this should be possible without disintegration of the compounds with bases affords another indication that free carboxyl-groups are not responsible for the union of proteins with bases. The same considerations probably will be found to apply to the coagulation by alcohol of the compounds of proteins with acids, but as yet these compounds have not been so thoroughly investigated from this standpoint as the compounds of proteins with bases. 170 COMPOUNDS OF THE PROTEINS COMPOUNDS OF PROTEINS WITH OTHER PROTEINS. When the Protamines, which, it will be recollected, are strongly basic proteins, are added to weakly alkaline solutions of other proteins, precipitates are formed which consist of compounds of the protamine and other protein employed. These compounds, once formed, are tolerably stable, and when precautions are taken to prevent admixture with excess of protamine they are found to be of very constant com- position. These compounds were investigated by Hunter who found that wiiile crystallized egg-albumin, casein, hemi-elastin, gelatin, edestin, heteroalbumose, protalbumose, "alkali albuminate" and histone sulphate yield a precipitate in alkaline solutions upon the addi- tion of the protamine Clupeine. Elastin-peptone, deuteroalbumose histo- peptone and several peptides fail to yield a precipitate. On digestion of these precipitates with Pepsin the protamine is set free, since the protamines are indigestible by pepsin, and the remainder of the compound is converted into proteoses and peptones. The compound of Clupeine with casein contains six per cent, of the protamine while the compound with hemoglobin contains five per cent of protamine. The compound of Salmine with edestin contains about ten per cent, of the protamine. When Globin and Casein are mixed in faintly acid solution a precipi- tate of globin caseinate is formed which is soluble in excess of acid or in dilute alkalies. The precipitate produced by admixture of an excess of globin with sodium caseinate in solution contains about 34.5 per cent, of casein. A compound of globin with deuteroalbumose has also been prepared by C. L. A. Schmidt. Thymus-histone combines with Hemoglobin, according to af Ugglas, in the proportion of one part of thymus-histone to two of hemoglobin, and with casein to form a compound containing about thirty per cent, of histone. A particularly interesting compound protein is the Hemoglobin Caseinate which has been prepared by af Ugglas. To a solution of casein in alkali an excess of hydrochloric acid is added until the precipi- tate of free casein which is at first formed is redissolved. The casein hydrochloride is precipitated from this solution by the addition of sodium chloride, and the precipitate redissolved and reprecipitated until the washings from the precipitate are perfectly neutral. A solution of this substance added to an excess of a solution of hemo- globin produces a precipitate containing 33 per cent, of casein and about 66 per cent, of hemoglobin. The commonly accepted molecular weight of hemoglobin, originally deduced from its content of iron, and now confirmed by a variety of measurements, is about 16,700. The minimal molecular weight of casein, calculated from the minimal quantity of an alkali which will just carry it into solution (see p. 154), is 8800. It seems evident, therefore, that casein and hemoglobin combine with one another in molecular proportions. If the same is COMPOUNDS OF PROTEINS WITH OTHER PROTEINS 171 true of the compounds of the various Protamines with such proteins as casein and hemoglobin, the low proportion of protamine which is present in these compounds would indicate that the protamines possess molecular weights in the neighborhood of 800; much lower, that is, than the weights of the majority of protein molecules. This corre- sponds with their less colloidal character, the compound Salmine with sulphuric acid, for example, being freely diffusible through parchment- paper, and with the relatively few amino-acids they yield on hydrolysis, reminding one of the peptones rather than of the more bulky and complex native proteins. From a variety of observations it appears extremely probable that many of the protein constituents which may be isolated from the various tissues and tissue-fluids do not preexist there, but represent fractions split off by chemical procedures from complex compounds of proteins with proteins which are present in the tissue or tissue-fluid. Thus W. B. Hardy has pointed out that in untreated Blood-serum no proteins exist which wander in an electrical field, but. as soon as the serum is acidified with acetic acid, a cloud appears, which is due to partial precipitation of "Insoluble" Serum-globulin (the globulin- fraction of serum which is insoluble in distilled water). On passing a current through this mixture the cloud moves over to the anode. If the serum be dialyzed until all of the serum-globulin has been precipi- tated the remaining protein is now found to be completely ionic and is precipitated, on passing a current, at the anode. Dialysis, therefore, or acidification of blood-serum evidently accomplishes the detachment of a fraction ("insoluble" serum-globulin) from the protein-complex which preexists in untreated serum. This fraction is electrically dissociated and so is the remainder from which it is split off, but the original protein-complex is not dissociated at all. Moreover, as Hardy has also pointed out, the globulin which we separate by dialysis or by acidification and dilution from blood serum possesses very different physical characteristics from any which are displayed by the proteins in unmodified blood-serum. In Hardy's words: "The globulin-fraction has an abiding characteristic. In all its solutions its molecular state is so gross as to cause the molecules to be arrested by a porous pot. They will not pass such a filter even under pressure. In this it is sharply distinct from the parent serum-protein, which is readily filtrable. If globulin be present as such in serum it is not only non-ionic, but the agent which dissolves it must be something more than alkali and salt, since either alone or together they will not produce so high a grade of solution." "The difference in the molecular grade of globulin when once separated, and the electrical homogeneity of serum-protein and of the fraction (still capable of further subdividion by salting-out) which remains after the alkaline globulin fraction which most readily appears, has been removed, suggests that serum-protein is a complex unit. If such a unit exists it is not saturated with globulin. Fresh ox-serum has 172 COMPOUNDS OF THE PROTEINS an extraordinary power of dissolving globulin, it will take up almost its own volume of the thick cake at the bottom of a centrifuge tube; and in ox-serum so saturated there is not a trace of alkali-globulin nor of any ionic protein." The phenomena observed by Hardy appear to admit of interpretation by the view that the protein-complex in serum is formed by the union of a number of alkali-protein compounds, the union taking place in a manner analogous to that which occurs between proteins and inorganic salts, protein-acid or protein-base compounds in this instance taking the place of the inorganic salts. The soluble compounds which are thus formed are non-ionic, as evidenced by lack of motion in an electrical field. It has been suggested that the tissues and tissue-fluids of the various species of the animal kingdom may owe their specific and individual character to a characteristic structure of the protein-complexes in their tissues or tissue-fluids. We shall have occasion to revert to this possi- bility in a subsequent chapter (Chapter XIV) . REFERENCES. GENERAL: Robertson: The Physical Chemistry of the Proteins. New York, 1918. MODE OP UNION OF AMINO-ACIDS AND FREE AMINO-GROUPS IN PROTEINS: Hofmeister: Ergeb. d. Physiol. 1 Abt., 1902, 1, p. 759. Levites: Zeit. f. physiol. Chem., 1904-5, 43, p. 202. Biochem. Zeit., 1909, 20, p. 224. Van Slyke and Birchard: Jour. Biol. Chem., 1913-14, 16, p. 539\ MODE or UNION OF ACIDS AND BASES WITH PROTEINS: Bugarszky and Liebermann: Arch. f. d. ges. Physiol., 1898, 72, p. 51. Hardy: Jour, of Physiol., 1905, 33, p. 251. Robertson: Jour, of Physical Chem., 1911, 15, p. 521. Blasel and Matula: Biochem. Zeit., 1913-1914, 58, p. 417. Pauli and Hirschfeld: Ibid., 1914, 62, p. 245. Osborne and Leavenworth: Jour. Biol. Chem., 1913, 14, p. 481. PRECIPITATION AND COAGULATION: Whetham: Theory of Solution. Cambridge, 1902, pp. 396 and 398. Pauli and Handovsky: Biochem. Zeit., 1909, 18, p. 340; 1910, 24, p. 239. Robertson: Jour. Biol. Chem., 1911, 9, p. 303. COMPOUNDS OF PROTEINS WITH OTHER PROTEINS: Hardy: Jour. Physiol., 1905-6, 33, p. 251, appendix p. 327. Hunter: Zeit. physiol. Chem., 1907, 53, p. 526. af Ugglas: Biochem. Zeit., 1914, 61, p. 469. Schmidt: Jour. Biol. Chem., 1916, 25, p. 63. Univ. of California Pub. Pathology, 1916, 2, p. 157. CHAPTER IX. THE NUCLEIC ACIDS AND THE NITROGENOUS BASES. THE DECOMPOSITION-PRODUCTS OF THE NUCLEIC ACIDS. The nucleic acids form the prosthetic group in an important series of conjugated proteins, the Nucleoproteins. These substances usually, but not invariably, occur in nuclear tissues and may be precipitated from tissue-extracts by acidification with acetic acid,, in excess of which they do not dissolve. The nucleoproteins dissolve, however, in dilute mineral acids and in dilute alkalies; they are not soluble in distilled water. Certain nucleoproteins designated the /3-nucleopro- teins, however, are soluble in boiling water and are extracted from tissues in this manner, leaving the other tissue-proteins in the form of coagula in the insoluble residue; the jS-nucleoproteirs are also pre- cipitable by acetic acid. When the alkali-compound of a nucleoprotein, dissolved in water, is heated, a portion of the protein is split off in a coagulated form, while the residue of the molecule, which still contains protein but is much richer in phosphorus than the original nucleoprotein, remains in solution. A similar cleavage is brought about by the Pepsin in gastric juice, which digests the protein fraction which is split off from the nucleoprotein, but leaves a residue undigested which still contains protein united to nucleic acid. This residue is designated Nuclein. By means of more intense hydrolysis with alkali the nucleins are split up into products of protein hydrolysis and the alkali salts of the nucleic acids. These salts may be precipitated from concentrated solutions by the addition of alcohol. Upon hydrolysis with acids all of the nucleic acids yield three widely differing groups of products. In the first place phosphoric acid is an essential constituent of the molecule, secondly a carbohydrate radical, which may be either a pentose or a hexose, and thirdly a nitrogenous base belonging to the group of Purine Bases or to the closely allied group of Pyrimidine Bases. The carbohydrate radical differs essentially in nucleic acids of different origin. In all of the plant-nucleic acids which have been investigated, the carbohydrate radical has been found to be a pentose d-ribose: CHO HCOH HCOH HCOH CH;OH 174 NUCLEIC ACIDS AND THE NITROGENOUS BASES which, until its discovery among the decomposition-products of nucleic acids, was unknown in nature. It is now not only recognized as the carbohydrate radical of plant nucleic acid, but also regarded as the only pentose which normally occurs in animal tissues. In two nucleic acids found in animal tissues, but possibly traceable to a vegetable origin, namely Inosinic Acid and Guanylic Acid, d-ribose also constitutes the carbohydrate radical, but the nucleic acid which is most charac- teristic of animal tissues, Thymus Nucleic Acid, so called because of the circumstance that it was first prepared in a pure condition from the tissues of the thymus, yields Levulinic Acid on hydrolysis by acids. Now levulinic acid, or /3-acetyl propionic acid: CH 3 CO.CH2.CH 2 .COOH is formed when Hexoses are boiled with mineral acids, Formic Acid being produced at the same time: C 6 Hi 2 O 6 = C 6 H 8 O 3 + HCOOH + H 2 O In the hydrolysis of thymus nucleic acid by mineral acids, formic acid is produced as well as levulinic acid. It is evident, therefore, that both of these products are derived from a Hexose radical in the nucleic acid and confirmation of this inference is supplied by the fact that on oxidation of thymus nucleic acid with nitric acid, Saccharic Acid is included among the products, and saccharic acid must have a hexose precursor: 2C 6 Hi 2 O 6 -}-3O 2 =2C 6 HioO 8 +2H 2 O Among the Nitrogenous Bases which result from the acid hydrolysis of nucleic acids, Guanine and Adenine, which are Purine Bases, are found in both animal and plant nucleic acids, but among the Pyrimidine Bases which are yielded by the two classes of nucleic acid, there is a difference, for while both types of nucleic acid yield Cytosine, the animal nucleic acid ("thymus nucleic acid") yields Thymine, and vegetable nucleic acid yields Uracil. The pyrimidine bases are heterocyclic compounds which are dis- tinguished by the possession of the following nucleus: I Pyrimidine itself has the formula: N HC DECOMPOSITION-PRODUCTS OF THE NUCLEIC ACIDS 175 It does not occur among the decomposition-products of the nucleic acids. Its derivatives Uracil, Cytosine and Thymine have the following formulae : HN CO N=C.NH 2 HN CO | I OC CH OC CH OC C.CH 3 I II HN CH HN CH HN CH Uracil. Cystosine. Thymine. Uracil is therefore dioxypyrimidine, cytosine is amino-oxypyrimidine and thymine is methyluracil. Cytosine is transformed into uracil by the action of nitrous acid. Each of these bases has been prepared synthetically; they are known to occur in Nature, however, only as decomposition-products derived from nucleic acids by hydrolysis. They are sparingly soluble in cold water, more soluble in hot water. Cytosine dissolves in alcohol, uracil with difficulty, and thymine not at all. Cytosine and thymine are precipitated by phosphotungstic acid. Uracil is not. On heating, thymine sublimes without decomposition, uracil partly decomposes and partly sublimes, while cytosine undergoes decomposition. Cytosine and uracil give the Weidel Reaction as follows: To a small quantity of solution chlorine water is added, and the mixture boiled. The solution is evaporated to dryness and then exposed while warm to the vapors of ammonia. A purple-red color develops. This reaction is frequently referred to as the Murexide Reaction because it is due to the formation of Ammonium Purpurate which is believed to be identical with the scarlet dye found in the mollusc murex which furnished the "purple" of the ancient Romans. An intermediate stage in the reaction is the formation of Alloxan. : HN CO oc co HN CO Alloxan. and the test is only given by such substances as can be made to yield alloxan by oxidation. The reaction is therefore not infrequently alluded to as the Alloxan Reaction. Nitric acid may be used in the place of chlorine as the oxidizing agent. Cytosine and uracil also give Wheeler and Johnson's Reaction: To the solution of the substance bromine water is added drop by drop until a permanent cloudiness appears. Baryta water is then added, 176 NUCLEIC ACIDS AND THE NITROGENOUS BASES when a purple or violet precipitate appears. The Purine Bases are formed by the union of a pyrimidine nucleus with an Iminazolyl radical : N=CH N=CH HC NHx HC CH N CH Purine. Pyrimidine. The purine bases which are obtained from the nucleic acids repre- sent only two members of a large group of substances which includes Uric Acid, Caffeine and Theobromine. For convenience of reference the carbon and nitrogen atoms in the central complex are often num- bered as follows: > r/ N 3 The purine substances which are most important from a physio- logical point of view are Uric Acid, Xanthine, Guanine, Hypoxanthine and Adenine, while Caffeine, Theobromine and Theophylline are also of importance from a medical and dietetic point of view. Their structure is as follows: Uric acid is 2, 6, 8, trioxypurine Xanthine " 2, 6, dioxypurine Guanine " 2, amino, 6, oxypurine Hypoxanthine " 6, oxypurine Adenine " 6, aminopurine Caffeine " 1, 3, 7, trimethyl, 2, 6, dioxypurine Theobromine " 3, 7, dimethyl, 2, 6, dioxypurine Theophylline " 1, 3, dimethyl, 2, 6, dioxypurine Thus the formula for Guanine may be graphically represented: HN co H 2 NC N Guanine while that of Adenine is as follows: N=CNH 2 HC DECOMPOSITION-PRODUCTS OF THE NUCLEIC ACIDS 177 The purine bases are all precipitable from acid solutions by phos- photungstic acid or from ammoniacal solutions by silver nitrate. Guanine is insoluble in water, alcohol or ether, but readily dissolves in dilute acids or alkalies (with the exception of ammonia). It does not give Weidel's reaction, but with nitric acid it yields, on evaporation, a yellow residue w T hich turns bluish- violet on heating with sodium hydroxide. With chlorine water guanine decomposes, yielding Guani- dine, Parabanic Acid and carbon dioxide: HN CO I I H 2 NC C NH\ /NH 2 HN CO / I + 3O + H 2 O = HN=C< + OC \ I I C0 2 X NH 2 HN CO Guanine. Guanidine. Parabanic acid. Free guanine is found in the scales and swimming-bladder of fishes. It also occurs occasionally in the form of concretions in the retinal epithelium of fishes and in the joints of pigs suffering from "guanine gout." It forms an important constituent in the excrement of spiders. Guanine is hydrolyzed by an enzyme, Guanase which is found in a variety of tissues, particularly those of the pancreas and thymus (but not the Spleen). The products are Xanthine and ammonia: HN CO H 2 NC C NH\ II II \ >CH + H 2 O = HN Guanine. Xanthine. Adenine undergoes an analogous change with the production of Hypoxanthine, but the enzyme which brings this about (Adenase) appears to be a different one from that which accomplishes the deamini- zation of guanine, since it occurs in the spleen, from which guanase is absent. Hence when the tissues of the thymus or pancreas are allowed to undergo Autolysis; that is to say, spontaneous hydrolysis by their own enzymes, the purines which are isolated from the mixture are the oxypurines, xanthine and hypoxanthine, instead of the amino- purines, guanine and adenine which result from hydrolysis by acids. Adenine is sparingly soluble in cold, but readily soluble in hot water; it is readily soluble in acids and alkalies. Adenine does not give Weidel's reaction. With nitric acid, on evaporation, it gives a nearly 12 178 NUCLEIC ACIDS AND THE NITROGENOUS BASES colorless residue which does not turn red or violet on heating with alkali. With hydrochloric acid and zinc and subsequent addition of alkali an adenine solution yields a ruby-red color which changes to a brownish tinge. Adenine has been obtained from certain pathological urines (leukemia) and it occurs in considerable amounts in tea-leaves. THE STRUCTURE OF THE NUCLEIC ACIDS. The nucleic acid of yeast appears to be identical with the nucleic acid of the wheat-kernel, Tritico-nucleic Acid. It yields, on complete hydrolysis, two purine bases and two pyrimidine bases, namely, guanine, adenine, cytosine and uracil. When yeast nucleic acid is heated in neutral solutions under pressure to 175 C. it splits off phosphoric acid and yields four different Nucleo- sides each consisting of a molecule of purine or pyrimidine base united to a molecule of a-ribose. These nucleosides are the following: Guanosine ..... ......... Adenosine .............. Cytidine ............... Undine ........... .... C B H9O4.C4H 3 N 2 O2 It follows that in the undecomposed molecule of nucleic acid the purine and pyrimidine bases must be attached directly to the a-ribose molecules. The nucleosides do not reduce Fehling's Solution and hence the carbohydrate radical must be united to the basic radical in such a way as to involve destruction of the actual or potential aldehyde structure of the sugar, in other words these compounds are analogous to the Glucosides. If, instead of hydrolyzing nucleic acid with the aid of heat or inor- ganic catalyzers, we employ extracts of various organs, such as the kidney, heart-muscle, liver, pancreas, or intestinal mucosa, or if we employ blood-serum or hemolyzed blood, all of which contain the enzyme Nuclease, the nucleic acid is split into four different Mono- nucleotides each of which, on intense hydrolysis, yields phosphoric acid, a carbohydrate which in the case of yeast nucleic acid is a-ribose, and one of the four different purine and pyrimidine bases which the original molecule contained. The molecule of nucleic acid is, therefore, a Tetranucleotid, built up out of the union of four mononucleotid radicals. Two mononucleotids are known to occur in animal tissues, they are Guanylic Acid, obtained by the partial hydrolysis of /3-nucleoproteins, those nucleoproteins which may be extracted from a variety of tissues by Boiling Water, and Inosinic Acid which exists as such in most extracts. When guanylic acid is completely hydrolysed by mineral acids it yields phosphoric acid, a-ribose and guanine. It yields no other purine base and no pyrimidine bases. By means of hydrolysis in STRUCTURE OF THE NUCLEIC ACIDS 179 neutral water under pressure, phosphoric acid may be split off from this substance and Guanosine or the nucleoside of guanine is produced. The /3-Nucleoproteins are, therefore, compounds of protein with a mononucleotid, while the normal or a-Nucleoproteins are compounds of protein with a tetranucleotid. Inosinic Acid is prepared from meat-extracts by converting it into the barium salt which is very sparingly soluble in water. On hydrol- ysis with acids it yields phosphoric acid, a-ribose and Hypoxanthine in molecularly equivalent proportions. It will be recollected that hypoxanthine may be derived from adenine by simple deaminization : N C N ' )CE + NH 3 N C r * Adenine. Hypoxanthine. so that inosinic acid is a derivative of a simple mononucleotid con- taining adenine. The fact that the mononucleotids in animal tissues yield a-ribose on hydrolysis while the tetranucleotid, Thymus Nucleic Acid, which is characteristic of animal tissues yields levulinic acid winch must be derived from a hexose radical, leads us to infer that the mononucleotids which are found in animal tissues are derived from a vegetable source and are possibly not synthesised by animal tissues at all, but formed by partial hydrolysis and subsequent modi- fication of plant nucleic acids received in the food. By very careful hydrolysis with acids, interrupting the process before it is complete, it is possible to split off hypoxanthine from inosinic acid, leaving a compound of phosphoric acid and pentose. On the other hand, by neutral hydrolysis under pressure, phosphoric acid is split off leaving the pentose combined with hypoxanthine. It is evident, therefore, that in this mononucleotid the carbohydrate radical occupies a middle position, linking together the phosphoric acid on the one hand and the purine base on the other. This will be clear from the following schema: by neutral hydrolysis Phosphoric aci d pentose hypoxanthine by acid hydrolysis we shall see that the arrangement of the radical in other mononucleotids is probably of the same type. 180 NUCLEIC ACIDS AND THE NITROGENOUS BASES There are three conceivable arrangements of the three constituent radicals of Guanylic Acid. They are: O 00 || II II HO P O.CsHsOs.CsI^NaO, C 5 H 9 O4.0 P C 5 H 4 N 5 O, C5H 9 O4.C 5 H3N 5 O P OH T k 9 Of these three arrangements II cannot be the one which actually occurs in guanylic acid, because on neutral hydrolysis under pressure it yields Guanosine C5H 9 O4.C5H4N 5 O, which would be impossible if, in the original molecule, the carbohydrate and basic radicals were separated by the interposition of phosphoric acid. Either I or III must be the correct formula. Now on comparing the rates at which phosphoric acid and guanine are liberated from guanylic acid by acid hydrolysis, it is found that guanine is liberated much more rapidly than phosphoric acid. This implies, of course, that during the progress of hydrolysis, while guanine is being split off, phosphoric acid is being held in combination with some other substance from which compound it is detached with relative difficulty. The only substance, guanine being excluded, with which the phosphoric acid can be combined is a-ribose. It follows, there- fore, that phosphoric acid is attached to the molecule through the pentose radical, and formula I must represent the actual arrangement of the groups in guanylic acid. The same reasoning applies to the adenine-uracil dinucleotid which may be split off from yeast nucleic acid by partial enzymatic hydrolysis. We infer, therefore, from these facts and from the general similarity of the various mononucleotids to one another that the arrangement of radicals in all of them is: Phosphoric acid carbohydrate purine or pyrimidine. It remains to be considered how these mononucleotid radicals are united together to form the tetranucleotids characteristic, respectively, of vegetable and animal tissues. Three alternative possibilities exist, namely, (a) that the mono- nucleotids are united to one another through their phosphoric acid groups, so that the tetranucleotid would be a substituted polyphos- phoric acid. This was the view originally propounded by Kossel and has claimed very general acceptance until quite recently; (6) that the mononucleotids are united to one another through their carbohydrate radicals and (c) that they are united to one another through their purine or pyrimidine radicals. Between the two latter alternatives it has not as yet proved possible to decide with certainty, but the first alternative, that the mononucleotids are united to one another through their phosphoric acid radicals, may be dismissed for the following reasons: STRUCTURE OF THE NUCLEIC ACIDS 181 Yeast nucleic acid is known to consist of the following four mono- nucleotids. H(X O = P O.C 6 H8O 3 .C 6 H4N5 / HO/ Adenine mononucleotid HCk O = P O.C 5 H 8 O 3 .C4H 3 N 2 O2 HO/ Uracil mononucleotid HOv O = HO/ Cytosine mononucleotid HOv O = P O.C 5 H8O3.CBH4N 5 O HO/ Guanine mononucleotid We know, also, that these mononucleotids are united to one another in the order indicated, for when yeast nucleic acid is heated with ammonia it yields adenine-uracil dinucleotid, so that the constituent mononucleotids of this substance must be united together in the unaltered nucleic acid molecule. On the other hand, when carefully heated with acids, yeast nucleic acid splits off adenine and guanine mononucleotids leaving uracil-cytosine dinucleotid. It is evident, therefore, that the uracil and cytosine mononucleotid radicals are united to one another in the yeast nucleic acid molecule, and that the adenine and guanine mononucleotids form the extremities of the molecule. Now the Adenine Uracil Dinucleotid might conceivably consist of two mononucleotids united by their phosphoric acid radicals, or they might be united in some other manner. If they were united by their phosphoric acid molecules, at least one of the hydroxyl-groups of the phosphoric acid radicles would disappear by neutralization. The total number of available hydroxyl-groups contained in the two phosphoric acid radicals is four, so that the maximum number of molecules of any base that adenin-uracil dinucleotid could combine with would be four. If any hydroxyl-groups were neutralized by union of phosphoric acid radicals with each other or with other parts of the associated mononucleotid the free hydroxyl-groups would be less than four, and the dinucleotid would, in consequence, neutralize less than four molecules of a base. Now adenine-uracil dinucleotid forms a compound with four molecules of Brucine. It follows, therefore, that 182 NUCLEIC ACIDS AND THE NITROGENOUS BASES the phosphoric acid radicals of adenine and uracil mononucleotids are not utilized in binding these constituents of the nucleic acid mole- cule together. When a Purine Nucleotid is heated with dilute sulphuric acid, phos- phoric acid is liberated rapidly and completely. On the contrary, when a Pyrimidine Nucleotid is similarly treated, phosphoric acid is split off slowly. Yeast nucleic acid yields one-half of its phosphoric acid rapidly, and the remaining half slowly. Now if we compare the relative rates of splitting off phosphoric acid by adenine-uracil dinu- cleotid and by the whole yeast nucleic acid when treated in this manner, we find the relative rates of yielding phosphoric acid are identical. Hence, so far as phosphoric acid is concerned, the nucleic acid molecule consists of two symmetrical parts. Union of the two dinucleotid fractions to form whole nucleic acid does not in the slight- est degree affect the rate of yield of phosphoric acid by the component dinucleotids, and hence, phosphoric acid cannot be concerned in their union, and the phosphoric-acid linkage (2) in the subjoined diagram evidentlv does not exist in nucleic acid. (i) (2) (3) The molecule of yeast nucleic acid having thus been shown to consist of two symmetrically constructed halves, so far as phosphoric acid is concerned, it follows that if linkage (3) exists, then linkage (1), which would unite the adenine, and uracil mononucleotids must also exist, but this linkage has been shown not to exist, by the compo- sition of the brucine salt of the adenine-uracil dinucleotid. Hence linkage (3) does not exist either and, in short, no phosphoric-acid link- ages exist which bind molecules of mononucleotid together to form the tetranucleotid yeast nucleic acid. Between the two remaining forms of linkage, by the carbohydrate or by the purine or pyrimidine radical it has not yet been possible to certainly decide. P. A. Levene, however, concludes that in Cystosine- uracil Dinucleotid only two possibilities exist, either constituent mono- nucleotids are connected by ribose to ribose, or else by uracil (not by cystosine) to ribose. W. Jones believes yeast nucleic acid to be constituted as follows: STRUCTURE OF THE NUCLEIC ACIDS 183 O = P O.C 5 H7O 2 .C 6 H4N 6 O = P O.C 5 H6O.C 4 H3N 2 O 2 HO/ O II O = P O.C 5 H 6 O.C 4 H 4 N 3 O HO/ HOv \ O = P O.C5H7O 2 .C 6 H 4 N 6 O Yeast nucleic acid. Tritico-nucleic Acid from the wheat embryo is identical in physical behavior and in the products it yields on hydrolysis, with yeast nucleic acid. They are therefore believed to be identical substances, and it is considered probable that this is the only vegetable tetranucleotid. Thymus Nucleic Acid, it will be recollected, is yielded by the partial hydrolysis of all nucleoproteins of animal origin. It contains a hexose radical which has not yet been positively identified and it yields thy mine instead of uracil. Levene and Jacobs consider that thymus nucleic acid probably possesses the following structure: HOv O = PO C 6 HioO 4 C 6 H4N 6 O / Guanine group 0/ HO O II PO C 6 H 8 O 2 C 6 H 6 N 2 O2 Thymine group O O = PO CeHsO:! C 4 H 4 N 3 O Cytosine group H O = PO C 6 Hio0 4 C 5 H4N B / Adenine group HO/ 184 NUCLEIC ACIDS AND THE NITROGENOUS BASES AMINES DERIVED FROM AMINO-ACIDS. The proteolytic enzymes, such as Trypsin and Erepsin, accomplish the conversion, by hydrolysis, of the proteins into their constituent amino-acids. The next step in the degradation of nitrogenous food- stuffs by animal tissues generally, appears to consist in Deaminization with the splitting off of ammonia and the oxidation of the remainder of the original amino-acid molecule to carbon dioxide and water. No intermediate stages in this process have been definitely established, and we have been unable to detect the presence in animal tissues of enzymes capable of producing nitrogenous bases other than ammonia from amino-acids. That such enzymes, perhaps highly localized, do actually exist in animal tissues may be regarded as exceedingly prob- able, from the variety and physiological importance of the nitrogenous bases which are found to occur in animal tissues and their significant chemical resemblance to certain of the amino-acids which are yielded by the digestion of protein. Bacteria and other Fungi, however, constitute a group of organisms which are able to rapidly produce from amino-acids a series of nitrog- enous bases which arise by Decarboxylization of the amino-acid molecule in accordance with the general equation: R -| , R CHNH 2 = CH 2 NH 2 + CO 2 COOH Amino-acid Amine At the same time that this is taking place, Deaminization is also proceeding, and is evidenced by the production of ammonia. The conditions determining the relative proportion of these two processes are complex and have not as yet been fully determined, but it has been observed that the presence of carbohydrates in a culture of bacteria or fungi greatly diminishes the production of ammonia, presumably because in the absence of carbohydrates the organisms utilize amino-acids as a source of energy as well as a source of nitro- gen, and consuming the carbon and hydrogen components for this purpose, split off ammonia as a by-product. In studying the decar- boxylization of individual amino-acids it has been found that the addition of Peptone to the bacterial culture increases the yield of amines, probably because the process of deaminization being shared between the amino-acid and the peptone, a greater proportion of the amino-acid remains available for decarboxylization. Decarboxylization may also, especially under anaerobic conditions, be accompanied by reduction, in which case Formic Acid is produced instead of carbon dioxide: R I R CHNH 2 | + H 2 = CH 2 NH 2 + HCOOH COOH Amino-acid Amine AMINES DERIVED FROM AMINO-ACIDS 185 The greatest importance of this process from a biochemical point of view arises out of the intense physiological activity of many of the products which originate in this manner, the resemblance of some of these products to the active principles of certain of the glands of internal secretion, and from the probability that some of them may reach the circulation, occasionally in injurious quantities, by absorp- tion from the large intestine wherein they are produced by bacterial activity. The following amines have been produced from the corresponding amino-acids by the action of putrefactive bacteria. It is possible, however, that the true source of methylamine in the putrefaction of fishes is not glycocoll, but choline (trimethyl oxyethyl ammonium hydroxide) which is the basic constituent of Lecithin. Amino-acid. Amine. Glycocoll, CH 2 .NH 2 .COOH Methylamine, CH 3 NH 2 Alanine, CH 3 .CH.NH 2 .COOH Ethylamine, CH 3 CH 2 NH 2 Valine, cH.CH(NH 2 ).COOH Isobutylamine, CH.CH 2 NH 2 CH; \ Leucine, \ CH.CH 2 .CH(NH 2 ).COOH Isoamylamine, ^>CH.CH 2 CH 2 .NH 2 CH/ CH 3 \ CH 3 v Isoleucine, NcH.CH(NH 2 ).COOH Ethylmethyl ethylamine \CH.CH 2 NH 2 c 2 H 6 / C Z H/ Phenylalanine, C6H 5 CH 2 .CH(NH 2 ).COOH Phenylethylamine, C 6 H 5 .CH 2 .CH 2 .NH 2 Tyrosine, HO.C 6 H 4 .CH 2 .CH(NH 2 ).COOH p-Hydroxyphenylethylamine (tyramine), HO.C 6 H 4 .CH 2 CH 2 NH 2 ,NH 2 Guanido-butylamine (agmatine) , /NH 2 NH.CH 2 .CH 2 .CH 2 .- CH(NH 2 ).COOH HN = Arginine, HN ^NH.CH 2 .CH 2 CH 2 CH 2 NH 2 Lysine, H 2 N.CH 2 .CH 2 CH 2 .CH 2 .CH(NH 2 ).- Pentamethylene-diamine (cadaverine), COOH H 2 N.CH 2 CH 2 .CH 2 CH 2 .CH 2 NH 2 Ornithine, H 2 N.CH 2 CH 2 .CH 2 .CH(NH 2 ).- Tetramethylene-diamine (putrescine), COOH Histidine, CH / \ N H 2 N.CH 2 .CH 2 .CH 2 .CH 2 NH 2 Iminazoylyl ethylamine (histamine, erga- mine), CH NH CH = C.CH 2 .CH(NH 2 ).COOH Tryptophane, C.CH 2 .CH(NH 2 ).COOH /\ C 6 H 4 CH \/ NH N CH NH C.CH 2 .CH 2 .NH 2 Indolethylamine, C.CH 2 CH 2 NH 2 /\ / \ C 6 H 4 CH \/ NH 186 NUCLEIC ACIDS AND THE NITROGENOUS BASES Pyrrolidine, which should be formed by decarboxylization from proline, oxypyrrolidine which should be formed from oxyproline, amino-ethyldisulphide which should be formed from cystine, and /3-hydroxyethylamine which should be formed from serin, have not yet been found possible to prepare by bacterial decarboxylization. While a wide variety of bacilli, especially anaerobes, are able to bring about the decarboxylization of amino-acids, this power would seem to be possessed in an exceptional degree by a specific organism, Bacillus aminophilus intestinalis which has been isolated by Bertrand and Berthelot. The production of these bases, many of which are definitely toxic is not necessarily accompanied by the production of the odor which is commonly considered to be indicative of putrefaction. The odor of putrefaction is due to Indol and Skatol or /3-methyl indol: CH C.CH 3 /\ ' /\ C 6 H 4 CH C 6 H 4 CH \/ . \/ NH NH Indol. Skatol. and these substances which are derived from Tryptophane are the products of a further stage of putrefactive decomposition, arising by combined decarboxylization and deaminization succeeded by partial (skatol) or complete (indol) oxidation of the aliphatic hydrocarbon chain of the tryptophane molecule. The bases which are derived in this way from the proteins dis- play the usual characteristic properties of the amines. They are very much more basic than the amino-acids from which they are derived, and yield crystalline salts with mineral acids. The aliphatic monamines (methylamine, ethylamine, isobutyl- amine, isoamylamine, dimethylaminobutane) exert a physiological action mimicking the effects of stimulation of the sympathetic nervous system, they are therefore termed by Barger and Dale ''Sympathomi- metic" bases. The lowest amine to produce a distinct rise in blood pressure on intravenous injection is, however, Isobutylamine ; the activity increases with increasing length of the aliphatic hydrocarbon chain up to Hexylamine, and thereafter declines as the number of carbon atoms increases. Very much more effective than mere increase in the length of the chain is, however, the introduction of a ring- structure as in the benzol and heterocyclic derivatives. Thus Phenyl- ethylamine is at least five times as active, physiologically, as any aliphatic amine. Two milligrammes of this substance when injected intravenously may increase the blood-pressure of a cat no less than six hundred per cent. (30 mm. to 180 mm.). The most active, however, of the monamines derived from the amino-acid cleavage-products of protein is parahydroxyphenylethylamine (Tyramine) which exerts an AMINES DERIVED FROM AMINO-ACIDS 187 effect upon blood-pressure about one-twentieth of that exerted by Adrenaline. When injected intravenously it causes a rapid and pro- nounced rise in blood-pressure which is somewhat more prolonged than the rise which is caused by injections of adrenaline. Unlike adrenaline, how r ever, tyramine does not cause any vasoconstriction when applied locally to mucous surfaces, and large doses fail to produce the glycosuria which results from adrenaline-poisoning. Tyramine, furthermore, has a decided action upon the uterus, causing the non- pregnant uterus to relax while the pregnant uterus is stimulated to contraction. The glands which are innervated by the sympathetic system are stimulated by tyramine. It has been considered possible that since tyramine may be produced in vitro from Tyrosine by the action of fecal bacteria, the presence of this substance in the large intestine and its absorption may be respons- ible for pathological conditions in which high blood-pressure is a leading symptom. As in the case of adrenaline, prolonged adminis- tration of tyramine leads to renal and vascular lesions similar to those which so generally accompany persistent arterial hypertension in man. Indolethylamine is not so potent as tyramine and differs from it in several details of its action, notably in giving rise to muscular tremors or even convulsions, due to a transient stimulation of the central nervous system. Indolethylamine has also a direct stimula- tory action on smooth muscle-fibers, which is especially marked in the arterioles of the iris and the uterus. Among the Diamines, Putrescine and Cadaverine are of historic interest as they were among the earliest putrefaction-bases to be isolated, definitely characterized and identified. They are, however, comparatively innocuous substances, having very slight physiological activity and in common with other diamines, but in contrast to the monamines, they cause a fall in blood-pressure when they are injected intravenously. They occur in the urine in cases of cystinuria, their presence indicating a defective power of the tissues to deaminize amino-acids. Agmatine has a direct action upon the muscular tissues of the uterus, inducing contractions; it is, however, very much less potent in this respect than Ergamine which, with Ergotoxine and Tyramine is the active principle of the pharmaceutical preparations of ergot. Ergot is a parasitic fungus, Claviceps purpurea, which grows on diseased rye, and has been employed from very ancient times to cause contractions of the uterus. The amines which it contains are undoubt- edly produced by this fungus, as they are by other fungi and bacteria, by decarboxylization of the corresponding amino-acids. Ergamine stimulates unstriated muscle-cells directly, inducing especially powerful contractions of the uterus, but also stimulating smooth-muscle fibers in other organs, for example the stomach and intestine and the con- strictor muscles of the pupil of the eye. When dissolved in physio- 188 NUCLEIC ACIDS AND THE NITROGENOUS BASES logical saline solution and perfused through excised bloodvessels the muscle-fibers of the vessels contract, causing a decrease in their diameter, but when er gamine is injected intravenously, the effect upon the majority of the vessels in situ is just the reverse, and the blood-pressure undergoes a profound decrease due to their dilation. The vessels of the lungs, heart and kidneys, however, are constricted. An exceptionally interesting action of ergamine is that of inducing spasmodic contractions of the Bronchioles when administered in relatively large doses. Thus 0.5 milligrams of ergamine intra- venously injected will kill a guinea-pig in a few minutes, and the cause of death is asphyxiation, which is due to closure of the bron- chioles, preventing the passage of air into or out of the lungs. Post- mortem examination shows that the lungs are permanently dilated (Emphysema). Now this is the condition which, in a milder degree, is responsible, in human beings, for the respiratory distress in Asthma. It may further be brought about by peptone-poisoning or by inducing Anaphylactic Shock. When a non-toxic foreign protein, for example egg-white, is injected hypodermically or into the circulation of an animal, if the first dose is followed within a few days by a second, that in a like period by a third, and so forth, no harmful results ensue, and the animal gradually acquires Immunity to the protein. If, however, after the injection of the first dose of protein a considerable period, e. g., three weeks, be allowed to elapse before the second is administered, if the second dose be sufficiently large, a condition of " anaphylactic shock" is induced which is frequently fatal. The cause of death is asphyxiation due to spasmodic contractions of the bronchioles and it is believed that the preliminary " sensitization" of the animal has endowed its tissues with the ability to so rapidly decompose the foreign protein that upon injection of the second dose dangerous quantities of toxic peptones or other products of protein decomposition are rapidly formed. The resemblance between the symptoms of ergamine poisoning, peptone poisoning, asthma and anaphylactic shock is so striking as to suggest a common cause and the view has been advanced that all of these phenomena are attributable to the liberation of j8-iminazolyl ethyl- amine in the blood or tissues, the source of the substance being the Histidine radical in proteins or peptones. On the other hand it has not been conclusively shown that peptones themselves or peptide- derivatives of /3-iminazolyl ethylamine may not produce like effects. At any rate a part of the symptoms of anaphylactic shock are not attributable to ergamine, because this substance does not render blood incoagulable, while incoagulability of the blood is one of the symptoms of profound anaphylactic shock and of peptone poisoning. The possibility of the formation of /3-iminazolyl ethylamine from proteins in the lower intestine by the action of fecal bacteria may enable us to trace certain forms of asthma to an intestinal source. The majority of cases appear, however, to be undoubtedly anaphy- THE BETAINES AND THE VITAMINES 189 lactic, the immediate origin of an attack being frequently traceable to ingestion of some protein to which the individual in question has become sensitized, e. g., the proteins in the sweat of horses, egg-white, the proteins in strawberries or in pollen, or possibly proteins pro- duced locally by bacterial infections. On the other hand asthmatic attacks, originally anaphylactic, may frequently be seen in early cases to pass through transitional stages into habitual reflexes, which are thereafter elicited by any unusual stimulus, e. g., emotional excite- ment or indigestion. The problem is therefore a many-sided one of which the several factors are frequently difficult or impossible to disentangle. Closely related to the amines which we have been considering are the co-Amino-acids in which the a-amino-group which is so characteristic of the amino-acids derived from proteins is absent, the ami no-group being attached to a carbon atom which is remote from the carboxyl- group. This results in greatly increased basicity of the amino-acid so that these compounds resemble the amines in chemical behavior rather than the amino-acids. They may be produced in putrefaction by partial deaminization of a diamino-acid, as in the production of 6-Amino-valeric Acid from ornithine: H 2 N.CH 2 .CH 2 .CH2.CH(NH 2 )COOH + 2H = H 2 N.CH 2 .CH 2 .CH 2 .CH 2 .COOH + NH 3 or they may result from partial decarboxylization of a dicarboxylic acid, as in the production of 7-Aminobutyric Acid, from glutamic acid: HOOC.CH(NH 2 ).CH 2 .CH 2 .COOH = H 2 N.CH 2 .CH 2 .CH 2 .COOH + CO 2 An important representative of this group of substances is Carnosine which, next to creatine, is the most abundant nitrogenous base in meat-extracts. It is present in horse-meat to the extent of 1.8 grams per kilo. On hydrolysis it yields Histidine and /5-Alanine in equi- molecular proportions. It is believed to be a dipeptide histidyl-/3- alanine: CH = C CH 2 .CH.COHN.CH 2 .CH 2 COOH I N NH NH 2 THE BETAINES AND THE VITAMINES. The Betaines are amino-acids in which the nitrogen atom is united to methyl-groups in the place of hydrogen atoms. These substances in the absence of water, form cyclic anhydrides which open up when they are dissolved in water or unite with acids. Thus Betaine itself, or Trimethylglycine has the formula: o (CH 8 ) 3 N< CH 2 190 NUCLEIC ACIDS AND THE NITROGENOUS BASES When dried at above 100; but when it is dissolved in water or combined with acids it is probably represented by the formula : OH Betaine occurs in the sap of the sugar-beet, Beta vulgaris, and is extracted together with the sugar, remaining in the molasses when the sugar is refined. It is non-toxic and is not utilizable by animals as a food, but it is stated that the creatine content of the muscles is per- ceptibly increased by administration of betaine. Trimethyl Histidine is found in edible mushrooms. The constitu- tion is: CH- x CH The corresponding betaine of tryptophane is Hypaphorine. Up to the present these betaines have only been found in plant-tissues. In putrefying meat we find a betaine which unlike those described above, has a powerful physiological action. This is 7-n-butyro-betaine, the betaine of 7-amino-butyric acid : It has an action upon nerve-endings resembling that of curare and when injected produces convulsions, dyspnea and paralysis. Carnitine is the a-hydroxy derivative of 7-butyro-betaine : it is found in meat-extracts and is almost devoid of immediate physio- logical actions. THE BETAINES AND THE VITAMINES 191 Trigonelline is the betaine of nicotinic acid and, therefore, unlike the betaines heretofore considered, is not obtainable from any amino-acid cleavage-product of proteins. Its constitution is as follows: / CH \ \ HC C.CO HC CH I CH 3 it is devoid of any obvious physiological action, but is of especial interest because in the first place of its wide distribution in a variety of vegetable tissues and in the second place because Nicotinic Acid, from which it is derived by methylation, occurs in the polishings from rice. In various parts of the Orient, but particularly Japan and the Philippines, where rice constitutes a very large proportion of the dietary, the introduction of milling methods which involve stripping off the pericarp, or "polishing" of rice has led to the widespread occur- rence of a disease known as Beri-Beri, the ravages of which were par- ticularly prominent in the Japanese army during the Russo-Japanese War. The disease is evidenced by general lassitude accompanied by anesthesia in certain areas of the skin, edema of the ankles and face, partial paralysis of the leg-muscles and, toward the termination of the disease, distress in breathing. These symptoms are traceable to a widespread peripheral neuritis, beginning in the nerve-fibers most remote from the central nervous system and travelling centripetally. The mortality is very high. It was pointed out by Eijkman in 1897 that beri-beri could be prevented by eating unpolished rice with the pericarp intact, and that it could furthermore be cured by the adminis- tration of rice-polishings ("rice-bran"). He discovered that a very similar disease, involving peripheral polyneuritis and ultimate death, could be induced artificially in pigeons by feeding them exclusively upon polished rice. The inference was plain that a preventive and curative substance is present in the pericarp of rice. The nature of this substance has been extensively investigated by C. Funk and many others. Funk has succeeded in isolating a curative crystalline substance from Yeast which is exceedingly potent, as little as two milligrammes restoring the power of movement within three hours to pigeons which have been completely paralysed by a diet of polished rice. Curative substances are also found in a variety of other foodstuffs and in animal tissues. They are soluble in water and in alcohol, but insoluble, or sparingly soluble in ether. An active curative substance is invariably found to yield a blue color when mixed with Folin and Macallum's "Uric Acid Reagent," which is a solution of sodium phosphotungstate containing a specified proportion of phos- 192 NUCLEIC ACIDS AND THE NITROGENOUS BASES phoric acid and sodium tungstate. A similar color is yielded by uric acid, alloxantin, dihydrophenylalanine, amino-tyrosine, and certain di- or tri-phenols, but not by purine or pyrimidine bases other than those mentioned, nor by tyrosine itself. The curative substances isolated by Funk have been termed by him Vitamines. From rice-polishings a crystalline curative fraction was obtained which, on fractional crystallization was separated into two substances. The one proved to be Nicotinic Acid, which, in the pure crystallized condition, is devoid of curative action, and the other an unidentified nitrogenous substance which tends to lose its curative power with successive purifications. The curative substance from yeast was similarly found to yield nicotinic acid and an unknown nitrogenous base. From the fact that some of the pyrimidine derivatives have a weak curative action on polyneuritis, it was at first thought that the vita- mines were probably pyrimidine derivatives. The more recent investigations of R. R. Williams indicate that the curative principles may be substances having a Betaine structure. Thus a-Hydroxy- pyridine has a definitely curative action upon artificially induced poly- neuritis, so long as it yields needle-shaped crystals, but these crystals spontaneously change, on standing, into crystalline granules which are quite devoid of antineuritic properties. Now a-hydroxypyridine may conceivably exist in a variety of chemical forms of which the following are examples : :H \ / CH \ \ / \ HC CH HC CH HC Cx HC COH The curative variety, yielding needle-shaped crystals, is probably the pseudobetaine form, resembling the betaines in containing the group: _N O I and the fact that this structure and the antineuritic properties of a-hydroxypyridine spontaneously disappear on standing is suggestive in view of the fact that the curative substances isolated from yeast and rice-polishings by Funk tend also to lose antineuritic power spontaneously on standing or on repeated purification. The betaines themselves, such as trimethylglycine or trigonellin are impotent to protect pigeons fed on polished rice from the develop- ment of polyneuritis. It is, however, characteristic of the betaines that the anhydride ring is very unstable and readily opens up, as, for THE BETAINES AND THE VITAMINES 193 example, when salts are formed with acids, and failure to obtain marked curative results with these substances may therefore be attrib- utable to absence of the above ring-structure in the preparations administered. The antineuritic substances in an aqueous extract of yeast may be removed therefrom by shaking up the fluid with fuller's earth. The fuller's earth then becomes "activated" and carries with it all the curative substances. An alkaline extract of this activated fuller's earth was found by Williams and Seidell to exert a marked curative effect, but on recrystallization the substance lost its antineuritic properties and then was identified as Adenine. On heating to 180 in sealed tubes with alcohol, a portion of the antineuritic activity was regained, and at the same time it acquired the power, which adeniue does not possess, of yielding a blue color with Folin and Macallums' "uric acid reagent." Williams and Seidell infer that the curative substances in this instance is an isomeric modification of adenine. The instability of the curative substances and the minute propor- tions in which they are present in antineuritic foodstuffs renders the attainment of any definite conclusions a matter of exceptional difficulty. In the meantime, however, and pending more exact knowledge of this subject, very great care should be taken to avoid confusion by grouping together essentially dissimilar substances of widely differing physio- logical significance as "vitamines." Such procedure can only lead to mystification, obscures the issue, and obstructs the progress of our knowledge. The term "vitamine" should be definitely restricted to those nitrogenous substances which are known to possess curative action upon Polyneuritis. While a variety of other substances are now known to exist, which, in relatively small amounts are essential to health or growth, yet to group them all together as "vitamines" simply deprives the name of its scientific significance. It is much better to use the descriptive term "Accessory Foodstuffs," invented by their discoverer, Gowland Hopkins, to include all dietetic factors which are essential constituents of the diet for purposes other than the provision of heat-units or the building-up of carbohydrates, fats and proteins. The hydroxy-acids and other substances in fruits and vegetables which act as Antiscorbutics or preventives of scurvy are therefore " accessory foodstuffs" but they are not vitamines. We shall make further reference to the various classes of accessory foodstuffs in later chapters. Another deficiency-disease which probably depends upon lack of vitamines, or of substances resembling those which are lacking in polished rice, is Pellagra, a condition which is very common in districts such as the Southern United States, where maize furnishes a large proportion of the diet. Milling methods which involve total removal of the pericarp of the grain are believed to be responsible for the disease. Maize deprived of its outer covering has been shown to cause polyneuritis in pigeons in the same way as polished rice. 1.3 194 NUCLEIC ACIDS AND THE NITROGENOUS BASES NITROGENOUS BASES DERIVED FROM GUANIDINE. Guanidine /NH 2 HN = C< X NH 2 is obtained from proteins by the employment of strong oxidizing rea- gents, its presence has also been detected in various vegetable tissues, among others in the sugar-beet. It is a strong base, yielding strongly alkaline solutions of very stable salts with acids. It is uncertain whether or not it occurs in traces in the blood and tissues. Methyl- guanidine /NH.CHa HN = C< X NH 2 is, however, a normal constituent of blood, muscular tissues and urine. Guanidine and methylguanidine have a very decided physiological action, two hundred milligrammes of methylguanidine being a lethal dose for a guinea-pig. The amount of methylguanidine in the urine is greatly increased by Anaphylactic Shock, but the symptoms of poisoning are nowise similar to those of anaphylactic shock. They consist in fibrillar twitchings of the peripheral muscles and an excitation of the spinal cord resembling in comparatively slight measure that produced by strychnine or by Curare when directly applied to the cord. In larger doses the myoneural junctions are paralyzed in the same way that they are by curare and the spinal centers are depressed. The fibrillar twitchings produced in muscles by small doses of guanidine or methyl- guanidine are suppressed by calcium salts and in this respect as well as in the character of the muscular excitation, the action of small doses of guanidine resembles the action of sodium salts upon nerves and muscles. The marked effect of methylguanidine upon neuromuscular tissues is of especial interest because a derivative of methylguanidine, Creatine, or methyl-guanidine-acetic acid: /N.CH 3 .CH 2 .COOH HN = C< X NH 2 is the most abundant nitrogenous base in muscular tissues. The per- centage of creatine varies in different muscles, being higher in voluntary (striated) than in involuntary (smooth) muscles. In given muscles the percentage of creatine varies in different species of animals, but is remarkably constant in different individuals of the same species. The following are the percentages of creatine found in the muscle of various animals by Myers and Fine. Per cent. Species. of creatine. Rabbit 0.52 Cat 0.45 Man 0.39 Dog . a. 37 NITROGENOUS BASES DERIVED FROM GUANIDINE 195 In the urine the anhydride of creatine, Creatinine, is an important constituent: /N.CH 3 .CH 2 .COOH /N.CHs.CHs HN = C< HN = C< I + H 2 O X NH 2 X NH CO As a rule creatine itself is not found in mammalian urine, although it replaces creatinine in the urine of birds and is a normal constituent of the urine of young children. In women creatine occurs in the urine immediately after menstruation and occurs in large amounts in the urine during the involution of the uterus which follows delivery. It is considered probable by Folin and others that the creatinine in urine is not derived from the creatine of the muscles but represents a product of the catabolism of protoplasm. Creatine administered by mouth in small doses does not appear in the urine either as such or as creatinine. Neither urinary creatinine nor the creatine in muscles is increased by muscular work, but the creatine content of muscles appears to be connected with their Tonus or degree of moderate contraction when at rest. Thus standing at "attention" in a military position increases the urinary creatinine while a long march does not. On the other hand if, as much of the evidence seems to indicate, urinary creatinine is not derived from the creatine of muscles but from the "wear and tear", of tissues this result may merely indicate that standing at "attention" involves more destruction of muscular tissues than the performance of muscular work. Creatine is one of the relatively few substances which stimulate the gray matter or Neurones of the cerebral cortex. The customary stimulants for nerve-fibers, calcium-precipitating substances, barium chloride and so forth, have no action upon nerve-cells. Creatine is devoid of stimulating action upon nerve-cells but when applied to the motor areas of the cortex, it throws the animal into convulsions. This may be connected with the fact that the convulsions which accompany Eclampsia, a metabolic disease of pregnancy, are heralded by a sharp rise in the creatine output in the urine. Creatinine may be detected by Jaffe's Reaction, which consists in the red color produced by creatinine in alkaline solutions when Picric Acid is added. The color is due to Picramic Acid which is formed by reduc- tion of picric acid. This reaction is employed for the quantitative estimation of creatinine. Creatinine also gives a ruby-red color with Sodium Nitroprusside in alkaline solution (Weyl's Reaction). Creatine may be converted into creatinine by boiling with dilute hydrochloric acid, or it may be determined directly by utilizing the pink coloration which it yields with Diacetyl. It should be noted that creatine is closely related to Arginine, which is the only product of protein hydrolysis that contains a guanidine radical : /CH 3 /NH.CH 2 .CH 2 .CH 2 .CH(NH 2 )COOH /N< HN = C< HN = C< X CH 2 COOH X NH 2 X NH 2 Arginine. Creatine. 196 NUCLEIC ACIDS AND THE NITROGENOUS BASES THE NITROGENOUS BASES DERIVED FROM THE PHOSPHOLIPINS. The saponification of the Lecithins by alkalies yields, besides soaps and the glycerophosphate of the alkali, a nitrogenous base, Choline or trimethyloxyethylammonium hydroxide. /CH 2 .CH 2 OH (CH 3 ) 3 i N< X OH It is a strong base, yielding alkaline solutions and forming a double salt with platinic chloride. By the saponification of Kephalins, how- ever, we obtain a different base, namely Amino-ethyl Alcohol. H 2 N.CH 2 .CH 2 OH from which choline is probably derived by methylation. There has been much discussion of the question whether or not a third and related base, Neurine, or vinyltrimethyl ammonium hydroxide : /CH:CH 2 * (CH 3 ) 3 i N< X OH is yielded by the hydrolysis of Protagon, but the consensus of opinion appears now to coincide with the view originally expressed by Gule- witsch, that neurine is in reality a putrefaction-product derived from choline by the action of bacteria. Thus perfectly fresh brain-tissue does not appear to yield neurine at all, unless the lecithins (or protagon) are boiled with strong alkalies which, even in pure solutions, results in a partial conversion of choline into neurine. Both choline and neurine exert the physiological actions which are typical of all the trimethylamine derivatives. The first symptom of poisoning is salivation, followed by intestinal cramps. There is a preliminary fall in blood- pressure succeeded by a rise. Death is ultimately due to arrest of the heart. These symptoms arise from stimulation of sympathetic nerve-endings in the glands or muscles affected and are prevented by the administration of Atropine which paralyzes these junctions. It was at one time thought that free choline might occur in the brain, particularly in degenerative changes of the central nervous system, and that under these conditions choline might be found in the cerebro- spinal fluid. The presence of choline in cerebrospinal fluid was, in fact, suggested as a means of detecting degenerative lesions of the brain. Since platinic chloride must be employed to detect the small quantities of choline looked for, however, and potassium and ammonium salts, both of which are also present, yield very similar crystalline platinichlorides, it is rather probable that the crystals obtained from cerebrospinal fluid are not in reality compounds of choline. The ACTIVE PRINCIPLES OF INTERNAL SECRETIONS 197 small quantities which are obtainable renders investigation of this question by direct analysis a very difficult one. The free choline alleged to have been detected in brain-tissue has been found to be a postmortem product arising from autolysis or putrefaction. The physiological action of neurine is much more intense than that of choline, being effective in about one twentieth of the dosage. By introducing radicals into the oxyethyl group, however, as in Acetyl- choline and the nitrous acid or nitric acid esters of choline, substances of very much more intense physiological activity than choline itself are produced. By the hydrolysis of the Cerebrosides, phrenosin and kerasin, a nitrogenous base, Sphingosine is obtained the constitution of which is at present unknown. Its percentage composition corresponds to the formula Ci7H 35 NO 2 , and it is a diatomic alcohol containing an amino- group. When sphingosine is heated with concentrated sulphuric acid and a sugar, it yields a purple- violet coloration. The cerebrosides, when similarly treated, first dissolve in the sulphuric acid, yielding a clear yellow solution, and then the sphingosine is split off and sepa- rates out in droplets which yield the reaction. The addition of sugar in this case is unnecessary because it is supplied by the galactose in the cerebroside. Regarding the physiological actions of sphingosine nothing definite is known. NITROGENOUS BASES FORMING THE ACTIVE PRINCIPLES OF INTERNAL SECRETIONS. Of these the best studied and, therefore, the best known is Adrenaline, the blood-pressure raising or Pressor principle of the Suprarenal Gland. Other names by which this substance is designated in current literature are Epinephrin, Adrenin and Suprarenin. The term adrenaline is that most customarily used although epinephrin is also frequently employed. Adrenaline is a derivative of Catechol and possesses the following constitutional formula : COH /\ HC COH HC CH \/ C CHOH CH 2 .NH.CH 3 Adrenaline. 198 NUCLEIC ACIDS AND THE NITROGENOUS BASES it is therefore, a methylamine and also a dihydroxybenzene derivative. The structure of adrenaline may be compared with that of tyrosine: COH /\ HC CH HC CH \/ : c CH 2 CH(NH 2 )COOH Tyrosine. from which it will be evident that adrenaline and tyrosine contain the same skeleton of carbon atoms. The immense physiological importance of the suprarenal gland was first demonstrated by Addison, in 1849, when he showed that the disease now named after him, was connected with degenerative changes of the suprarenal bodies. A few years later it was also discovered that the suprarenal glands contain a " chromogenic substance" which yields a vivid green color with ferric chloride and a red color with iodine. Strangely enough, however, the remarkable effect of suprarenal extracts upon blood-pressure was not discovered until 1894, and the positive identification of the pressor-substances with the " chromogenic substance" was not established until some years later. Pure adrenaline crystallizes in colorless spherules; it is sparingly soluble in water and almost insoluble in most organic solvents, it will however, dissolve in glacial acetic acid, ethyl oxalate or benzaldehyde. Adrenaline may be prepared from fresh suprarenal glands by extract- ing the minced tissue with water, coagulating the proteins by heat or trichloracetic acid, concentrating the extract and adding ammonia which causes the adrenaline to separate out. The readily oxidizable catechol (orthodihydrobenzol) nucleus in adrenaline is responsible for a variety of color reactions which it yields. The following are among the most characteristic: Ferric Chloride Reaction. In neutral or faintly acid solutions adrena- line yields with ferric chloride a vivid grass-green color, which changes to violet, reddish violet and red on rendering the solution alkaline. This will detect about one part of adrenaline in thirty thousand, but the addition of Sulphanilic Acid, while changing the color to reddish brown, also renders the test much^more sensitive. Iodine Reaction. With iodine or iodic acid adrenaline yields a red color. The excess of iodine may be removed by shaking up the mixture with ether. Mercuric Chloride. With mercuric chloride, in the presence of Calcium Salts, which act as catalyzers, solutions of adrenaline yield a red color. ACTIVE PRINCIPLES OF INTERNAL SECRETIONS 190 Persulphate Reaction. This is the most delicate of all the tests for adrenaline, detecting one part of adrenaline in five million of solution. Potassium persulphate is added to the solution to the extent of one- tenth of a per cent., and the test-tube is then heated by immersion in boiling water. Phosphotungstic Acid Reaction. With Folin and Macallum's "uric acid reagent," which consists of a mixture in specified proportions of sodium tungstate and phosphoric acid, adrenaline yields the blue color which Uric Acid, Alloxantin, certain Dihydrophenols, Aminotyrosine and other substances including the vitamines also yield. The test will detect one part in 3 million of adrenaline. Adrenaline constitutes about one-tenth of a per cent, of the fresh tissue of the suprarenal gland. One bullock's gland, weighing about ten grams, should therefore yield about ten milligrams. The origin of adrenaline is unknown. The close relationship to Tyrosine would suggest this as the parent substance, but the trans- Normal A 20 mg.Tethelin 20 mg.Tethelin 1 cc. Split Product -* Minutes > FIG. 6. Tracing showing effect on the uterus (guinea-pig) of split products of tethelin. formation of tyrosine into adrenaline would involve a series of changes not merely decarboxylization, but also the introduction of two hydroxyl- groups, one of them in the benzol-ring, followed by methylation of the resultant compound. We are not familiar with any mechanism which could bring about this rather complicated series of transforma- tions. There are some indications, however, that the suprarenal glands may contain precursors of adrenaline which are devoid of pressor-action, and yet yield a coloration with oxidizing-agents. The important physiological actions of adrenaline will be separately discussed (cf. Chapter XVI). The posterior lobe or Infundibulum of the Pituitary Body contains a nitrogenous substance which exerts an action upon the uterus as distinctive as that of ergot and has also the peculiar property of exciting the secretion of the Mammary Glands. The structure and even the composition of the active substance are unknown, but since it yields a red color with Diazobenzene Sulphonic Acid (see Histidine), it probably 200 NUCLEIC ACIDS AND THE NITROGENOUS BASES contains an iminazolyl radical and is, therefore, related to Ergamine. The active substance which, in aqueous solutions, is known by the trade name of Pituitrin, gives the Biuret-reaction and its activity is rapidly destroyed by trypsin; it is consequently believed to be a Peptamine or an amine derived from a polypeptide containing a histidine radical. Several synthetic peptamines have been prepared but their actions have hitherto been found to be much weaker than those of the simpler amines. A parent-material, which yields pituitrin, or at least a substance resembling pituitrin in its action upon the uterus after hydrolysis by acids or alkalies (Fig. 6), is found in the Anterior Lobe or glandular portion of the pituitary body. This substance, which is a water- soluble phospholipin, has been designated Tethelin. The histological structure and anatomical relationship of the two parts of t he pituitary body are such as to suggest that the anterior lobe furnishes some material to the posterior lobe, and it is therefore, possible that the posterior lobe manufactures pituitrin from tethelin supplied to it by the anterior lobe. A nitrogenous base of unknown composition appears to be the active principle in acidified aqueous extracts of intestinal mucosa which stimulates the secretion of Pancreatic Juice when these extracts are injected intravenously. The substance which is known as Secretin, is insoluble in neutral water, soluble in dilute acids, and precipitable by mercuric chloride and by picric acid. REFERENCES. GENERAL: Jones: The Nucleic Acids. London, 1914. Barger: The Simpler Natural Bases. London, 1914. THE NUCLEIC ACIDS: Jones and Rowntree: Jour. Biol. Chem., 1908, 4, p. 289. Jones and Richards: Ibid., 1914, 17, p. 71. Wells: Ibid., 1916-1917, 28, p. 11. Jones and Read: Ibid., 1917, 29, pp. Ill and 123; 1917, 31, pp. 39 and 337. Read: Ibid., 1917, 31, p. 47. Read and Tottingham: Ibid., 1917, 31, p. 295. Levene: Ibid., 1917, 31, p. 591. VlTAMINES: Funk: Ergeb. d. Physiol., 1913, 13, p. 125. Folin and Macallum: Jour. Biol. Chem., 1912, 11, p. 265. Funk and Macallum: Biochem. Jour., 1913, 7, p. 356. Williams: Jour. Biol. Chem., 1916, 25, p. 437; 1917, 29, p. 495. Williams and Seidell: Ibid., 1916, 26, p. 431. Voegtlin and Myers: U. S. Pub. Health Service. Reprint No. 471. Pub. Health Reports, Washington, 1918. PHYSIOLOGICAL ACTIONS OF THE BASES. Sollmann: A Manual of Pharmacology. Philadelphia, 1917. Cushny: A Text-book of Pharmacology and Therapeutics. Philadelphia, 1918. Schafer: The Endocrine Organs. New York, 1916. Maxwell: Jour. Biol. Chem., 1907, 3, p. 359. Guggenheim: Biochem. Zeit., 1913, 51, p. 369; 1914, 65, p. 189. Schmidt and May: Jour. Lab. and Clin. Mod., 1916-17, 2, p. 708. CHAPTER X. THE HYDROLYZING ENZYMES. GENERAL CHARACTERISTICS OF THE ENZYMES. Disaccharides in aqueous solution are hydrolyzed by mineral acids in accordance with the equation: Ci2H 22 On + H 2 O = 2C 6 Hi2O6 Any acid will act upon any disaccharide, but the intensity or Velocity of Hydrolysis varies somewhat with the nature of the acid and of the disaccharide. The law which connects the time and the extent of the hydrolysis of cane-sugar by acids was first formulated by Wilhelmy in 1850, who showed that at every instant the same percentage of the hitherto unchanged sugar is hydrolyzed per second. Thus, if to begin with we have 100 parts of sugar, and of this 5 parts are hydrolyzed in a given interval of time, we have now 95 parts of unchanged sugar left, and in the succeeding interval five hundred ths of this will be hydrolyzed. The transformation, therefore, proceeds in the following manner: Number of cane- Number of sugar- sugar molecules. molecules hydrolyzed. First interval of time . . . . . 100.00 T ^ 7 X 100.00 = 5.00 Second interval of time . . . . . 95.00 Y^ X 95.00 = 4.75 Third interval of time . . . . . 90.25 T fo X 90.25 = 4.51 Fourth interval of time .... 85.74 T ^ X 85.74 = 4.29 Fifth interval of time ..... 81.45 T fo X 81.45 = 4.07 In other words, unit-mass of sugar, or one hundred molecules of sugar, always decomposes at the same rate, no matter how much or how little sugar, i.e., how many units or what fraction of a unit, is present in the given solution at the moment. If a gram-molecule of sugar be present, just the same percentage of sugar-molecules will be undergoing transformation per second as when five gram-molecules of sugar are present, but in the latter case the total amount of trans- formation observed per second will be five times as great as in the former. 202 THE HYDROLYZING ENZYMES The rationale of this law, which is known as Wilhelmy's Law, may be made clear in the following way: In every instant innumerable collisions are taking place between sugar-molecules and water-molecules. Only a small proportion of these collisions are effective in accomplishing the breaking up of a disaccharide molecule. The proportion of effective collisions is, however, the same no matter how many sugar-molecules may chance to be present. We may picture to ourselves, without seeking to employ the analogy too literally, the effective collisions as "head-on" collisions, the ineffective collisions being "glancing." Each sugar-molecule is independent of all the rest, and its chance of achieving an effective collision with a water-molecule is the same as that of all the rest. Suppose every thousandth collision is effective, i. e., one tenth of a per cent, of the total collisions per second. If the solution of sugar be 2-molecular, the total number of collisions per cubic centimeter per second will be twice as great as when the solution is 1 -molecular, because there are twice as many molecules of sugar in a given space. In each solution the percentage of effective collisions is the same. Out of one thousand collisions in a 2-molecular solution one will be effective, and out of a thousand collisions in a 1-molecular solu- tion one will also be effective. But as there are twice as many collisions per second in the former as there are in the latter solution, there will also be twice as many effective collisions per second, and the amount of sugar transformed in a second, i. e., the Velocity of Hydrolysis in the 2-molecular solution must be twice as great as it is in the 1-molecular solution. This very simple relationship may also be expressed in an algebraical formula : Velocity of hydrolysis = k(a x) where (a x) is the mass of unaltered sugar at any instant, "a" being the initial amount and "x" the quantity which has already undergone hydrolysis at the moment of observation. The constant "k" expresses the constant ratio which, as we have seen, subsists between the mass of unhydrolyzed sugar which is present and the velocity with which hydrolyzed sugar is making its appearance. It is, in fact, the velocity of hydrolysis when (a x)=l, that is, when the mass of unconverted sugar is unity, one gram-molecule, or one gram, or whatever mass we may arbitrarily choose as a unit, provided we measure all the quantities in the equation in the terms of the same unit. Also, and this is very important to notice, the constant "k" is a direct measure of the percentage of effective collisions between sugar-molecules a ad water-molecules, for if the percentage of effective collisions be doubled by any means then the velocity of hydrolysis must obviously be doubled also. In the equation : Velocity = k(a x) GENERAL CHARACTERISTICS OF THE ENZYMES 203 if the term "velocity" is doubled in magnitude, while (a x) remains unaltered, then k must have been doubled in magnitude. When the percentage of effective collisions is doubled, therefore, k is doubled, and so forth. We have hitherto not considered the part which the Acid plays in bringing ahout the hydrolysis. Neutral water only hydrolyses sugar extremely slowly, so slowly that the velocity is negligible in comparison with the velocity of hydrolysis in acid solutions. At the completion of hydrolysis the acid is unaltered and is available for bringing about further and, apparently, unlimited hydrolysis. The acid does not, therefore, communicate any energy to the system; it merely increases the Percentage of Effective Collisions of molecules of sugar with mole- cules of water. Such an action is termed a Catalytic Action, and the agent which brings about the acceleration, in this instance the acid, is termed a Catalyzer or Catalyst. The Mechanism of Catalysis is, in this instance, not perfectly clear, but judging from the analogy afforded by the mode of action of many other catalysts, we may conclude that a compound of the disaccharide with acid is formed and that it is this compound which actually undergoes hydrolysis. In many cases of catalysis such compounds of the catalyst with the substance undergoing decomposition, or Substrate, have been isolated and identified, so that we feel justified in assuming that if such compounds are not readily detectable in an instance of catalysis such as that afforded by the hydrolysis of cane-sugar by acids, the reason is that only a minute trace of the compound of the catalyst and the substrate is present in the mixture at any moment The quantity of the compound of the substrate with the catalyst which is present at any moment in the mixture, must, however small, be proportional to the concentration of the catalyzer and also to the concentration of the substrate, for if two substances A and B combine to form a third AB the quantity of this compound formed must be determined, as the Guldberg and Waage Mass-law requires, by the equation : Mass of A X mass of B = constant X mass of AB If, then, the concentration (=mass per unit- volume) of the catalyzer be kept constant, the quantity of the substrate-catalyzer compound, in this instance the compound of cane-sugar with acid, must be directly proportional to the concentration of the still unaltered cane-sugar and decrease as it decreases. Since, for all practical purposes of measure- ment, it is only the molecules of sugar-acid compound which are under- going hydrolysis it follows that the velocity of hydrolysis must, if the same proportion of these compound molecules is decomposed in each instant, fall off in direct proportion to the concentration of still un- altered sugar, or in other words the equation : Velocity of hydrolysis = k(a x) 204 THE HYDROLYZING ENZYMES must hold good for a catalyzed as for an uncatalyzed hydrolysis, so long as the concentration of the catalyzer is kept unaltered. Since the quantity of the acid-sugar compound is also proportionate to the concentration of the acid, it follows that with varying concen- trations of acid the velocity of hydrolysis must also vary directly with the concentration of acid. In other words the value of "k" in the above equation is directly proportional to the concentration of catalyst, or: Velocity of hydrolysis = kf (a x) where "f" is the concentration of the catalyzer. In fact so accurately does this relationship obtain that the ratio of the velocity of the hydrolysis of cane-sugar to its concentration is very commonly employed as a means of measuring the quantity of free acid (= hydrogen ions) which is present in a solution. So much for the hydrolysis of cane-sugar and of other disaccharides by acids; but cane-sugar is also hydrolyzed by an Enzyme, to wit, Invertase. This enzyme is found in yeasts, in certain moulds and bacteria, in green leaves and young twigs, in some fruits and in germi- nating grains. In mammalia, it is sometimes found in the human gastric juice, but not in the gastric juice of cows. It is also found in weak concentration in other organs. It may be extracted from yeast- cells with water, provided the cells have previously been subjected to the action of some Plasmolyzing Agent, or agent which will break up or enhance the permeability of the limiting membrane of the cell, such as alcohol or ether. The invertase can then be precipitated from its watery solution by alcohol, and this precipitate, in nearly neutral solutions in which hydrolysis would otherwise be excessively slow, rapidly decomposes cane-sugar into its constituent hexoses. It should be clearly understood that, as in the case of the other enzymes, we possess no clue which enables us to decide whether there is only one or whether there are many invertases. We do not know anything whatever concerning the chemical composition of invertase. Our only means of recognizing this enzyme is by the property which it possesses of hydrolyzing cane-sugar, giving rise to Glucose and Fructose. Any agent which can be extracted from living tissues which does this and is inactivated by high temperatures we call invertase. It is quite conceivable that many different substances can accomplish this hydrolysis. The activity of an invertase preparation is no guide to its individuality, because in the absence of any knowledge of the chemical properties of invertase, we cannot estimate the purity of any prepara- tion. We can recognize certain impurities, such as phosphates, pro- teins and so forth in a preparation of invertase, and we can remove some of them, wholly or partially. But when easily recognizable impurities have been removed, we cannot tell whether the residuum is a pure material, that is, a chemical individual, or a mixture of different GENERAL CHARACTERISTICS OF THE ENZYMES 205 substances of which perhaps one is active or, perhaps, many. Could we discover any chemical resembling invertase in its solubilities and in its sensitiveness to temperature and so forth, and possessing the action of invertase, we might be inclined to claim an identity between the two and then analysis would give us a criterion of the purity of any given invertase preparation. But at present we have no such criterion, and we cannot say whether a given preparation of invertase is a single or a multiple preparation, or whether it contains 99 per cent, of inver- tase and 1 per cent, of impurities or, on the contrary, 1 per cent, of invertase and 99 per cent, of impurities. In this connection the properties and peculiarities of invertase may be regarded as illustrative of the peculiarities which distinguish nearly all of the enzymes. The same difficulties are encountered in the study of each of the enzymes in turn. It might be imagined that the problem of extracting an enzyme in pure condition from a crude preparation of proved activity could be attacked in a manner analogous to that employed in the original discovery of radium; by employing a variety of precipitant s and solvents to fractionate the crude preparation, reject- ing the inactive and retaining the active fraction in each successive stage of the process. Unfortunately, however, it is found that almost every chemical procedure which we may employ results in some loss of activity by the enzyme. If a fraction of the original crude pre- paration be precipitated out from the rest it may be found, for example, to contain an amount of active ferment corresponding to fifty per cent, of the amount which was present in the crude preparation, while the residue, after the separation of the precipitate, may be found to contain none of the ferment whatever. In this way successive processes of puri- fication involve successive losses, until the activity of the preparation ultimately disappears altogether. It is for this reason that the more impure preparations of the various soluble enzymes are usually more active than those preparations which are relatively "pure," i. e., contain a smaller variety of substances. We have referred to the fact that many different enzymes may be mistakenly regarded as one if they chance to possess a common action. As an illustration of how an enzyme regarded as a single chemical individual may become, with increased acquaintance, recognized as multiple, we may cite the Trypsins : enzymes which have this in com- mon that they hydrolyze proteins and peptones in faintly alkaline solutions. Until recently no means of distinguishing between dif- ferent trypsins were known, and trypsin was tacitly assumed to be one ferment and only one. Now the investigations of Emil Fischer and of Abderhalden have shown us that there are many trypsins, which differ from one another in, the relative ease with which they attack different peptide-linkages. The enzymes are, as a rule, destroyed, or, at least, inactivated by high temperatures. But this is by no means a rule without exceptions. 206 THE HYDROLYZING ENZYMES Thus several of the oxidizing enzymes, or Oxidases regain their activity, lost by heating, when the solution is allowed to stand for some time at ordinary temperatures. According to Gramenetski the same phe- nomenon may be displayed by certain Diastases or starch-splitting enzymes and even in some measure by Trypsin. The vegetable Proteases or protein-splitting enzymes sometimes withstand higher temperatures than the corresponding enzymes of animal origin and Karl Meyer has drawn attention to the rather extraordinary fact that the Trypsin which is produced in culture-media by Bacillus prodigiosus will withstand heating for fifteen minutes to 100 C, although it is destroyed within thirty minutes at 56 C. This looks rather as though a trypsin-splitting ferment also existed in the culture-medium for which the optimum temperature is about 56, and which is destroyed by higher temperatures more rapidly than trypsin itself. It would, therefore, be very unsafe to infer because a substance does not lose its characteristic activity of some type or other when it is heated that it is therefore not an enzyme. It would be still more unsafe, of course, to infer that it is an enzyme simply because it is "inactivated" by heat. Yet both of these inferences, unfortunately, have not infrequently been made in biological and biochemical investi- gations. In deciding whether or not a substance or material should be classed as an enzyme we should be guided, rather, by its quantitative relationship toward the particular activity which it displays. The enzymes are usually effective in relatively minute concentration. It has been estimated, for example, that a certain Rennin or milk-coagulat- ing enzyme preparation will convert no less than 500,000 times its weight of casein into the coagulating form, paracasein, and a prepara- tion of pepsin has been obtained which will hydrolyze to peptones 100,000 times its weight of fibrin. The excessive amount of change which may thus be brought about by relatively minute proportions of enzymes almost compels the assumption that they are not consumed during the progress of the changes which they accelerate, for otherwise the enzyme would probably be "used up" long before so immense a proportion of change had been accomplished. It is, however, impos- sible at the present stage of our knowledge to submit this supposition to vigorous investigation, because the various hydrolyzing enzymes, at all events, are themselves chemically unstable substances and undergo spontaneous transformation resulting in inactivation on standing in aqueous solution. They are carried down together with any precipitates which may be formed in the digestion-mixture and are not infrequently partially bound by or combined with not only the substrate, but also the products of hydrolysis. Any chemical procedure designed to isolate and recover the enzyme from a digest in which it has been operating would involve loss or impairment of the enzyme even if it had been dissolved in distilled water instead of in a solution of the substrate which it attacks and of the products of its hydrolysis. No QUANTITATIVE RELATIONSHIPS IN HYDROLYSIS 207 attempt to re-isolate an enzyme after it has acted upon a measured amount of substrate, in order to determine the loss of activity it may or may not have sustained during the reaction, can possibly be success- ful, therefore, in the present inadequate state of our knowledge and our manipulative technique. A variety of the hydrolyzing enzymes are not only inactivated, temporarily or permanently by heat, but also by exposure to light and particularly to Ultraviolet Light. Solutions of enzymes are also temporarily inactivated by intense agitation of the solution of such a character as to give rise to excessive formation of foam. It is then found that the enzyme has become concentrated in the foam and is restored to the solution when the foam subsides. A portion of the enzyme also, under these circumstances, becomes temporarily attached to the surface of the containing vessel. This phenomenon is not peculiar to enzymes, however, for it is exhibited, in greater or less measure, by all those substances which, when dissolved in water, reduce the Surface-tension of an air-water interface. For example it is dis- played to a striking extent by the various Saponins or by Bile-salts. (See Chapter XIII.) This liability to become concentrated at liquid surfaces is probably the explanation of the striking tendency, to which reference has already been made, of the various hydrolyzing enzymes to be carried down in association with precipitates which form in their solutions. They are similarly "adsorbed" by such substances as animal charcoal or by insoluble proteins. THE QUANTITATIVE RELATIONSHIPS IN HYDROLYSIS BY ENZYMES. We have seen that when cane-sugar is hydrolyzed by acids the relationship between the amount of unaltered sugar in the system and the velocity of change is rather a simple one. The two quantities, the amount of unaltered sugar and the velocity of decomposition, are simply proportional to one another and stand in a constant ratio to one another throughout the reaction. The case is not so simple when the hydrolysis is brought about by Invertase. It will be recollected that we regard the rapid hydrolysis of cane-sugar by acids as being due to the formation of a compound between the cane-sugar and the acid, this compound being very easily attacked by water. At any instant the percentage of acid thus combined is almost infinitesimal. The amount of this compound is, as usual in such cases, directly propor- tional to the concentrations of its components, the acid and the sugar, so that we have: Concentration of sugar-acid compound = constant X concentration of acid X concentration of sugar. 208 THE HYDROLYZING ENZYMES The velocity of hydrolysis is proportional, at every instant, to the concentration of that portion of the sugar which is actually undergoing hydrolysis, i. e., the sugar-acid compound, and so we have: Velocity of hydrolysis = constant X concentration of sugar-acid compound. Combining the two equations we find that : Velocity of hydrolysis = k X concentration of acid X concentration of sugar. For any given concentration of acid, therefore, since the acid is not consumed at all during the reaction, we have : Velocity of hydrolysis = k(a x) where (a x) is the concentration of unhydrolyzed sugar at any instant. The case would be very different, however, if more than a trace of the catalyst were combined with the sugar. Suppose, for example, that in the early stages of the reaction, all of the catalyst were combined with the sugar; then, so long as there were enough sugar present to combine with all of the catalyst the amount of the catalyst-sugar compound would always be the same, namely the chemical equivalent of the amount of the catalyst in the mixture. But since this is the only portion of the sugar which hydrolyzes at any measurable rate, the velocity of hydrolysis would in that event be constant. This will, of course, only hold good so long as the sugar which is still unconverted is sufficient to combine with all of the catalyzer. As the reaction proceeds, however, a point will be reached at which the amount of sugar is insufficient to bind all of the catalyzer. After this point in the reaction is reached, the amount of the catalyst bound by the sugar, and therefore the velocity of hydrolysis of the sugar, will become progressively less as the conversion proceeds. In fact it will obviously be equal to the quantity of sugar which is still unconverted, and again we shall have the relationship: Velocity of hydrolysis = k X concentration of unhydrolyzed sugar for any given proportion of acid present in the mixture. These complications, as has been implied above, are not observed during the hydrolysis of cane-sugar by acids because the proportion of acid which is at any instant bound by the sugar is so small that it has not as yet been quantitatively estimated. But when Invertase is the catalyst instead 'of acid, we meet with precisely the conditions which we have outlined. When the proportion of sugar to invertase is high, all of the invertase is bound by the sugar, and the portion of the sugar which is thus combined is the only portion which undergoes hydrolysis at a perceptible rate. Under such conditions, for a given concentration of invertase, the rate of hydrolysis of the sugar is constant, while for varying amounts of invertase, the velocity of hydrolysis is proportional to the concentration of invertase employed. Algebraically: Velocity = kF QUANTITATIVE RELATIONSHIPS IN HYDROLYSIS 209 where "k" is a constant and "F" is the concentration of the invertase. As the proportion of sugar to enzyme falls off, however, a point must be reached at which the remnant of sugar is insufficient to bind all of the invertase. At this stage of the reaction, therefore, or if the pro- portion of sugar to enzyme is small to begin with, the reaction-velocity begins to fall off as the reaction proceeds, in accordance with the formula : Velocity = kF(a x) the numerical value of "k" being different in the two cases because, in the latter case, the "k" includes the equilibrium constant for the reaction : Sugar + catalyst *~j sugar-catalyst compound. Turning now to the question of the relationship of the Quantity of Substrate hydrolyzed to the Time of Hydrolysis it is at once manifest that if the relationship Velocity = kF holds good, then, the quantity of sugar decomposed in each unit of time being the same, the quantity "x" decomposed after time "t" must be given by the equation : x = kFt In the more general case in which the relationship obtains: Velocity = kF(a - x) the relationship between the mass of substrate transformed and the time occupied in the transformation may be found by a simple opera- tion of the integral calculus to be : loge -^- = kFt The following results are illustrative of these two types of relation- ship: Invertase on cane-sugar (A. J. Brown) Concentrated Substrate: Grams of cane sugar per Grams of cane-sugar 100 c.c. inverted in 60 minutes. 40.02 . . . . : . . . .V. . 1.076 0.179 29.96 ........... 1.235 0.206 19.91 ........... 1.355 0.226 9.85 ........... 1.355 0.226 4.89 ........... 1.230 0.206 Invertase on cane-sugar (A. J. Brown) Dilute Substrate: j Grams of cane- 10 s a sugar per Grams of cane-sugar 1(PK = -- logio 100 c c. inverted in 60 minutes. t (2.00) ........... (0.308) (132) 1.00 ........... 0.249 219 0.50 ........... 0.129 239 0.25 ........... 0.060 228 14 210 THE HYDROLYZING ENZYMES Saliva-Diastase on Starch (A. E. Taylor) ; a = 0.25 per cent. Starch: 10K = logic Time in minutes. t a x 30 . . . '; . . . .'* . .- . . , 490 45 465 60 455 75 470 90 465 120 .455 150 ; v 460 180 . . : . ..... . , 455 Trypsin on d-alanyl-d-alanine (Abderhalden and Koelker) : 103 X K = - Time in minutes. t 50 ' v 380 65 .400 7.5 . 400 16.0 369 22.0 368 28.0 368 30.0 ,-.-.' 314 38.0 ; 332 Trypsin on Sodium Caseinate (E. H. Walters) : 10* a 104 K = logio Time in minutes. t a x 15 18. 30 16. 45 : 15. 60 -....,,...., 14. 75 13. 90 ..... ^ ........... 12.5 105 12. 120 . 12. 135 12. 150 13. 165 .13. 180 12.5 210 ................. 13. 240 '..'- 12.5 270 . . \ ;..... .-..:.- Y ... ... ... 12. 300 .. . . . . . . . . . . . . :. . . . 13.5 330 ' . . 13. 360 . . . . . ... . . . . . ... . .14. 420 . \ .'.-. V . . . 14.5 480 ;' . . . . . ". . '. " '.. . 14. 540 . . ... .... . . . 14. In certain instances yet another type of relationship between the amount of transformation and the time may be observed. In the clotting of milk by Rennin and in the hydrolysis of proteins by Pepsin we find that the mass "x" of the substance transformed is connected with the time "t," the initial concentration of the substrate "a," and the concentration of the enzyme "F" by the relationship: k\Art QUANTITATIVE RELATIONSHIPS IN HYDROLYSIS 211 which is known as the Schiitz-Borissoff Rule. The rule only holds good, however, during the earlier stages of the hydrolysis and before about fifty per cent, of the substrate has been hydrolyzed. Arrhenius has found that an exactly similar relationship obtains between the time and the extent of the transformation in the hydrolysis of Ethyl Acetate by ammonia. He accounts for it by the fact that in this instance, and presumably also in pepsin-digests, the catalyzer is bound and inactiv- ated by one of the products of the hydrolysis; thus ammonia combines with the acetic acid which is liberated by the hydrolysis of ethyl acetate, forming ammonium acetate which exerts no catalytic action. If we differentiate the above algebraical expression of the Schiitz- Borissoff rule we obtain: dx k 2 aF = velocity of hydrolysis = dt 2 x from which it is evident that the velocity of hydrolysis is inversely proportional to its extent. We can account for this by supposing that the enzyme combines with a product of the hydrolysis to form an inactive compound according to the equation: Free enzyme + product = inactive compound and that the concentration of enzyme is so small compared with that of the substrate that in the first few moments after digestion has begun the concentration of the inactive compound is nearly equal to the whole of the initial concentration of the enzyme. 1 The trace of free and active enzyme which then remains will be given by: initial concentration of enzyme Concentration of free enzyme = constant X - 7: 7 1 concentration of product or, expressed algebraically: kF Concentration of free enzyme = The actual velocity of hydrolysis must be proportional to the con- centration of active catalyst and also to the concentration of uncon- sumed substrate; hence we have: kF Velocity of hydrolysis = -- k*(a x) where "k 1 " is a proportionality-factor which differs in meaning and magnitude from "k." 1 These conditions obviously do not hold in a mixture of ethyl acetate and ammonia, but the progressive modification of the electrolytic dissociation of the residual ammonia by the ammonium acetate which is formed during the reaction brings about very similar quantitative relationships. The conditions depicted, however, correspond much more closely to those which we may reasonably expect to obtain in a mixture such as that furnished by a pepsin-digest than to those which actually obtain in a mixture of ammonia and ethyl acetate. 212 THE HYDROLYZING ENZYMES The integration of this equation would lead to the relationship loge - - - x = kFt which can be shown in many instances to be the relationship which actually does obtain. When x is small, however, that is, in the early stages of the digestion, "a x" is nearly equal to "a;" the velocity equation becomes: kFa Velocity of hydrolysis = - and the integrated expression becomes: x = \/2kFat which is the Schiitz-Borissoff rule. The following is an instance of the applicability of these relationships: DIGESTION OF EGG-ALBUMIN BY PEPSIN FOLLOWED BY THE CHANGES IN THE ELECTRICAL CONDUCTIVITY OF THE MIXTURE (J. SJOQVIST). log x Protein ka = ks = Hours. digested /y/t 2 . . , ~ . . . 10.5 3.0 7.5 4 . . . '. . .-:,. 16.4 3.8 8.2 6 19.9 3.8 8.1 8 . . . . . . . 22.7 3.8 8.0 12 27.0 3.7 7.7 16 ....... 30.4 3.6 7.6 20 ...'.... 33.7 3.7 7.5 32 . . . . . . . 40.0 3.4 7.1 48 ........ 45.1 3.2 6.5 64 ... . . .-. . 50.8 3.1 6.3 96 ....... 57.4 2.8 5.9 Both formulae give tolerably uniform values for the constants, but those obtained by the complete logarithmic formula are more nearly uniform than those obtained by the employment of the partial expres- sion, the Schiitz-Borissoff rule. Whatever may be the relationship between the extent of transforma- tion and the time which may chance to obtain in a given instance of an enzymatic hydrolysis, one quantitative relationship remains invariably true, namely, that the time required to attain a given amount of trans- formation of the substrate is inversely proportional to the concentration of the enzyme. There appears to be no deviation from this rule which is not immediately explicable by decomposition of the enzyme or such adventitious factors as fluctuation of temperature, reaction and so forth. The following are instances of the applicability of this, the only universal rule which has been found to govern the action of the hydrolyzing enzymes : j INFLUENCE OF TEMPERATURE UPON ENZYMES 213 TIME REQUIRED TO LIQUEFY GELATIN HARDENED BY THYMOL, WITH VARYING QUANTITIES OF TRYPSIN (MADSEN AND WALBUM.) F = Concentration t =Time required ' of Trypsin. in hours. Ft. 0.105 0.5 0.052 0.050 1.0 0.050 0.027 2.0 0.054 0.020 3.0 0.060 0.015 4.0 0.060 0.011 5.0 0.055 0.009 6.0 0.054 0.0072 8.0 0.058 0.0060 10.0 0.060 0.0037 16.0 0.059 0.0032 18.0 O.C58 0.0027 20.0 0.054 0.0025 22.0 0.055 0.0022 24.0 0.053 COAGULATION OF MILK BY RENNET (MADSEN AND WALBUM). F = Concentration t =Time in of Rennet. minutes. Ft. 8.00 4 32 5.00 6 30 3.30 9 30 1.90 12 23 1.30 20 26 0.70 30 21 0.70 35 25 0.50 50 25 0.40 70 28 0.32 80 26 0.28 100 28 0.25 120 30 0.185 180 33 0.167 200 40 THE INFLUENCE OF TEMPERATURE UPON ENZYMES. The general effect of increasing temperature upon the hydrolyzing enzymes is to accelerate their action. This favorable influence is, however, limited by the fact that after the temperature exceeds a certain optimum the auto-inactivation of the enzyme, which always takes place at a perceptible rate in enzyme-solutions, even at ordinary temperatures, becomes so rapid as to more than counterbalance the acceleration of its hydrolyzing action. The Optimum Temperature for enzyme action was formerly supposed to be a characteristic of each enzyme, distinguishing it more or less sharply from other enzymes. We now recognize, however, that while in some measure the optimum temperature does characterize certain groups of enzymes, yet it is greatly influenced by a variety of factors other than the nature of the enzyme itself, such as the reaction (acidity or alkalinity) of the medium in which the enzyme is dissolved; the concentration of the enzyme itself and the nature and concentration of the substrate upon which it is acting. It is, of course, impossible to render all of these conditions 214 THE HYDROLYZING ENZYMES comparable in experiments with different enzymes, and we are there- fore left frequently in uncertainty whether the observed differences in temperature-optimum are in reality due to specific differences between the enzymes investigated or are not simply attributable to the circum- stances attending their action. In the very great majority of cases, however, it is found that the temperature-optimum from hydrolyzing enzymes lies very slightly above the body-temperature of the warm- blooded animals, namely between 40 and 45 C., the normal tempera- ture of man being 37.8 C. and the temperature of birds about 41 C. This remarkable correspondence is certainly not accidental, and we may infer that the processes which have brought about the evolution of the warm-blooded animals from cold-blooded ancestors has consisted essentially in an improvement of the adaptation of the more recent forms to the properties of their enzymes, whereby swifter transforma- tions and exchanges of material are rendered possible without at the same time incurring the wasteful expenditure of catalysts which would be involved by still higher bodily temperatures. The factor which determines the bodily temperature of the warm-blooded animals is certainly not the coagulation-temperature of their tissue-proteins, for that lies very considerably above the maximum body-temperature which is observed in any species. The cold-blooded animals and plants, therefore, are handicapped by a disharmony between the properties of their enzymes and the temperature of their tissues. Whether or not this is in some instances compensated for by greater specific activity of their enzymes or by the production of enzymes in greater quantity is a question which the data at present in our posses- sion do not enable us to answer. In a few exceptional cases the temperature-optimum lies far above the usual level. We have seen that some enzymes, especially certain oxidizing enzymes and the proteolytic enzymes derived from certain bacteria (Bacillus prodigiosus, for example) will withstand the tem- perature of boiling water. Two vegetable proteolytic enzymes, namely the Papain from the pawpaw, or fruit of Carica papaya and the Bromelin in pineapples act best at about 60 C. Generally speaking, the more nearly neutral the solution of the enzyme and the higher the concentration of substrate it contains, the higher is the optimal tem- perature. It would therefore appear that acids and alkalies accelerate the inactivation of enzymes and that their substrates protect them, probably owing to the fact that they combine with them. Exposure of an enzyme to moderately high temperatures, for example 60 C. for some hours generally results, not only in loss of hydrolyzing power, but in the acquirement of a power to inhibit the very hydrolysis which the active enzyme normally accelerates. This phenomenon has been attributed by Bayliss to the formation of "Zymoids" which, he believes, combine with the enzymes from which they are derived to form an inactive compound. In some instances, however, it appears that the retarding influence of heated enzymes arises not so INFLUENCE OF TEMPERATURE UPON ENZYMES much from inactivation of the unheated enzyme as in preferential acceleration of the resynthesis of the substrate, thus opposing the reaction which the unheated enzyme accelerates. If this actually occurs then it is evident that a shift in the equilibrium of the reaction : Substrate + water ^ products of hydrolysis must be brought about by the heated enzyme, of such a nature as to increase the proportion of the substrate in equilibrium with its products. Such a shift of equilibrium would, of course, necessitate a consumption of energy and a corresponding alteration in the material bringing it about, i. e., in the heated enzyme. When heated enzymes are allowed to stand in aqueous solution at room-temperatures they may undergo spontaneous Reactivation. This phenomenon has been more especially studied in connection with the Oxidizing Ferments ; but Gramentzki has observed a similar phenomenon in heated solutions of Diastase, Invertase and pancreatic Trypsin. The following are illustrative data obtained with the commercial starch-splitting enzyme Taka-diastase, obtained from Aspergillus oryzGB. The enzyme-solution was heated to 95 and then immediately cooled and allowed to stand at room temperatures. REACTIVATION OF HEAT-INACTIVATED TAKA-DIASTASE (GRAMENTZKI). Solution tested. Hydrolyzing power. Unheated enzyme '. 12.0 Heated, immediately after cooling 0.6 Heated, twenty-five minutes after cooljng 4.2 Heated, seventy-five minutes after cooling 5.5 Heated, six hours after cooling 8.2 Heated, five days after cooling 12.0 The accelerative influence of raising the temperature upon the hydrolysis of the substrate by an enzyme has been frequently investi- gated quantitatively, and it has been found that the relationship between the temperature and the velocity of hydrolysis is that which commonly pertains in chemical reactions. It may be expressed as follows : to\ f -i- = e 2 \ tito V where "\ r i" is the velocity of the hydrolysis at the temperature "ti;" "v " is the velocity of hydrolysis at the temperature "to;" "e" is the base of the natural or "Napierian" logarithms (2.71828 ) and " n" is a constant which is characteristic for the specific reaction, and is expressive of the degree of effect which temperature exerts upon it. The temperature is measured in "absolute" units, that is to say in degrees centigrade above zero plus 273. The relationship only holds good, however, so long as the temperatures employed do not 216 THE HYDROLYZING ENZYMES exceed the "temperature-optimum/' otherwise the secondary inactiva- tion of the enzyme introduces a disturbing factor. It is more simple, however, although less accurate, to estimate the effect of temperature by the change in velocity produced by a rise of 10 C. It is found, as a very general rule, excepting in the case of photochemical reactions, that the value of ^ for chemical transforma- tions is of such a magnitude (10,000 or over) that a rise of 10 at ordi- nary room- or incubator-temperatures doubles or more than doubles the velocity of transformation. The "Temperature-coefficient," or ratio: Velocity at T + 10 Velocity at T for chemical reactions is therefore 2 or over, while for purely physical processes, such as changes in viscosity or capillarity or for photo- chemical reactions the value of the coefficient generally only slightly exceeds unity or, in the case of capillary phenomena, may be less than unity. The following are illustrative values of "/*" for various hydrolyses brought about by enzymes. For comparison the value of n for the hydrolysis of cane-sugar by acids is included. Process. At Hydrolysis of cane-sugar by acids 25,600 Hydrolysis of cane-sugar by invertase 11,000 Hydrolysis of starch by amylase 12,300 Hydrolysis of triacetin by lipase 16,700 Hydrolysis of egg-albumin by pepsin . . . . . . . . 15,570 Hydrolysis of casein by trypsin 37,500 Inactivation of rennet 90,000 Inactivation of pepsin 75,000 Inactivation of invertase 72,COO Inactivation of trypsin 62, 000 On comparing these various figures it will be seen that the effect of temperature upon enzymatic hydrolyses is of the same general order as its effect upon other chemical reactions. The Inactivation of an enzyme by heat, however, is exceptionally accelerated by rise of temperature, the coefficients for all inactivations being very much higher than those for the hydrolyses which the enzymes accelerate. This accounts for the relative "steepness" with which the curve of enzymatic activity falls off after the temperature has passed the opti- mum; at this point the inactivation of the enzyme is very much accelerated by a rise in temperature sufficient only to produce a slight modification of the velocity of the hydrolysis which the enzyme is accomplishing. Enzymes are also inactivated by exposure of their solutions to Light, and especially to the Ultra-violet Rays. The inactivation by ultra- violet light occurs in the absence of oxygen, but the visible rays of light, especially in the presence of fluorescent dyes such as Eosin, are INFLUENCE UPON HYDROLYSES BY ENZYMES 217 also able to inactivate enzymes provided oxygen be present. Evi- dently two different types of change in enzymes may be brought about by light, involving different parts of the spectrum. THE INFLUENCE OF REACTION UPON HYDROLYSES BY ENZYMES. The great majority of the enzymes are very decidedly influenced by the reaction, or H + or OH" ion concentration of the medium in which they act. For each enzyme or group of enzymes there is a certain range of H + or OH~ concentrations within which they work best and below or above which their activity is impeded. The upper limit of H+ or OH~ concentrations is set by the destruction of the enzyme which rapidly occurs in solutions which are too acid or alkaline. The factor which sets the lower limit is not so easy to perceive since the stability of the enzymes in neutral solutions is often greater than it is in the faintly acid or alkaline solutions in which their hydrolyzing activity is most favorably displayed. The most striking dependence upon reaction is shown by the Proteo- lytic Enzymes, Pepsin and Trypsin. Pepsin acts best in a faintly acid, while trypsin acts best in a faintly alkaline medium. A slight excess of alkali rapidly destroys pepsin, while an even slighter acidity inacti- vates trypsin. Both of these enzymes will hydrolyze proteins in neutral solutions, but their activity is much inferior to that which they will display in a medium of favorable reaction. So far as pepsin is concerned it is not difficult to infer that the need for a slight acidity of the medium arises from the fact that a compound of the pepsin with the acid is formed which possesses much greater proteolytic power than the uncombined pepsin. This is evidenced by the fact that not all acids are equally efficient in promoting the hydrolysis of protein by pepsin, there being a marked specificity in the relationship of pepsin to Hydrochloric Acid. While other acids will accelerate the hydrolysis of proteins by pepsin, their accelerative influence is far inferior to that of hydrochloric acid, and the favoring action of different acids, instead of running parallel to their degree of electrolytic dissociation, as we should expect if it were an effect purely due to hydrogen ions, bears, in fact, no relationship to the "strength," i. e., dissociation of the acid. Lactic Acid, for example, in equimolecular solutions, has a more favorable effect than sulphuric acid. In the case of Trypsin the accelerative action of alkalies runs strictly parallel to their dissociation, so that here we are left in doubt as to whether the effect is due to the formation of a compound of the trypsin with the base employed, or whether the alkali does not, on the con- trary, act as an accessory catalyzer, so altering the substrate as to render it more susceptible to attack by the enzyme. There are certain observations, however, which seem to show that in this case also a compound is formed of the trypsin and the added 218 THE HYDROLYZING ENZYMES base which exerts a much more intense proteolytic action than trypsin itself. For if we follow the hydrolysis by trypsin, of an alkaline solu- tion of casein by means of the gas-chain (potentiometer) so that we obtain a measure of the changes in the actual hydroxyl ion concentra- tion as the hydrolysis proceeds, we find that the alkalinity of the digest progressively diminishes at a uniform rate corresponding to the formula : Velocity = k(a x) until a certain Critical Alkalinity is reached, which lies in the N neighborhood of 10~ 6 N or nnn nnn , below which the velocity of 1,UUU,UUU hydrolysis diminishes very much more rapidly than Wilhelmy's law would indicate. For a given concentration of trypsin the critical reaction is exactly the same when a basic protein such as protamine is employed as when the acid protein, casein, is the substrate. Evi- dently, therefore, this sudden falling off in the velocity of hydrolysis is not due to any relationship of the Substrate to the free alkali in the digest, but rather to a relationship of the Enzyme to the free alkali. The result is, in fact, exactly what one would expect to obtain if the actual catalyst were a compound of trypsin with the alkali. The concentration of the catalyst would remain constant at all alkalinities below those destructive of the enzyme, provided there was a sufficient amount of unneutralized alkali present to combine with all of the trypsin. Directly the concentration of free alkali fell below this limit, however, the concentration of active enzyme would diminish in pro- portion to the diminution of alkalinity, that is to say, in proportion to the extent of hydrolysis, and the velocity of hydrolysis would fall off correspondingly rapidly. Since in these two instances we have experimental ground for the belief that the favorable influence of dilute acids or alkalies upon the activity of the enzyme is due to the formation of compounds with the enzyme, we may infer that the mechanism of the acceleration by acids or alkalies is probably the same in other cases. THE SPECIFICITY OF THE HYDROLYZING ENZYMES. The various enzymes which hydrolyze Disaccharides and Glucosides are highly specific in their action, that is to say, a given enzyme will hydrolyze a particular disaccharide or a particular type of glucoside and no other. A very beautiful example of the specific relationship which subsists between the structure of a glucoside and the nature of the enzyme which attacks it is that afforded by the enzymatic hydrolysis of the various Methyl Glucosides. Four of these glucosides are known, namely a-methyl-1-glucoside, and /3-methyl-l-glucoside, SPECIFICITY OF THE HYDROLYZING ENZYMES 219 a-methyl-d-glucoside and /3-methyl-d-glucoside. Their structures are represented below: o o, HCOCHa \ HCOH\ HOCH HCX I HCOH CH 2 OH a-methyl-d-glucoside. CHsOCH CH 3 OCH !\ HCOH :o o; CH 2 OH a-methyl-1-glucoside. HOCH | / HC/ HCOH CH 2 OH /3-methyl-d- glucoside. HCOCH 3 HOCH HCOH H I HOCH CH 2 OH /8-methyl-l- glucoside. Of these neither a- nor /3-methyl-l-glucoside are acted upon by enzymes. The a-methyl-d-glucoside is hydrolyzed by the Maltase in yeast, but the j3-methyl-d-glucoside is not hydrolyzed by yeast; it is, on the other hand, hydrolyzed by the enzyme Emulsin which is found in the kernels of stony fruits such as' the almond and in the tissues of the fungus Aspergilhis niger. But emulsin is without action on the a-methyl-d-glucoside. Similarly, Invertase, which hydrolyzes cane-sugar to glucose and fructose, will not hydrolyze maltose; maltase, which hydrolyzes maltose to two molecules of glucose, will not attack cane-sugar or lactose; lactase, which hydrolyzes milk-sugar, will not hydrolyze maltose or cane-sugar. Since these various disaccharides differ from one another only in the arrangement of the various groups about the central carbon-skeleton, the high degree of specific interrelationship with the enzymes which attack them which they display, led Emil Fischer to the view frequently alluded to as the Lock-and-key Hypothesis, whereby the enzyme is supposed to possess a structure which fits a particular disaccharide or glucoside as the grooves of a key fit the wards of a lock. Indeed there is no other way in which we can imagine a mechanism which will so precisely pick out a particular arrangement of atoms and decompose that one and no other. The phenomenon affords, in fact, a striking confirmation of the view that these enzymes accomplish the hydrolysis of the disaccharides through the formation of intermediate compounds. The fat-splitting ferments, or Lipases do not exhibit such extremely preferential specificity. Nevertheless some measure of specificity is displayed in certain instances. Thus Dakin found that in a mixture of the menthyl esters of d- and 1-Mandelic Acid the menthyl-d-mandelate is hydrolyzed by pancreas-lipase much more rapidly than the menthyl- 1-mandelate, so that the mandelic acid which results from the hydrolysis is strongly dextrorotatory. 220 THE HYDROLYZING ENZYMES Among the Proteolytic Enzymes we again meet with a series of specific relationships between the enzymes and the substances which they hydrolyze. This specificity is not revealed when we act upon Proteins with various Trypsins since every protein which is soluble contains some of the linkages which are susceptible to attack by any given trypsin. Digestion of a protein w r ill therefore proceed with trypsin from any source, and if the linkages in the molecule which are attacked differ with the type of trypsin employed, we have at present no certain means of ascertaining that fact in so complicated a molecule as that of a protein. It was for this reason that the specificity of different trypsins was until recently totally unsuspected, and it was tacitly assumed that all of the enzymes which hydrolyze native proteins to amino- acids in faintly alkaline solutions were identical. The employment of synthetic Peptides of known structure and configuration as substrates, however, has recently revealed to us a heretofore unsuspected multi- plicity of protein-hydrolyzing enzymes. The trypsin in pancreatic juice was found by Fischer and Abder- halden to hydrolyze certain synthetic peptides while others remain unattacked. The various peptides which they investigated were distributed between these two classes as follows: HYDROLYZED. *Alanyl-glycine *Alanyl-leucyl glycine *Alanyl-alanine Dialanyl-cystine *Leucyl-isoserine Dileucyl-cystine Glycl-1-tyrosine Tetraglycl-glycine Leucyl-1-tyrosine Triglycl-glycine ester *Alanyl-glycyl-glycine d-alanyl-d-alanine *Leucyl-glycyl-glycine d-alanyl-1-leucine *Glycyl-ieucyl-alanine 1-leucyl-l-leucine l-leucyl-d-glutamic acid NOT HYDROLYZED. Glycyl-alanine Leucyl-proline Glycyl-glycine Diglycyl-glycine Leucyl-alanine Triglycyl-glycine Leucyl-glycine Dileucylglycyl-glycine Aminobutyryl-glycine d-alanyl-1-alanine Valyl-glycine 1-alanyl-d-alanine Glycyi-phenylalanine 1-leucyl-d-leucine d-leucyl-1-leucine It will be observed that especially among the various dipeptides formed by the union of d- and 1-alanine with d- and 1-leucine the specificity of the enzyme is very strongly marked. It will be noted also that mere length of the pep tide-chain confers upon it susceptibility to attack. Thus diglycyl-glycine and triglycyl-glycine were not attacked, while tetraglycylglycine was hydrolyzed. The compounds marked with an asterisk were racemic, and in every case only one of the optical anti- podes was attacked, in every case also the isomer which was hydrolyzed was that which occurs in the native proteins. SYNTHETIC ACTION OF HYDROLYZING ENZYMES 221 The trypsin which is contained in red blood-corpuscles was found to hydrolyze glycyl-1-tyrosine, in this respect resembling the trypsin of pancreatic juice. It also hydrolyzed diglycyl-glycine, however, and therefore it cannot be identical with the trypsin of pancreatic juice. Blood-serum will not hydrolyze glycyl-1-tyrosine and the trypsin which it contains therefore differs both from pancreatic trypsin and red-blood- corpuscle trypsin, yet it will hydrolyze d-1-alanyl-glycine, diglycyl- glycine and tri-glycyl-glycine. The existence of three different trypsins is thus demonstrated, and from these and similar experiments we can infer that the variety of animal trypsins is very great and possibly coextensive with the number of different types of tissue which may comprise the body of a multicellular animal. THE SYNTHETIC ACTION OF HYDROLYZING ENZYMES. When we hydrolyze such a substance as Ethyl Butyrate with the aid of a non-enzymatic catalyzer, we find that the transformation into ethyl alcohol and butyric acid is never complete, but stops short when, in dilute solutions, about two- thirds of the ester is decomposed. No matter what catalyzer we may employ, or if we allow spontaneous hydrolysis to occur, or bring about hydrolysis by means of a fat-split- ting enzyme, the transformation comes to a standstill when about one- third of the ester still remains undecomposed. 1 On the other hand, if we mix ethyl alcohol and buytric acid, and by the agency of cata- lyzers or otherwise, bring about their combination, we will find that here also the transformation is never complete, but that it comes to a standstill when about one-third of the alcohol and buytric acid have combined to form the ester. Evidently, therefore, from whichever end of the process we start we reach a mixture of the same composition. We cannot suppose that either reaction has then ceased to occur, but we can readily see that in the mixture which no longer alters in com- position and is at equilibrium the forward and reverse actions are pro- ceeding at the same rate: C 3 H7COOC 2 H B + H 2 O ; C 3 H 7 COOH + C 2 H 6 OH In a variety of hydrolyses the same phenomenon is observed, but in the majority of instances the station of equilibrium lies further to the right or left than in the instance chosen for illustration. Thus in the hydrolysis of cane-sugar it lies so far to the left that at equilibrium the hydrolysis is, so far as all practicable measurements are concerned, absolutely complete. Now a true catalyzer is, as we have seen, not consumed at all during the progress of the process which it accelerates, and, this being the case, it cannot communicate any Energy to the system. Any shift of equilibrium in a chemical reaction which absorbs or liberates heat must involve the consumption or absorption of a quantity of energy equivalent to the heat of reaction. But equilibrium, as we have seen, 1 The exact proportion depends, as we shall see, upon the dilution of the solution, i. e., upon the mass of water in the reacting mixture, 222 THE HYDROLYZING ENZYMES occurs only when the forward and reverse velocities are equal, and hence if the forward reaction, or reaction of hydrolysis, is accelerated by a catalyzer, the reverse reaction or reaction of synthesis must also be accelerated and to an exactly equal degree. If, then, the hydrolyz- ing enzyme's are analogous to other catalyzers and are not consumed during the progress of the reactions which they affect, they must accelerate the resynthesis of the substrate from its products no less than the hydrolysis of the substrate itself. The prediction, based upon the above premises, made by the Dutch chemist Van't Hoff in 1898 that enzymatic syntheses might prove possible was -verified experimentally in the same year by Croft Hill, who succeeded in this manner in synthesizing a disaccharide by acting upon a highly concentrated solution of glucose with the enzyme Maltase obtained from yeast. The synthetic disaccharide was, very naturally, assumed to be Maltose, but further investigation showed that the prediction of Van't Hoff had not been so completely verified as was at first supposed, for Emmerling in 1901 showed that the disaccharide which was actually produced in Croft Hill's experiment was not maltose, or glucose-a- glucoside, but a disaccharide which yields a predominating proportion of /3-glucose on hydrolysis, namely glucose-/3-glucoside, or Isomaltose. Now isomaltose is not hydrolyzable by maltase, so that the synthetic activity of the enzyme, instead of reversing the reaction of hydrolysis, produces a disaccharide which it cannot hydrolyze. It would appear that a shift of equilibrium is actually occasioned by the enzyme, but as there is no difference of energy-content between optical isomers, the shift in the station of equilibrium caused by the enzyme is not, so far as the production of isomaltose instead of maltose is concerned, of such a character as to require consumption of the enzyme to accomplish the liberation or absorption of energy. Isomaltose is, however, hydrolyzable by the enzyme Emulsin which occurs in different situations from those in which maltose is found-. It became at once a matter of great interest to ascertain what synthetic products would result from the action of emulsin upon concentrated solutions of glucose. This experiment was carried out by E. F. Armstrong, who found that the product resulting from the synthetic action of emulsin was not isomaltose, but Maltose. Each enzyme, therefore, synthesiezs that enzyme which it cannot hydrolyze. These relationships may be schematically represented by the following diagram : Emulsin Maltose < Glucose Glucose Isomaltose Maltase SYNTHETIC ACTION OF HYDROLYZING ENZYMES 223 Similarly the Lactase in kephir yeast was found to synthesize, not Lactose which it hydrolyzes, but Isolactose, which it does not hydrolyze. Since the publication of Croft Hill's fundamental observation a great number of enzymatic syntheses have been accomplished. Among carbohydrates, substances resembling Starch and Glycogen have been synthesized through the action of Diastases, while Triacetyl Glucose has been formed from acetic acid and glucose under the influence of pancreas-extract. Among the fats Ethyl Butyrate has been synthesized from ethyl alcohol and butyric acid, glyceryl butyrate, amyl butyrate, methyl oleate, glyceryl triacetate and glyceryl trioleate have all been synthesized from their components through the reversed action of various Lipases. In the case of methyl oleate it has been shown that the pancreas-lipase employed to bring about its synthesis definitely does not affect the final equilibrium which is attained, for the proportion of ester formed after a sufficient lapse of time is independent of the quantity of enzyme employed, only the Speed with which the equilib- rium is attained being affected. The following are illustrative data: SYNTHESIS OF METHYL OLEATE (POTTEVIN). Quantity of pancreas- extract employed. 1 Percentage of ester formed. 1 day. 8 2 days. 56 66 66 74 20 days. 84 82 84 85 2 12 5 21 10 . .... 43 Quantitative data of this description are very important because the whole question whether the enzymes act as true catalyzers or, on the contrary, enter into and affect the equilibria of the reactions which they accelerate, turns upon the question whether the final proportion of substrate to products is at all influenced by the presence of the enzyme. The results of Pottevin indicate that there is no such influ- ence in the case of pancreas-lipase synthesizing methyl oleate, because if there were, then doubling the amount of enzyme should double the displacement of equilibrium and since no measurable effect upon the equilibrium results from doubling or even multiplying by ten the quantity of enzyme employed, it follows that the single unit of enzyme also did not affect the station of equilibrium. Similarly A. E. Taylor has shown that the station of equilibrium in the hydrolysis of glyceryl triacetate by lipase is exactly the same as that which is obtained when sulphuric is used as the catalyzer. Such measurements are, however, usually lacking in enzyme studies, frequently because of the technical difficulty of measurements extending over the long periods required to attain final equilibrium. In the case of Glyceryl Trioleate, which differs from the glyceride studied by Taylor in being insoluble in water, H. C. Bradley has attained results which point rather clearly toward a decided displace- ment of equilibrium by the enzyme, for he finds it impossible to procure 224 THE HYDROLYZING ENZYMES any appreciable synthesis of Triolein from oleic acid and glycerol in the presence of fifty per cent, of water, although when we start with triolein in this proportion of water an appreciable amount of triolein remains unhydrolyzed at the end of prolonged hydrolysis. The presence of the enzyme in this case appears to selectively accelerate hydrolysis, and if this is the case then the enzyme must be consumed in the process. Among the proteins the synthesis of a protamine, Salmine, from a concentrated solution of its digestion-products has been accomplished by A. E. Taylor, who employed an unusually stable Trypsin obtained by extracting the liver of a mollusc (Schizothoerus nuttalli) with glycerol, and adding this in very large amounts to a saturated solution of the amino-acids which finally result from the hydrolysis of protamine. Only a small proportion, about one-half of a per cent., of the original protein was recovered and that only after a lapse of five months. The identity of the synthetic protein with salmine was deduced from analysis and general physical behavior. When Sodium Caseinate in neutral solution is subjected to hydrolysis by Pepsin a group of infraproteins results, which are collectively termed Paranucleins, and which subsequently undergo further hydrolysis, with the production of proteoses and peptones. The paranucleins resemble casein in being soluble in dilute alkalies and precipitable by acetic acid, but are less soluble in dilute mineral acids than casein itself. When to the concentrated solution obtained by evaporating down the final products of the prolonged peptic hydrolysis of casein, a very large proportion of fresh pepsin is added, after a comparatively brief period (forty-eight hours) at 40 C. a precipitate is formed in the mixture which appears to be identical with paranuclein. In this case the identity has been confirmed by immunological methods. The antiserum to casein or paranuclein produced by repeatedly injecting these substances into the circulation of rabbits yields no precipitate either with the products of the complete peptic hydrolysis of casein or with pepsin, but it does yield a precipitate with the synthetic para- nuclein obtained in the manner outlined, and this precipitate binds the antibodies to casein and paranuclein which the serum contained. Since the Antibodies which appear in the circulation of animals as a result of immunizing them against proteins are in the highest degree specific, yielding precipitates only with the protein employed in the immunization or with inf raprotein derived from it by partial hydrolysis the synthetic product may be considered to be thus clearly identified with the infraproteins which are the first cleavage-products of casein. In this case, however, rather definite indications were obtained that the Synthesizing Enzyme is not identical with pepsin itself, for it proved possible to bring about the synthesis a great deal more rapidly at 70 C. than at 40 C., while the hydrolyzing activity of pepsin is inhibited altogether at this temperature. Moreover not every preparation of pepsin will yield the synthesis. It has been suggested that the active SYNTHETIC ACTION OF HYDROLYZING ENZYMES 225 agent in accomplishing the synthesis is a modification of pepsin, arising from it by loss of water : Hydrolyzing Pepsin ^ Synthesizing Pepsin + H2O and that in bringing about the hydrolysis of protein the hydrolyzing form may partially lose water and be transformed into the synthesizing form and vice versa, high temperatures, as usual, favoring the forma- tion of the anhydride or synthesizing form. If this were so, of course, pepsin would be far from being a true catalyzer, since it would enter into and be modified by the reactions which it accelerates. Such modification has, however, not yet been shown to occur. It will be seen, therefore, that while in some instances the hydrolyzing enzymes appear to act as genuine catalyzers, in other instances their behavior appears to be inconsistent with this view. In any case, the synthetic activity of the hydrolyzing enzymes, so far as it has yet been demonstrable in vitro is very inferior in point of speed and completeness to the synthetic processes which actually and continually occur in living tissues. As we shall see, glucose is converted into glycogen almost as rapidly as it can be absorbed and transported, dissolved in minute concentration in the. blood, to the cells of the liver. Fat is synthesized from glycerol and fatty acids in the intestinal mucosa within a few moments after absorption, there are strong reasons for the belief that the synthesis of protein from amino-acids in the tissues is not much less rapid. The contrast betweea these phenomena and the prolonged periods of action and high concentrations both of the enzyme and the products required to resynthesize the substrate by a hydrolyzing enzyme, and the fragmentary yield which results, point very strongly to the existence, in living tissues of a decisively different synthesizing mechanism to that involved in the reversed action of catalyzers. Only two alternatives appear to be open to us in interpreting this dispro- portion. Either the tissues employ enzymes which selectively accele- rate syntheses and therefore are consumed by the syntheses which they accomplish, or else the syntheses in living tissues, composed though they are of over eighty per cent, of water, take place under conditions approximating to almost complete desiccation. This latter alternative is not so inconceivable as it might appear, because the reactions in question may possibly take place at the surface of lipoid granules which are emulsified in the protoplasm, but which, being insoluble in water, afford a medium which is almost water-free. The most serious difficulty attaching to this view, however, is that so many of the products so rapidly synthesized in living tissues are as insoluble in oils as water is itself, for example the proteins. On the other hand if the former alternative be adopted we are faced with the difficulty that the hypothetical synthesizing enzymes have never been obtained apart from living tissue, so that either their action is intimately bound up with the uninjured Structure of the protoplasm, or else we have not yet hit upon the right methods of extracting them from living tissues and conserving their synthetic activity. ' 15 226 THE HYDROLYZING ENZYMES ANTIENZYMES. Like the proteins and the poisonous products of bacterial metabolism, the various enzymes, when injected into the circulation of living organisms, give rise to specific Antibodies, or substances in the circula- tion of the immunized animal which combine with the enzyme which has been injected. The Antienzymes thus produced are highly specific and bind only the enzyme employed for immunization. Normal blood, however, contains appreciable amounts of antitrypsin and also of antirennet, which latter, however, is stated not to be identical with the antirennet produced by immunization. The antienzymes appear as a rule to be very resistant to heat, withstanding for some time a temperature of 70 without losing their power of inhibiting digestion of the enzymes which they bind. Antipepsin and Antitrypsin also occur in notable quantities in the tissues of Intestinal Worms, and it is to this that their immunity to digestion is attributed. The immunity of the tissues of the stomach to digestion by the gastric juice which they produce, and of the tissues of the intestine to digestion by pancreatic juice is similarly attributed to the normal presence, in these tissues, of antienzymes. The antigenic property of the enzymes rather strongly points toward their ultimate protein nature, for up to the present no substance has been found to produce antibodies on injection which has not been a protein, or a substance possibly contaminated by a protein. REFERENCES. GENERAL: Taylor: Fermentation, Univ. of California Pub. Pathology, 1907, 1, 87. Bayliss: The Nature of Enzyme Action. London, 1914. Euler: General Chemistry of the Enzymes. Trans, by Pope. New York, 1912. Arrhenius: Quantitative Laws in Biological Chemistry. London, 1915. Effront: Biochemical Catalysts in Life and Industry. Trans, by Prescott. New York, 1917. Robertson: The Physical Chemistry of the Proteins. New York, 1918. INFLUENCE or TEMPERATURE: Arrhenius: Immunochemistry. New York, 1907. INFLUENCE OF REACTION: Kanitz: Zeit. f. physiol. Chem., 1902-3, 37, p. 75. Berg and Gies: Jour. Biol. Chem., 1906-7, 2, p. 489. Robertson and Schmidt: Ibid., 1908-9, 5, p. 31. Loeb: Biochem. Zeitsch., 1909, 19, p. 534. SPECIFICITY : Armstrong: The Simple Carbohydrates and the Glucosides. London. 2d ed. Fischer and Bergell: Ber. d. d. chem. Ges., 1903, 36, p. 2592; 1904, 37, p. 3103. Dakin: Jour, of Physiol., 1904, 30, p. 253; 1905, 32, p. 199. Fischer and Abderhalden: Zeit. f. physiol. Chem., 1903, 39, p. 81; 1905, 46, p. 52; 1907, 51, p. 264. Abderhalden and collaborators (Bergell, Rona, Samuely, Teruuchi, Babkin, Hunter, Kautzsch, Schittenhelm, Koelker, Gigon, Deetjen, McLester, Manwaring, Lussana, Rilliet, Strauss, Dammhahn, Pringsheim, Pincussohn, Weichardt, Heise, Medigre- ceanu, Walther): Zeit. f. physiol. Chem., 1903, 39, p. 9; 1905, 46, pp. 176 and 187; 1906, 47. pp. 159, 346, 359, 391, 466; 1906, 48, pp. 537 and 557; 1906, 49, pp. 1, 21, 26, 31; 1907, 51, pp. 294, 311, 334; 1907-8, 54, p. 363; 1908, 55, pp. 371, 377, 384, 390, 395, 416; 1908, 57, p. 332; 1909, 59, p. 249; 1909, 60, p. 415; 1909, 61, p. 200; 1909, 62, pp. 120, 136, 145, 243; 1910, 66, pp. 265, 277; 1910, 68, p. 471. ANTIENZYMES 227 SYNTHETIC ACTION: Croft Hill: Jour. Chem. Soc., 1898, 73, p. 634. Cremer: Ber. d. d. chem. Ges., 1899, 32, p. 2062. Berninzone: Atti. del. soc. ligi. di. scien. nat. e. geograph., Genoa, 1900, 11, p. 327. Kastle and Loevenhart: Am. Chem. Jour., 1900, 24, p. 491. Hanriot, C. R.: Acad. des. Sci., 1901, 132, p. 212. Acree and Hinkins: Am. Chem. Jour., 1902, 28, p. 370. Armstrong: Proc. Roy. Soc. London, B; 1904, 73, p. 500. Taylor: Univ. of California Pub. Pathology, 1904, 1, p. 33. Jour. Biol. Chem., 1906-7, 2, p. 87. Bodenstein and Dietz: Zeit. f. Elektrochem., 1906, 12, p. 605. Poltevin: Bull. Soc. China., 1906, 35, p. 693. Ann. Inst. Pasteur, 1906, 20, p. 901. Taylor: Jour. Biol. Chem., 1907, 3, p. 87. Robertson: Jour. Biol. Chem., 1907, 3, p. 95; 1908-9, 5, p. 493. Robertson and Riddle: Ibid., 1911, 9, p. 295. Gay and Robertson: Ibid., 1912, 12, p. 233. Harden and Young: Biochem. Jour., 1913, 7, p. 630. ANTIENZYMES: Hildebrandt: Virchows Arch., 1893, 131, p. 12. Achalme: Ann. Inst. Pasteur, 1901, 15, p. 737. Weinland: Zeit. f. Biol., 1903, 44, pp. 1 and 45. Cathcart: Jour. Physiol., 1904, 31, p. 497. Hedin: Ibid., 1905, 32, p. 390. Saiki: Jour. Biol. Chem., 1907, 3, p. 395. Robertson and Hanson: Jour. Immunology, 1918, 3, p. 131. CHAPTER XI. THE DIGESTION AND ASSIMILATION OF THE FOODSTUFFS. THE DIGESTION OF THE CARBOHYDRATES. The Starch in our diet is converted by cooking into "soluble starch" which is much more readily hydrolyzed by the starch-splitting enzymes or Amylase 1 than the uncooked material. The first enzyme to en- counter the foodstuffs upon their introduction into the alimentary canal is the amylase or Ptyalin of saliva. This enzyme energetically hydrolyzes the starch to maltose, and it is for this reason that starch, when it is held in the mouth, presently begins to taste sweet. There is no amylase in the Gastric Juice, but nevertheless the diges- tion of starch or glycogen continues for some time in the stomach, because the optimum reaction for amylase is a very faint acidity. The gastric juice itself is strongly acid, in fact, far too acid to permit the action of amylase if this enzyme were received directly into unneutral- ised and undiluted gastric juice. But various constituents of the diet, and especially the proteins, combine with the Hydrochloric Acid of the gastric juice and partially neutralize it, so that the contents of the stomach during the partaking of a meal and the earlier periods of digestion are either neutral or only faintly acid. Long before the acidity of the gastric contents approaches that of pure gastric juice, the pyloric sphincter opens and permits the passage of the semi- digested foodstuffs in small portions at a time into the lumen of the small intestine. The maltose which is thus formed by the digestion of starch is not normally absorbed from the stomach either as such, or in the form of its further cleavage-product, glucose. Under normal conditions there is little or no absorption of maltose or other sugars from the stomach. If the pylorus be ligated, some absorption of sugar will then be found to occur, but only under conditions involving abnormal dilatation. No carbohydrate-splitting enzymes are found in the gastric juice of man. It is stated that Lactase may often be found in the gastric juice of the calf, but not in the adult animal. After the foodstuffs have remained in the stomach for a sufficiently long period to allow the Chyme to become faintly acid through admix- ture with an excess of gastric juice, the pyloric sphincter opens and permits the passage of the chyme into the upper part of the small 1 The amylases are frequently referred to as Diastases. In French scientific litera- ture the word "Diastase" is used as a generic term to include all types of enzymes. DIGESTION OF THE CARBOHYDRATES 229 intestine. Here the foodstuffs are very soon met by the alkaline Pancreatic Juice, which reaches the intestine, in man, through the common duct of the liver and the pancreas. The pancreatic juice contains an Amylase which completes the work of the salivary amylase and furthermore, a Maltase which converts the maltose, derived by the action of amylase from starch, into Glucose. The glucose which is thus formed is very rapidly absorbed into the portal circulation, and carried to the liver where it is converted into Glycogen. The rapidity of this conversion is very great. Thus the quantity of glucose derived from the polysaccharides in a single meal may very readily exceed one hundred grams. Dissolved in all of the blood in the body, which cannot exceed seven liters in a man of seventy kilos, this would give a glucose concentration of no less than 1.5 per cent. As a matter of fact even at the height of absorption during a meal rich in carbohydrates the concentration of glucose in the blood of a person in normal health never exceeds one-tenth of this. As rapidly, therefore, as the glucose is taken to the liver by the portal circulation, it is transformed into the colloidal anhydride, glycogen, and held in reserve for future consumption. When, however, an extraordinary load is thrown upon this mechan- ism, by the excessive ingestion of diffusible sugars, some slight degree of Glucohemia or excess of sugar in the blood may nevertheless occur and in these cases the glucohemia is relieved by the passage of sugar into the urine. This type of glycosuria is known as Alimentary Glyco- suria. The sugar which is found in the urine is usually Glucose, but when cane-sugar or sweets made of cane-sugar have been ingested in large quantities, Levulose may also be found in the urine, together with traces of unhydrolyzed cane-sugar. Lactose is somewhat more readily absorbed and excreted as such than cane-sugar. If either of these sugars be injected intravenously, they appear unaltered and quantitatively in the urine. It is an exceedingly remarkable fact that whereas amylase and maltase are both present in the digestive juices, Lactase and Invertase are usually completely absent, or if lactase is present its action is inconspicuous. We have seen that the unaltered disaccharides, if absorbed as such, are not utilized but are as promptly as possible ejected from the circulation by the kidneys. Yet lactose is the sole carbohydrate nutriment of suckling infants and cane-sugar is an exceed- ingly important item in the dietary of modern peoples. As a matter of fact, although the hydrolysis of these disaccharides cannot be accomplished to any important extent by the secretions which are poured into the alimentary canal by the various digestive glands, yet the consequence of their ingestion is actually the appearance of in- creased glucose in the portal circulation and enhanced storage of glycogen in the liver. When partaken of in reasonable amounts they are furthermore fully utilized for maintenance and the production of energy in the body. At some point during their passage through the 230 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS intestinal epithelium they are evidently broken down into simple sugars or monosaccharides, but the modifications induced by the intestinal epithelium go even further, for the hydrolysis of cane-sugar yields Levulose as well as glucose, and the hydrolysis of lactose yields Galactose as well as glucose. We are compelled to assume that the levulose and galactose fractions of these molecules are converted, either in the intestinal mucosa or else in the liver epithelium, into glucose. In the case of levulose this presents little theoretical difficulty, for the partial conversion of levulose into glucose can be brought about in vitro by the prolonged action of dilute alkali. We are ac- quainted with no mechanisms, however, which will accomplish the direct transformation of d-galactose into d-glucose, much less with any enzyme which will bring it about. That the converse process, the transformation of glucose into galactose may be brought about in living tissues is shown by the Glycosuria which immediately succeeds extirpation of the mammary glands in milch-cows and goats. The sugar that appears in the urine is glucose, and glucose only, although the lactose for the manufacture of which the excess of glucose had previously been utilized, is a compound of glucose and galactose. The remarkable feature of this transformation is that it involves disruption of the oxide-ring of glucose and its reformation upon the opposite side of the molecule: HCOH HOCH HCOH [OCH / \ / HCOH \ HOCH / \ HOCH HC/ HCOH HCOH CH 2 OH CH 2 OH ot-d-Glucoae. d-Galactose. The normal circulating form of hexose is therefore d-Glucose and d- glucose only. Whatever form of hexose or polysaccharide derived from a hexose may be ingested, if it is absorbed at all, it appears under normal circumstances as Glycogen in the liver, having either reached the liver-cells in the form of glucose, or else been transformed by them into glucose as a preliminary step in the formation of this colloidal reserve-carbohydrate. From the liver the carbohydrate material is redistributed over the body as the need arises, being broken down to glucose again before it makes its appearance in the circulation. The determining factor which regulates the discharge of this carbohydrate reservoir is probably the concentration of glucose in the blood. This DIGESTION OF THE CARBOHYDRATES 23i normally lies between 0.5 and 0.15 per cent, and we may suppose that when the consumption of carbohydrate in distant tissues results in a certain degree of depletion of local stores, and of the circulating glucose, the equilibrium between the glycogen stores in the liver and the glucose in the fluids bathing the liver-cells is disturbed, and glycogen breaks down in order to restore it. (C 6 HioO 5 )n + nH 2 O -> nC 2 Hi2O 6 ) Glycogen Glucose We must suppose that Pentoses, in so far as they form constituents of animal tissues, namely in the d-Ribose radical of guanylic and inosinic acids, are derived from pentoses originally contained in the diet. It will be recollected that guanylic and inosinic acids represent fragments derivable by partial hydrolysis from vegetable nucleic acid, and catalytic agents capable of bringing about this cleavage are found widely distributed in animal tissues and tissue fluids The mono- nucleotids are not improbably absorbed as such, the adenine mononucle- otid being transformed by direct deaminization into the corresponding hypoxanthine derivative. An important proportion of the dietary of herbivora, however, is furnished by Pentosans, or polysaccharides derived from pentoses. We have no evidence of the existence, either in the digestive juices or in the epithelial wall of the intestine, of any enzymes capable of trans- forming these substances into glucose. Yet the experimental fact remains that pentoses can be utilized by animals, and energy derived from them for the performance of work and the maintenance of the body. Whether they are absorbed and oxidized in the tissues as such or as glucose is not known, but the administration of Rhamnose to a diabetic whose urine has been made sugar-free by the exclusion of carbohydrates from the diet, leads to reappearance of glucose in the urine. The Celluloses in the dietary are indigestible by any of the enzymes produced by the digestive glands or the intestinal epithelium. Never- theless they are partially utilized, as much as forty per cent, of young and tender cellulose, such as that occurring in lettuce, being utilizable for the production of energy by human beings. We owe this ability to the digestive activities of the bacteria which inhabit the lower intestine. The bacterial flora is particularly abundant in the lower intestine of the herbivora, and as much as seventy per cent, of cellulose may be dissolved in vitro by the intestinal juices of a horse. The products of this digestion are not monosaccharides, but carbon dioxide, methane and fatty acids of which the latter only, of course, are avail- able for nutrient purposes. The most important function of the celluloses in the diet, however, is that of communicating bulk to the intestinal contents, and promot- ing peristalsis by affording a favorable consistency and volume for propulsion with a minimum of muscular effort. This function of the 232 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS celluloses is most especially important in those animals, such as the herbivora, which have very long intestines. In man, however, too large a proportion of indigestible carbohydrate, as for example in the dietary of vegetarians, may lead to incomplete absorption of the digestible foodstuffs and promote, in this way, bacterial activities to an undesirable extent. The Digestion of the Fats. Very slight lipolytic action is exerted by the Gastric Juice. Not only is the lipase-content of gastric juice low, or the lipase weak in action, but the fats, while they remain in the stomach, being insoluble in water and in the form of relatively large masses, present only a limited surface of contact with the gastric juice, and the Lipase which it contains can hydrolyze the fat-masses only at their surface. However, the slight action which is exerted by the gastric lipase is probably of no little importance, for it ensures that upon the entry of the fats into the upper part of the small intestine they contain a small admixture of fatty acid, which greatly promotes their rapid emulsification by the alkaline fluids with which they here come into contact. The path of Absorption of the fats is quite different from that which is followed by the carbohydrate. Instead of passing into the blood- stream, after having traversed the epithelial lining of the intestine, they are deflected into the lymphatics and carried thence into the thoracic duct. After a meal rich in fats, the numerous small lymph- atic vessels coming away from the small intestine are full of milky fluid and stand out distinctly from the surrounding tissues, by reason of their whiteness and opacity, whereas under resting conditions, when digestion is not proceeding, they are transparent and difficult to distinguish. It is, therefore, possible to follow the absorption of fat from the small intestines by naked-eye examination, and it was in this way that Claude Bernard in 1846, discovered that the Pancreatic Juice is essen- tial for the digestion and absorption of fats, for no absorption is evidenced by the appearance of fat in the lacteals until the foodstuffs have reached the point at which the pancreatic duct opens into the duodenum. In man the ducts from the liver and the pancreas join to form a common channel of discharge, in the dog the two ducts enter the intestine very close together, but in the rabbit a considerable interval separates the openings of the two ducts, the Bile from the liver being discharged into the intestine at a point considerably above that at which the pancreatic duct opens. In the space between these two ducts no absorption of fat whatever is to be observed after a meal, but immediately below the pancreatic duct the lacteals are seen to be filled with emulsified fat. Not only the pancreatic juice, but also the bile is essential for the absorption of fat, however, for if by surgical procedures the bile-duct be made to open into the intestine below, instead of above the pan- creatic duct, the lacteals in the space between the ducts, notwithstand- DIGESTION OF THE CARBOHYDRATES 233 ing the admixture of pancreatic juice with the foodstuffs, are again seen to be clear and free from fat, while immediately below the new point of entry of the bile-duct into the intestine active absorption of fat is evidenced. Moreover, partial or total occlusion of the bile- ducts through catarrhal conditions or tumors is not uncommon, and this invariably leads to very defective absorption of fat, a large proportion of unabsorbed fat passing into the feces, even when the discharge of pancreatic juice into the intestine has not been interfered with at all. Two separate factors are therefore essential for the proper absorp- tion of fats, namely the bile and the pancreatic juice. The fat-split- ting enzyme, Lipase, is contained in the pancreatic juice and not in the bile. The essentiality of the bile in this process arises not from any power of digesting fats which it possesses itself, but from the facili- tation of the digestion of fats by pancreatic juice which it brings about. The fats differ from the other nutritive constituents of the diet in their insolubility in water. The enzyme, lipase, which accomplishes their digestion is, however, not only soluble in water, but secreted and poured into the intestine in a watery medium. To secure contact of these substances of diverse solubilities some special mechanism is required and this is supplied by the Emulsification of the fat, partly by the alkaline carbonates contained in the pancreatic juice and the bile, but especially by the Bile-salts, sodium glycocholate and tauro- cholate. By the action of alkalies and alkaline salts upon partially hydrolyzed fat containing a little fatty acid, Soaps are formed, by combination of part of the base in the alkaline salt with the fatty acid. Na 2 C0 3 + CnHssCOOH = NaHCOs + CnHfcCOONa Sodium carbonate Stearic acid Sodium bicarbonate Sodium stearate. The presence of a small amount of soap facilitates the formation of an emulsion of fat with water because the soap tends to collect in a film at the surfaces of the oil-droplets and impedes their coalescence into larger drops. The concentration of the soap at the surfaces of the droplets is brought about by the fact that they lower the Sur- face-tension of the oil-water interface, so that, a film having once been formed, if a discontinuity should appear in it, the surface of oil which is exposed will have a greater tension than the surface of soap which covers the remainder of the droplet. The effect of this is to cause the exposed surface, where the film is broken, to contract more forcibly than the remainder and thus pull the edges of the film together again. The emulsification of the fats in the foodstuffs is thus initiated by the alkaline carbonate in bile and pancreatic juice, forming soaps with the trace of fatty acid arising from lipolytic action of the gastric juice. The emulsifying-power of the soaps is, however, far inferior to that of the bile-salts, which reduce the tension of the oil-water 234 DIGESTION AMD ASSIMILATION OF THE FOODSTUFFS interface a great deal more than soaps do, and the alkaline salts of the pancreatic juice, unaided by the sodium glycocholate and taurocholate of the bile, are unable to bring about sufficiently rapid and complete emulsification to permit digestion and absorption to occur with the necessary rapidity to ensure total utilization of the fats in a meal. Furthermore the bile-salts, in some way which is not yet fully under- stood but which also probably arises from reduction of surface-tensions, facilitate the Absorption of the fatty acid and glycerol by the intestinal epithelium after the digestion of the fat has been completed. The emulsification of the fats enormously enhances the area of fat and, therefore, the number of fat-molecules which are exposed simul- taneously to the action of lipase. Thus one cubic centimeter of oil floating upon the top of water in a test-tube which is one centimeter in diameter will be in contact with the water. over an area of 0.785 square centimeters. The same volume of oil, broken up into ten thousand droplets and distributed through the water would expose a surface of no less than a hundred square centimeters to contact with substances dissolved in the water. Hence the droplets formed by emulsification are rapidly eroded and finally consumed and converted into fatty acids and glycerol by the Lipase in the pancreatic juice. The carbohydrate and the proteins are broken up by the digestive ferments and the intestinal epithelium into their simple constituents, and these are absorbed and carried to the liver as such, to be subse- quently distributed therefrom over the body. The fats, on the con- trary, reach the blood through the lymphatic circulation without preliminary elaboration or reassortment by the liver. Corresponding to this fact we find that they are thrown into the circulation, not in the form of their simple components, but in the comparatively elaborate form of Neutral Fat. The fatty acid and alcoholic radicals of the original fat are, in fact, quantitatively recombined within the brief period of their passage through the substance of the intestinal epithe- lium, and the work of digestion is completely undone again before the fat appears in the lacteals. It was inevitable that the appreciation of this fact by investigators should suggest the question whether and to what extent the preliminary hydrolysis of fats by lipase is essential. If the hydrolysis of fat is completely reversed within so brief a period and distance, is the hydrol- ysis itself a necessary prerequisite to absorption? Much investigation has been devoted to this problem, and as a result we are in possession of a variety of results arising out of different methods of attack. These results indicate that notwithstanding the fact that the hydrolytic splitting of the fats is so temporary it is never- theless essential. Thus fats which are not hydrolyzed by lipase, and cholesterol esters, waxes and hydrocarbon oils which simulate fats in their solubilities and other physical characteristics, but are not decom- posed by lipase, are not absorbed to any measurable extent. Even when vaseline or liquid petrolatum are administered in emulsions, DIGESTION OF THE CARBOHYDRATES 235 formed by mixing them with small quantities of an emulsifiable fat or oil, over ninety-five per cent, of the 'hydrocarbon is recoverable, unaltered, in the feces. The same is true of Lanoline which consists of a mixture of cholesterol esters of fatty acids and is not saponifiable by lipase. These esters are not absorbed; they pass into the feces unaltered, although free cholesterol itself can be shown to undergo absorption. Fatty acids which are normally foreign to animal tissues are absorbed, but only when the fat is previously split into its constituents by lipase. This has been most conclusively shown by the following very beautiful experiment of Bloor's. Bloor prepared the fatty-acid compounds of the polyatomic alcohols derivable from sugars by reduction. Among these Dilaurate of Isomannitol has a high dextrorotation, while the normal body-fats are optically inactive. On administering this substance to dogs in their food and collecting the chyle from the thoracic duct a large proportion of fat was obtained which yielded Laurie Acid on hydrolysis. Laurie acid isnot found normally in animal fats, and it must therefore have been derived from the isomannitol dilaurate which had been administered. But the fat obtained from the thoracic duct was also Optically Inactive; therefore, it cannot have consisted of isomannitol dilaurate. The accuracy of the method was sufficient to have detected the absorption of 0.5 per cent, of the isomannitol dilaurate which had been administered. Not more than this proportion therefore can have been absorbed without previous hydrolysis by the pancreatic lipase. The carbohydrates, and, as we shall see, the proteins are absorbed in the form of the simplest components into which they can be con- verted by the hydrolyzing enzymes, and are distributed to the tissues after having passed through the liver. The fats, on the contrary, are thrown directly into the circulation in a comparatively complex form. Corresponding to this difference in the method of distribution we find that the composition of the tissue-fats is very much more dependent ' upon the varying nature of the diet than the tissue-carbohydrates or the tissue-proteins. Thus Erucic Acid, C 2 iH 4 iCOOH, is never normally present in the tissue-fats of dogs. Yet if rape-seed oil, which contains notable quantities of erucic acid glyceride, be given to starving dogs, this fatty acid may subsequently be isolated from their tissues. The normal melting-point of dog-fat is 20 C. Munk allowed a dog to fast for nineteen days, until the tissues were presumably free of reserve- stores of fat. The dog then weighed sixteen kilograms. It was now fed for fourteen days with mutton tallow. The weight of the animal increased during this period by seventeen per cent. On "trying out" the tissues 1100 grams of fat were obtained and its melting-point was 40 C. The administration of the fat of high melting-point had, under these conditions, led to an abnormally high melting-point of the fats laid up in the tissues. The results which we have quoted were obtained with starving animals. Under normal conditions, however, when the tissues are not 236 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS depleted of fat-reserves, they are able to exert some measure of control over the nature of the fats which they assimilate. Munk investigated a patient who was afflicted with a fistula from which it was possible to collect the chyle before it entered the blood-stream. When this patient was fed upon a diet containing no other fat than mutton -fat, the chyle-fat had nevertheless a lower melting-point than mutton-fat. When the patient was fed with no fat at all but Cetyl Palmitate, which melts at 55, the chyle-fat was found to melt at 36 and to consist of a mixture of Glyceryl Tripalmitate and glycerides of Oleic Acid to the extent of fourteen per cent, of the mixture, adjudged by the absorption of iodine by the mixed fats. Hence under normal conditions a measure of control is exerted by the intestinal wall itself and a proportion of glycerol and oleic acid may be furnished to supplement the deficiencies of the dietary. This of course becomes impossible if the glycerides of oleic acid which are present in normal tissues have been previously depleted by starvation. The fats may also be modified in the opposite direction and the proportion of oleic acid glycerides reduced during their transmission through the intestinal epithelium. Thus when Cod-liver Oil, which contains a great excess of unsaturated fatty acids, is administered to dogs, the iodine number of the fats after absorption is less than that of the fat in the food. The absorption of fat leads to a temporary increase in the fat content of the blood, where it is held in a finely emulsified condition. The ingestion of fat-rich foods, as for example, cream, may result in an increase of the fat-content of the blood to no less than six times the normal concentration during the intervals between absorption. Under such circumstances the blood-serum obtained by centrifugalizing defibrinated blood is often cloudy with suspended fat and globules of fat may not infrequently be found floating upon the top of the column of fluid in the centrifuge-tube. Ultimately the excess of fat disappears from the blood, the neutral fats having been built up into the fatty connective tissues. The greater part of the fat-reserve is contained in special fat-cells, in which the fat appears, at first in small globules, and later in larger globules which coalesce until the accumulation of fat forces the protoplasm into the periphery of the cell, so that it presents an annular appearance on cross-section, with the flattened nucleus forming a slight thickening of the ring at one side. Occasion- ally such cells disintegrate bodily, it being in this way that the solid constituents of Milk are formed in the mammary glands. Upon allowing fat-rich blood to stand in laboratory-glassware at body-temperatures a proportion of the fat becomes diffusible. The nature of the change which occurs is not yet understood, nor is it certain whether or not this, or a similar change in the properties of circulating fat precedes its absorption by the tissues. Under certain pathological conditions, and particularly in Diabetes, the percentage of fat in the DIGESTION OF THE CARBOHYDRATES 237 blood is greatly increased above the normal, a condition which is known as Lipemia. It is stated that in these cases the power of the blood to render fats diffusible is diminished, the excess of fat being present wholly in the emulsified, or non-diffusible condition. The Lecithins are very readily and rapidly split by lipase into fatty acids and Glycerophosphoric Acid. This latter compound, however, is not split by the digestive juices. It is not known whether hydrolysis necessarily precedes the absorption of the lecithins. The rapidity with which they are split by lipase indicates that at any rate a large proportion of the lecithins must inevitably be hydrolyzed before absorption can be completed. On the other hand the extreme solu- bility of lecithins in solutions of bile-salts encourages the view that a part at least of these substances may be absorbed without prelimi- nary digestion, and this view is further supported by the remarkable effects of egg-lecithin upon the growth and development of animals and upon the nitrogenous balance, when it is administered by mouth, effects which, as yet, have not proved possible to evoke by the adminis- tration of the constituent parts into which it disintegrates upon hydrolysis. It is, however, possible that these effects of administering lecithin may be attributable, not to lecithin itself, but to impurities which are commonly associated with crude preparations of lecithin. Cholesterol has been definitely shown to be absorbed as such. If an abnormal quantity of cholesterol be administered by mouth to animals, the excess is laid up in certain tissues, particularly those of the liver, spleen and suprarenal gland, and serious lesions may result from the accumulation of these deposits. Certain organs, e. g., the kidneys, remain free from cholesterol deposits even when cholesterol is administered in very great excess, but if lesions arise from some other cause, for example if Nephritis is induced by the administration of uranium salts, then cholesterol tends to become deposited in the injured tissues. In rabbits, but, so far as has yet been ascertained, not in other species of animals, the administration of excess of cholesterol is followed by the formation of large deposits in the intima of the arterial walls, particularly in the wall of the aorta, leading to the formation of atheromatous lesions resembling those which are observed in cases of Arteriosclerosis. The Cholesterol Esters, however, are not saponifiable by lipase and are not absorbed. Hence Lanoline, administered by mouth, is recover- able quantitatively in the feces. The Bile-salts, which serve as a vehicle and adjunct in the absorption of the fats, undergo a partial circulation in the body, for after entering the small intestine through the channel of the bile, they are partially reabsorbed during their passage with the foodstuffs down the intestine. Thus Glycocholic Acid is nearly absent from the bile of carnivora, but on administering this bile-acid to carnivorous animals, it appears in important quantities in their bile. 238 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS THE DIGESTION OF THE PROTEINS. Until a very few years ago it was generally held that the proteins were absorbed from the stomach and intestine in the form of Peptones, and, indeed, prior to the first years of the present century, it was be- lieved in many quarters that not merely peptones, but even unaltered protein or at least infraproteins resulting from the very earliest cleav- age of the native protein molecule might be absorbed without further hydrolysis. This view of protein-absorption stood in rather striking contrast to our knowledge of the absorption of other foodstuffs which were at a comparatively early date known to undergo complete or nearly com- plete hydrolysis prior to their absorption. Moreover, the elaborate machinery of enzymes for the splitting of proteins not only to peptones, but to amino-acids which is provided by the digestive organs would appear, if the older view were correct, to exist without any necessary purpose or function. Considering these facts it may appear strange that so exceptional a view of protein-absorption should ever have gained general acceptance; but, as usual in the historical development of science, a misinterpreted experiment furnished the foundation for an extensive edifice of erroneous hypothesis. The observation which led us astray was the outcome of an experi- ment by Voit, who, in 1869, showed that undigested proteins, unmixed with gastric or pancreatic juice, rapidly disappear when they are introduced into a ligated loop of small intestine, while a little later it was further found by Hofmeister that proteoses and peptones similarly disappear when introduced into an isolated loop of intestine. The latter of these observations received its correct interpretation when Cohnheim, in 1901, showed that the Succus Entericus which is secreted by the mucous membrane of the small intestine, contains an enzyme, Erepsin, which hydrolyzes proteoses and peptones to amino-acids, leaving, however, native proteins with the exceptions of casein and the protamines, unattacked. The disappearance of peptone from an intestinal loop is therefore accounted for by its hydrolysis by erepsin into amino-acids. The disappearance of native proteins such as egg- albumin from isolated loops of intestine is, however, a more difficult matter to interpret, and it cannot yet be said to have been completely elucidated. It is, however, certain that under normal conditions unaltered proteins never reach the circulation by absorption from the intestine for the following reasons: In the first place, when native proteins are injected into the circu- lation, a proportion of the protein thus introduced appears in the urine. Evidently it is treated as a foreign constituent of the blood and dis- charged, in so far as that is possible, by the kidneys. Another portion of the protein is discharged by the kidneys in a non-coagulable form which is still, nevertheless, a protein. At the same time there is a marked increase of proteose-like substances in the blood and some increase of the urea-output. DIGESTION OF THE PROTEINS 239 To some extent, but a very limited extent therefore, parenterally introduced protein, that is, protein injected directly into the circu- lation, may be utilized by the tissues, since a proportion of the protein is evidently converted into a normal product of protein catabolism, namely, urea. But it is also evident that the utilization of protein thus introduced is imperfect, that it is abnormal because the urine contains protein which is not the case when proteins are absorbed from the digestive tract, and that the utilization of the protein which does occur is preceded by hydrolytic cleavage. Moreover it is not even certain that the additional urea-output which results from the injection of foreign proteins is due to utilization of the protein itself, since it has been found by Mendel and Rockwood that the intro- duction of a foreign protein, such as Edestin or Casein into the cir- culation leads to a considerably more than proportionate increase of the nitrogenous secretion, in other words to actual destruction of tissue-proteins. In the second place, the intravenous or subcutaneous injection of proteins which are foreign to the tissues of the animal receiving them, results in the production of a variety of specific Antibodies or substances appearing in the circulation which have the property of precipitating or otherwise modifying the protein employed for injection. If the injections are repeated, and successive injections are separated by only a few days from one another, the result after some weeks is the production of a specific Precipitin which circulates in the blood of the immunized animal, so that if the blood-serum of the animal be now mixed with a solution of the protein which was employed for injection, that protein, but no other, is precipitated. If a single injection be made and then a second only after a considerable interval, e. g., three or four weeks, the effect of the second injection is to induce Anaphy- lactic Shock, a condition which so strikingly resembles peptone-poison- ing that many investigators are of the opinion that it is due to the development in the tissues of the sensitized animal of an enzyme having the specific ability to rapidly break down the particular protein employed and to convert it into proteoses or peptones. Now it has been shown that even after the introduction of excessive amounts of native protein into an isolated loop of intestine, although the protein disappears and would seem to have been absorbed, yet no evidence is obtainable of the development of antibodies in the circula- tion of the animal so treated, nor is there any sensitisation, so that a second dose of the protein, after a considerable interval, does not give rise to symptoms of anaphylactic shock. There can be little doubt therefore that proteins are not absorbed without previous hydrolysis, and there is much ground for supposing that even that proportion of parenterally introduced protein which is utilized by the tissues, is utilized simply because it has been excreted into the intestine and reabsorbed therefrom after digestion. The case against the direct absorption 1 of peptones from the intestine 240 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS is an even stronger one, for a considerable proportion of the proteoses and peptones arising from the incomplete hydrolysis of proteins are definitely toxic, and the absorption of this type of protein digestion- product would lead to incessant food-intoxication. It is true that peptones may be absorbed from a ligated stomach, and peptones and proteoses resulting from secondary degenerative changes in the intes- tinal epithelium may be absorbed after prolonged ligation of an intes- tinal loop, and may produce severe symptoms of peptone intoxication. These, however, are circumstances not in the least comparable with those attending normal digestion, for a ligated and distended stomach becomes permeable, as we have seen, to carbohydrates which are normally not absorbed until they reach the intestine, so that the permeability of the gastric mucosa is evidently deranged by this proc- ess. The absorption of toxic proteoses from an intestinal loop is attained only after very prolonged ligation, and while this condition may be comparable with that prevailing in severe intestinal stasis, it is certainly not comparable with the normal phenomena of digestion and absorption. In certain cases, which must be admitted to be exceptional, individ- uals may display phenomena of Anaphylaxis after the ingestion of particular proteins toward which the individual has an idiosyncrasy. Thus, hyper sensitiveness to the proteins of horse-serum is not unusual and is responsible for the majority of cases of Serum-sickness and deaths arising from the use of Diphtheria Antitoxin prepared from horse-serum. It is especially frequently displayed by chronic asthmatics. A smaller proportion of individuals are hypersensitive to the proteins in the white of egg and betray symptoms of anaphylactic shock, such as Asthma, when they have partaken of eggs. Others, again, are hyper- sensitive to the protein in strawberries, others to the proteins in shell- fish and so forth. In all of these cases we must assume, from the char- acter of the symptoms, that the absorption of a proportion of unaltered, native protein is responsible for the disorder. The proportion of pro- tein absorbed which would suffice to account for these effects, however, is excessively small. Thus Wells has shown that the injection of such a minute amount as one millionth of a gram of crystallized egg-albumin will sensitize a guinea-pig, so that a subsequent intravenous injection of no more than one-tenth of a milligram of the same substance will lead to fatal shock. The comparative rarity of these phenomena of protein-intoxication, and the minute proportion of absorption of unaltered protein which would evidently suffice to evoke them, afford eloquent testimony to the difficulty with which native proteins and peptones are absorbed without preliminary digestion. We are thus brought indirectly and by the exclusion of other possi- bilities, to the conclusion that the proteins of the diet must be com- pletely broken down into Amino-acids prior to their absorption. Direct evidence of the correctness of this view has, however, been obtained in recent years in a variety of ways, of which the following are the more important. DIGESTION OF THE PROTEINS 241 In the first place it has been ascertained, thanks to the researches of London, that during the normal digestion of proteins in the intestine large proportions of amino-acids are actually formed, and progressively absorbed subsequently to their formation. The experiment consisted in establishing a number of fistulae at intervals along the intestinal tract, so that samples of the intestinal contents could be withdrawn and examined after varying periods of intestinal digestion. The animal was fed with measured amounts of pure proteins and the diges- tion-products obtained from the successive sectors of the intestine (Fig. 7). It was found that these samples contained notable amounts of amino-acids and, moreover, that the relative proportions of the amino-acids arising from the dietary protein differed in different sec- FIG. 7. Dog with intestinal and glandular fistulse. (After London.) tions of the intestine. Thus when the animal received the protein Gliadin. the duodenum contained 0.75 grams of tyrosine to 2.5 grams of glutamic acid; the jejunum contained only 1.1 grams- of tyrosine per 20.9 grams of glutamic acid and the ileum contained only a trace of tyrosine as contrasted with 33 grams of glutamic acid. Evidently therefore, not only are amino-acids formed in the normal intestinal digestion of proteins, but they are absorbed selectively, e. g., in the particular case in point, tyrosine was absorbed from the intestine much more rapidly than glutamic acid. In the second place it has been shown by Folin and Denis, that if amino-acids are introduced into a loop of intestine the non-protein nitrogen of the blood is decidedly increased during the period that absorption might be supposed to be occurring, and the origin of this 16 242 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS increase was subsequently demonstrated by Abderhalden, who, by employing an enormous volume, fifty liters of blood, succeeded in demonstrating the presence therein of the amino-acids glycine, alanine, leucine, valine, proline, aspartic acid, glutamic acid, arginine, histidine and lysine. It has furthermore been shown by Abderhalden, Henriques and others that animals may be maintained in perfect nitrogenous equilib- rium for prolonged periods on a diet containing no other source of nitrogen than amino-acids. It is necessary, however, to include in this diet all of the amino acids-which contribute to the composition of the various tissue-proteins. The omission of Tryptophane, for example, leads to daily loss of weight and, in effect, to nitrogen-starvation. Nitrogen equilibrium and even nitrogen retention, i. e., accretion of tissue, was secured by Abderhalden in a dog to which a diet was ad- ministered containing the following admixture of amino-acids as the sole source of nitrogen: Glycocoll 5 grams, d-alanine 10 grams, 1-serine 3 grams, 1-cystine 2 grams, d-valine 5 grams, 1-leucine 10 grams, d-iso- leucine 5 grams, 1-aspartic acid 5 grams, d-glutamic acid 15 grams, 1-phenylalanine 5 grams, 1-tryosine 5 grams, 1-lysine 5 grams, d-arginine 5 grams, 1-proline 10 grams, 1-histidine 5 grams, 1-tryptophane 5 grams. The daily ration of amino acids therefore weighed 100 grams and con- tained 13.87 grams of nitrogen. It approximately resembled in com- position the mixture of products which results from the hydrolysis of the proteins of muscular tissue. We have seen therefore: (1) That amino-acids are formed in impor- tant proportion in the intestinal digestion of proteins. (2) That amino- acids may be absorbed from the intestine and, (3) that amino-acids suffice to supply the nitrogenous needs of the body. We may infer that the absorption of amino-acids is a normal and probably the only normal method whereby the materials for -the synthesis of proteins are conveyed to the tissues. The difficulty of demonstrating the presence of amino-acids i,n the blood after the absorption of the digestion-products of protein arises from two sources: firstly the slowness of absorption and the rapidity of circulation, which results in extreme dilution of the amino-acid products which enter the portal venous system, and secondly the rapidity with which the amino-acids in the blood are absorbed from it by the tissues. The amino-acids are therefore present in the blood even during the height of absorption only in very small concentrations and, to add to the difficulty of the problem, the blood is a fluid which is very rich in nitrogenous substances, proteins, which interfere to a serious extent with the chemical manipulations which were formerly necessary for the determination of small concentrations of amino-acids. In recent years the development of our technical knowledge has simplified and enhanced the accuracy of our methods of estimation and, in particular, the development of the nitrous-acid method of estimating amino-nitrogen immediately enabled us to detect with DIGESTION OF THE PROTEINS 243 _ ease and certainty the accumulation of amino-acids in the blood which accompanies absorption after a meal rich in protein. The absorption of amino-acids from the intestine and their conse- quent presence in the blood has also been very beautifully demon- strated by Abel, employing his method of Vividiffusion. This method consists in deflecting a fraction of the blood-stream and causing it to pass through a series of collodion-tubes before returning to the general circulation. The collodion-tubes are immersed in a salt solution of the same concentration as the inorganic salts in the blood, so that dif- fusible substances other than inorganic salts dialyze out of the blood into the saline solution. By renewal of the saline solution considerable quantities of the diffusible substances in blood may be collected and, among others, various amino-acids (Fig. 8). FIG. 8. Abel's vividiffusion apparatus. (After Macleod.) It is thus evident that the protein constituents of the dietary are absorbed into the circulation in the form of their amino-acid compon- ents, some selection of the amino-acids transmitted to the blood being exercised by the intestinal epithelium. The question now arises, where, and in what way, these amino-acid fragments are resynthesized into protein. Amino-nitrogen determinations show that the excess of amino-acids which accumulates in the blood after a meal very rapidly disappears, while coincidently a considerable increase in the free amino-acids is found to have taken place in the tissues. The amino-acids are there- 244 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS fore stored up in the tissues. There is a limit, however, to the capacity of the tissues to retain amino-acids and this upper limit of capacity varies with different tissues. Thus the upper limit in the case of muscular tissues is about 75 milligrams of amino-nitrogen per hundred grams of tissue. This characteristic limit cannot be overstepped and if the quantity of amino-nitrogen brought to the tissues exceeds their Assimilation-limit the excess of amino-acids in the blood is destroyed by Deaminization, the nitrogen being split off in the form of ammonia which is converted by the liver into Urea. The Liver plays a leading part in this process and continually and rapidly desaturates itself by deaminization of the free amino-acids which it contains; doubtless other tissues share this ability, but the power of the liver to deaminize amino-acids is certainly in excess of that of other tissues, because, although other tissues do not show any greater avidity than the liver for amino-acids, and do not reach a higher saturation-limit of amino- nitrogen, yet within a few hours after the saturation of all the tissues with amino-acids which succeeds a protein-rich meal, or injection of amino-acid mixture, the other organs all contain more amino-acid material than the liver. When we consider, also, that the liver is an exceedingly bulky organ, its possession of a high deaminizing power ensures its overwhelming predominance in this process. When, for any reason such as that afforded by degenerative changes, the deaminizing power of the liver is impaired, this mechanism for dis- posing of undue excess of amino-acids may prove insufficient and the kidneys may assist by excreting unaltered amino-acids. If, at the same time, the introduction of amino-acids into the blood is unnaturally rapid, as for instance by rapid injection of large amounts of amino-acids in solution, the mechanisms for their disposal may prove to be entirely inadequate and serious symptoms of intoxication, or even death may ensue. The absorption of amino-acids from the blood is never complete, and free amino-acids are still present in the blood even when the amino-acid concentration in the tissues is far below the saturation- limit. Evidently, therefore, we are dealing here with an equilibrium, somewhat resembling the partition of a dissolved substance between two immiscible solvents, increase in the amino-acid content of the blood leading to increase, up to the saturation-limit, of the amino-acid con- tent of the tissues, while the loss of tissue-protein which occurs in Starvation indicates that diminution of the amino-acid content of the blood may also lead to desaturation of the tissues by the passage of amino-acids into the blood. The same mechanism, also, permits trans- fer of particular amino-acids from one tissue to another and explains the otherwise surprising fact that certain tissues, for example malignant growths, may grow at the expense of other tissues, and also, in part, the fact that the loss of weight of the various organs and tissues in starvation is very unequal, certain tissues losing very heavily while others retain their weight very nearly undiminished until death ter- DIGESTION OF THE PROTEINS 245 minates the process. It will be recollected that a similar equilibrium between the tissues and the blood obtains in the case of glucose and its anhydride, glycogen, a subnormal glucose concentration in the blood leading to the splitting up of glycogen and liberation of glucose by the liver to replenish the blood and thereby the muscular tissues to which it carries the carbohydrate fuel which furnishes the energy-equivalent of muscular work. In the same way we may suppose that the amino- acids in the tissues stand in a relation of equilibrium to the amino-acids in the blood, on the one hand, and to the proteins of the tissue on the other. The proteins which are found in the various tissues of the body are highly specialized and characteristic of the tissue-elements in which they occur. The proteins in the various Connective Tissues are especi- ally diverse in their composition and characteristics. Thus the pro- teins of fibrous tissue are extraordinarily rich in glycocoll, and those of elastic tissue are especially rich in glycocoll and also in glutamic acid. Among other highly specialized proteins may be mentioned the keratin of horny epidermal tissues which is exceptionally rich in cystine, the protamines which are exceptionally rich in diamino-acids, and the mucins which contain an amino-carbohydrate radical. In a less degree the proteins of every type of tissue and cell betray, either in biological or physical behavior or directly, in chemical composition, evidence of distinctive architecture. The question of the locality of Protein Synthesis has evoked a very great deal of discussion and prompted a variety of investigations. Arguing that only the normal blood-proteins, the serum-albumins and serum-globulins could be tolerated in the circulation, and assuming "that the amino-acids were not, as we now know that they are, absorbed as such, Abderhalden supposed that the amino-acids which result from digestion are synthesized into protein in the intestinal epithelium, just as the fatty acids and glycerol are synthesized into fats during their passage through the intestinal wall, but with this difference, namely, that whereas the fats which are thus synthesized bear a very close relationship to the fats which were present in the diet, the protein which was presumed to be synthesized must be limited to the blood- proteins characteristic of the species. This hypothesis, however, made it necessary to view the process of protein synthesis as a very roundabout and uneconomical one, for since the proteins of the tissues differ so markedly from one another and also from the blood-proteins, the blood-proteins evidently could not be built up directly into tissue-proteins, but must first be broken down in the tissues themselves, their amino-acids resorted and rear- ranged, and resynthesized into the characteristic proteins of the tissue in question. Thus the synthetic work of the intestine would have to be undone again in each of the tissues. Moreover in many of the tissues the process of redegradation and resynthesis would involve an extraordinary amount of waste of ami no-acid material. For example, 246 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS the proteins of connective tissues could not be synthesized at all from serum-albumin, because it contains no glycocoll, and even their syn- thesis, from serum-globulin would involve a great deal of wastage, because, whereas serum-globulin contains only 3.5 per cent, of glyco- coll, the connective-tissue proteins contain about twenty per cent., so that not less than six molecules of serum-globulin would have to be destroyed to build up one molecule of connective-tissue protein, and the greater part of the remaining amino-acids in these six molecules would not be needed. What, then, is to become of them? They might be locally deaminized, but the predominant deaminizing tissue is that of the liver. In order to reach the liver the rejected amino-acids would generally have to travel thereto in the circulation. If, on the contrary, the amino-acids rejected by the connective tissues are utilized by another tissue, then in order to travel from the one tissue to another they must again enter into the circulation. Whichever hypothesis we adopt we are therefore compelled to revert to the presence of amino- acids in the blood. The discovery that the amino-acid products of digestion are actually absorbed into the blood-stream as such, and are absorbed from the blood by the tissues, removes these complexities from our interpre- tation of the process of protein synthesis. It seems most reasonable to suppose that each tissue synthesizes its own individual proteins, and that it is able to utilize for this purpose all of the amino-acids which it absorbs for the reason that the characteristic composition of each individual tissue-protein is already determined by the character- istic admixture and proportion of the various amino-acids which that tissue absorbs and holds in equilibrium with the blood, on the one hand, and with the tissu -protein itself on the other. The individual characteristics of the proteins of the various tissues are therefore determined, in ultimate analysis, by the relative Permeability of the tissue in question for various amino-acids, i. e., by the relative ease with which the amino-acids traverse the boundaries which demarcate the tissue. It is not unlikely that this mechanism of two-sided equilibrium is limited in its powers and that it is for this reason that it is safeguarded or assisted by a degree of preliminary selection by the Intestinal Epi- thelium. It will be recollected that the experiments of London show that if a protein differing very widely from animal tissue-protein be administered, certain amino-acids are absorbed selectively. Thus from Gliadin, tyrosine is absorbed much more rapidly than the glut- amic acid which it contains in notable excess. The composition and general nutritional standard of the tissues is therefore determined by the following interrelated factors which are severally in equilibrium: (1) The selective absorptive activities of the intestinal epithelium. (2) The general average concentration of food-products in the blood, i. e., the abundance of the dietary. (3) The deaminizing activity of DIGESTION OF THE PROTEINS 247 the various tissues and particularly of the liver. (4) The "saturation- limit" of the tissues for amino-acids. (5) The relative velocities of the opposed processes of protein synthesis and degradation in the several tissues of the body. Of these factors, two main groups may be recog- nized. Absorption and deaminization on the one hand, determining the abundance of nutrient material in the circulating medium, and the excess or defect of the velocity of synthesis in comparison with that of degradation in the tissues, on the other, determining the rapidity with which the available nutrients are utilized. The former factors are largely subject to environmental influence, for example that of the abundance of the dietary. The latter factors are individually char- acteristic of the organism, and in turn of the several tissues of which the organism is composed. Two main groups of factors, therefore, contribute to determine the nutrition, composition and growth of organisms, an Environmental Group and an Internal Regulatory Group. We shall see when we come to the consideration of the problem of Growth (Chapter XX), that the diverse significance of these two groups of factors may very clearly be recognized in the processes of development. We have seen that the intestinal digestion of proteins leads to the production of amino-acids, and that these are absorbed into the blood- stream as such. A considerable degree of preliminary digestion of proteins is, however, achieved by the Pepsin in the gastric juice, and the question therefore arises as to whether any digestion-products of proteins are absorbed from the stomach? This question may be answered in the negative. We have already seen that under normal conditions neither carbohydrates nor fats are absorbed from the stomach and, analogously, protein digestion- products are not absorbed from the stomach. It is true that carbo- hydrates may be absorbed from a li gated stomach and so, also, may proteoses and peptones, but this constitutes a condition which is nowise analogous to the conditions which pertain in normal digestion. The non-absorption of protein digestion-products from the stomach is in the first place guaranteed by the fact that the products of protein hydrolysis by pepsin are proteoses and peptones, not amino-acids. It would not be altogether safe, of course, to argue from the inability of pepsin to digest peptones in vitro to a similar inability upon the part of the stomach in situ, but the studies of London have shown that the production of proteoses and peptones is, in actuality, the main result of gastric digestion. This investigator has established in animals a fistula opening into the intestine immediately below the pyloric sphinc- ter of the stomach. From this fistula it is possible to collect samples of the stomach contents the moment after the completion of gastric digestion, and their ejection into the intestine. The samples not only failed to contain any amino-acids, but the larger proportion of the nitrogen was present in the form of Proteoses, and only a lesser proper- 24S DIGESTION AND ASSIMILATION OF THE FOODSTUFFS tion in the form of the further cleavage-products, the Peptones. The following are typical results obtained: Percentage of proteoses Protein in on completion of the diet. gastric digestion. Egg-albumin 72.5 Gliadin 67.7 Edestin 60.3 Casein 59.1 Gelatin 50.6 Serum-albumin 46 . 1 the remainder of the protein having reached the peptone-stage of cleavage. With varying quantities . of the same protein a definite proportion of proteoses is always formed, as the following results illustrate: Quantity of fdiadin Percentage of proteoses in a meal. on completion of Grams. gastric digestion. 25 80.8 50 86.1 75 . . 86.5 100 84.9 The significance of gastric digestion lies in the preparatory work which it accomplishes for the intestinal and pancreatic enzymes. The hydrolysis of proteins by Trypsin is much more rapid and complete if the protein has been subjected to preliminary digestion by pepsin, and the hydrolysis of proteins to the peptone and proteose stage, furthermore, converts the protein foodstuffs into forms open to attack by the Erepsin in the succus entericus. The superior velocity and thoroughness of intestinal protein digestion to the digestion of pro- tein in vitro by pancreatic trypsin is attributable in large measure to the fact that the various proteolytic enzymes act in conjunction or succession upon the protein foodstuffs in the alimentary canal, and also to the fact that the products of digestion are removed almost as rapidly as they are formed. In addition to the conversion of proteins into proteoses and peptones, the gastric juice has the special property of converting the casein of milk into Paracasein. 1 Paracasein has recently been shown to be derived from casein by partial hydrolytic cleavage, the paracasein molecule representing one-half the casein molecule. Paracasein resembles casein very closely in its general properties and behavior, but its calcium salt is rendered insoluble by a very slight excess of calcium ions at a much lower temperature than the corresponding salt of casein itself. If a sufficiency of calcium chloride, for example, be added to a solution of calcium caseinate, the protein salt will be 1 In British scientific literature these substances are termed, respectively, Caseinogen and Casein. The word casein, therefore, means the unmodified protein of milk, in American literature, and the infraprotein derived therefrom by the action of Rennin, in British literature. The American nomenclature is to be preferred because it possesses the claim of priority, and is that generally employed in other languages. DIGESTION OF THE PROTEINS 249 precipitated at ordinary temperatures. If a little less calcium chloride be employed, it will remain in solution at ordinary temperatures, but will form a curd on elevating the temperature. Even in the absence of free calcium ions a solution of calcium caseinate becomes markedly opalescent on heating to 45 C. The calcium salt of paracasein is, however, for a like concentration of free calcium ions, clotted or curdled at a lower temperature than calcium caseinate. The presence of free calcium ions is therefore necessary to permit the clotting of milk by gastric juice or extracts of the gastric mucosa. They are not necessary, however, for the conversion of the casein into paracasein, which occurs just as readily in a medium free from calcium ions as in one which contains them; but visible evidence of the change which has occurred is lacking until calcium salts are added. Thus if excess of ammonium oxalate be added to milk, the free calcium ions are removed, through the formation of calcium oxalate. The calcium combined with the casein is unaffected because it is not ionized. On adding Rennet (extract of gastric mucosa) or gastric juice in small amount and warming the mixture to body temperature no visible change in the milk occurs. The mixture may be heated to boiling to destroy the enzyme without causing any precipitation or clotting of the milk, but on adding soluble calcium salts, after cooling, clotting of the milk instantly occurs. The calcium is necessary, therefore, merely to render the product of the enzyme action insoluble; not to enable the enzyme to act upon the casein. The part played by calcium in this process is therefore sharply in contrast to the part which it plays in the coagu- lation of the blood. It has long been supposed that this change in the properties of casein which is brought about by gastric juice is due to a special enzyme, which is termed Rennin or Chymosin. Evidence has accumulated in recent years, however, tending to show that rennin is, in fact, identical with Pepsin and that rennet preparations which are devoid of power to digest proteins other than casein represent merely pepsin, weakened so greatly as to have lost ability to hydrolyze the majority of proteins at any appreciable speed. Thus, although pepsin and rennin are found in a great variety of situations both in the animal and in the vegetable kingdoms, yet they are invariably found to be associated with cne another in the same tissue or tissue-fluid. The close relation- ship of rennin action to pepsin action is also shown by the following experiment of Morgenroth's. If mixtures of calcium caseinate con- taining free calcium ions and rennet are kept at low temperatures no coagulation occurs, but slow digestion of the casein (proteose pro- duction) does occur. If, however, these mixtures be heated to 20 C. they clot immediately. Thus the process which underlies the clotting, has taken place during the digestion which occurs at low temperatures, but it cannot be visibly evidenced by clotting until the temperature is raised. 250 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS THE TIME- AND MASS-RELATIONS OF DIGESTION AND ABSORPTION. The Intestine is an extraordinarily efficient organ of absorption. As much as seventy or eighty per cent, of the total length of the jejunum and ileum may be removed and, provided fats be not too abundant in the diet, absorption still remains practically complete. If the food contains a large proportion of fat, however, over twenty- five per cent, of the fat may, under these conditions, be discharged unabsorbed in the feces as contrasted with four or five per cent, in normal animals of similar kind and dimensions. The greater part of absorption takes place in the upper part of the small intestine and absorption is practically complete before the contents of the small intestine are discharged into the cecum. There are, however, certain exceptions to this rule, mainly furnished by dif- ficultly digestible foodstuffs. Thus uncooked white of egg is digested with great difficulty and as much as seventy per cent, of this protein may pass undigested and unabsorbed into the large intestine where it may be presumed to afford a favorable culture medium for putrefactive bacteria. Then, also, when proteins very diverse in composition from the normal tissue-proteins of animals, such as certain vegetable pro- teins, are partaken of, the selective absorption which occurs may result in a proportion of an amino-acid which is present in undue excess remaining unabsorbed until its passage into the large intestine. The Stomach, as might be imagined from the nature of the part it plays in digestion, is not essential to the absorption of foodstuffs. Excision of the stomach is followed by a good utilization even of proteins, the digestion being accomplished by the trypsin of the pan- creatic juice and the erepsin of the succus entericus. Provided the stomach be left in situ, moreover, efficient digestion and absorption of proteins may still continue when the pancreatic duct is ligated, so that pancreatic juice cannot enter the intestine. In this case digestion is effected by erepsin after preliminary cleavage by the pepsin in the gastric juice. The absorption of Fats, however, is very seriously interfered with by the exclusion of pancreatic juice from the intestine. An important relationship subsists between the amount of food which is partaken of at a meal and the quantity of digestive juices secreted on the one hand, and the time occupied in digestion on the other hand. In the case of the Gastric Juice the quantity of the digestive fluid secreted may be estimated by forming a diverticulum or pocket in the stomach which is connected with the exterior by means of a fistula. The juice secreted by this diverticulum is found to be a constant proportion of the fluid which is secreted by the whole area of the gastric mucosa and the total secretion of gastric juice during digestion may therefore be estimated in terms of the volume of the secretion furnished by the diverticulum. It has been observed that the quantity of gastric juice which is secreted during the digestion RELATIONS OF DIGESTION AND ABSORPTION 251 of a meal is very closely proportionate to the quantity of food of any one kind which is ingested. The following are results obtained by Klugine, the "calculated" figures being estimated on the assumption of strict proportionality between the volume of secretion and the mass of the given type of food which was ingested. Quantity Quantity of gastric juice: of food. Observed. Calculated. Type of Food. grams. c.c. c.c. Raw meac ......*.. 400 106 99 Raw meat . . . .- .'_.-. . 200 41 50 Raw meat . '.....". ". 100 27 25 Boiled meat . . . . . . . -. 200 42 42 Boiled meat ........ 100 21 21 Milk I...!. 600 56 53 Milk 500 41 44 Milk 200 17 18 Soup of oats and meat 600 43 41 Soup of oats and meat . . ..... 300 20 21 Meat, bread and milk . . . . . -. 800 83 90 Meat, bread and milk . . . . . 400 41 45 It is evident that the calculated figures agree as closely as could be desired with those actually observed. Evidently, then, there is not a constant amount of gastric juice secreted for each meal, but the amount furnished is proportionate, for any one kind of food, to the mass ingested. As Arrhenius has pointed out, this would appear at first sight to be an uneconomical arrangement, since a very small quantity of a digestive enzyme is capable, in time, of digesting a very great excess of foodstuff. The length of time required for diges- tion, however, if the mass of enzyme available for each meal were a limited, fixed quantity, would be so extremely variable that the economy of the tissues could not be adjusted to so irregular a method of furnishing their needs. For instance if about four and a half hours are requisite for the gastric digestion of 100 grams of raw meat by a given amount of pepsin then it may readily be calculated from the Schiitz-Borrissov Rule, which pepsin obeys, that no less than 70 hours would be requisite for the digestion of 400 grams and eighteen days for the digestion of a kilogram. As a matter of fact, however, the process of gastric digestion is carried out in successive portions of the foodstuffs, a fresh supply of gastric juice being furnished for each portion of food that comes into contact with the surface of the gastric mucosa. In this way much more rapid and uniform digestion is secured than would otherwise be possible. The hydrolysis of foodstuffs in the alimentary canal appears to follow the same quantitative laws as the hydrolyses by the corre- sponding enzymes in vitro. Thus Gastric Digestion follows the Schiitz- Borrissov rule, while the hydrolysis of protein in the small intestine by Pancreatic Juice follows the monomolecular logarithmic formula: log ^r~ = kt which holds good for the action of this enzyme in glassware. 252 DIGESTION AND ASSIMILATION OF THE FOODSTUFFS It is a rather noteworthy fact that the Rate of Absorption of digestion- products from the intestine does not appear, in so far as it has been quantitatively investigated, to follow the logarithmic rule, as we should expect if the rate of absorption depended solely upon the concentration, i. e., osmotic pressure, of the substance undergoing absorption. On the contrary, for the absorption of glucose at all events, a square-root rule seems to hold good, i. e., the quantity absorbed in a given time is proportional to the square-root of the concentration of the material which is being absorbed. It is, however, perfectly evident, even apart from these measurements, that the proc- ess of absorption cannot, be purely a question of the diffusion of sub- stances into and through the wall of the intestine in simple propor- tion to their osmotic pressures, for otherwise no Selective Absorption would be possible. We have seen that certain amino-acids are absorbed preferentially, others being absorbed with relative slowness even when they are present in excess. This implies that besides the forces of osmotic pressure, phenomena of solubility in the absorbing tissue-elements or of chemical affinity therewith play an important or decisive part in determining the relative rates of absorption and the types of material absorbed. From the Large Intestine, as we have seen, the products of bacterial decomposition of foodstuffs may be absorbed, sometimes with physio- logically undesirable results. A considerable Absorption of Water occurs here also. During digestion and absorption in the stomach and small intestine the contents of the alimentary canal retain a watery consistency which is favorable to the rapidity of hydrolysis, and to the thorough admixture of the digestive secretions with the foodstuffs and the absorption of the products of digestion. In the large intes- tine, however, a large proportion of this water is absorbed, so that the water-content of the feces is normally considerably less than that of the contents of the small intestine. In case the feces are expelled with undue rapidity, however, and before the absorption of water is complete, as when a cathartic is administered, then the feces have a watery consistency and thirst is engendered through insufficient absorption of the water which has been partaken, and which has also been furnished to the intestinal contents by the various digestive fluids. Not only water and products of bacterial action may be absorbed from the large intestine, however, but also foodstuffs if they chance to find entry therein without previous absorption. Thus, it is not an uncommon procedure in medical practice to furnish nutrition to very weak individuals or to persons who are unable to swallow, by Rectal Feeding, or the introduction of enemas containing fully hydro- lyzed foodstuffs, such as glucose. The substances thus administered are found to be absorbed, and to be normally utilized for the mainten- ance of the tissues, and the provision of energy. RELATIONS OF DIGESTION AND ABSORPTION 253 REFERENCES. ABSORPTION OF CARBOHYDRATES: Woodyatt, Sansum and Wilder: Jour. Am. Med. Assn., 1915, 65, p. 2067. MacLeod and Fulk: Am. Jour. Physiol., 1917, 42, p. 193. ABSORPTION OF FATS: Bloor: Jour. Biol. Chem., 1912, 11, pp. 141 and 429; 1913, 15, p. 105; 1913-14, 16, p. 517; 1914, 17, p. 377; 1914, 19, p. 1; 1915, 22, p. 133; 1915, 23, p. 317; 1916, 24, p. 447. Bloor and Knudsen: Ibid., 1916, 27, p. 107. Bloor: Ibid., 1916, 25, p. 577; 1916, 26, p. 417. ABSORPTION OF PROTEINS: Mendel and Rockwood: Am. Jour. Physiol., 1904-5, 12, p. 336. Wells: Proc. Soc. Exp. Biol. and Med., 1908-9, 6, p. 1. Whipple: Jour. Am. Med. Assn., 1915, 65, p. 476. Cathcart: The Physiology of Protein Metabolism. London, 1912. Folin and Denis: Jour. Biol. Chem., 1912, 11, pp. 87 and 161; 1912, 12, pp. 141, and 253. Abderhalden: Zeit. f. physiol. Chem., 1913, 88, p. 478. Van Slyke and Meyer: Jour. Biol. Chem., 1912, 12, p. 399; 1913-14, 16, pp. 197, 213 and 231. Van Slyke: Ibid., 1913-14, 16, p. 187. Abel, Rowntree and Turner: Jour. Pharm. Exp. Therap., 1913-14, 5, p. 611. Turner, Marshall and Lamson: Ibid., 1915, 7, p. 129. Underhill: The Physiology of the Amino-acids. Newhaven, 1915. Abel: The Mellon Lecture. Science, N. S., 1915, 43, p. 135. TIME AND MASS RELATION OF DIGESTION AND ABSORPTION: Erlanger and Hewlett: Am. Jour. Physiol., 1901-2, 6, p. 1. Arrhenius: Quantitative Laws in Biological Chemistry. London, 1915. PART II. THE PROPERTIES OF PROTOPLASM CHAPTER XII. PROPERTIES CONFERRED BY THE DIFFUSIBLE CONSTITUENTS. THE OSMOTIC PRESSURE OF THE TISSUE FLUIDS. The diffusible constituents of living matter and of the media which bathe it, play a leading part in determining the movements and dis- tribution of the most abundant constituent of living cells, namely Water. Water is a very essential constituent of protoplasm, for a variety of reasons. In the first place it is a solvent for the majority of the protoplasmic constituents, and thus permits their mobility and promotes, by reduction of internal friction and cohesion, the free and rapid interplay of chemical reactions which characterizes the unstable equilibria of life. Then, again, water is the most efficient ionizing solvent, and thus permits electrical forces to come into play, and that notable increase in chemical reactivity which accompanies the ionization of dissolved substances. The low internal friction of water permits the changes of form, and rapid displacements of sub- stance which render the mobility of living matter possible. The high surface-tension of water is essential in the conservation of the boun- daries of the cell, and their restoration after displacements due to motion, and this, in turn, conserves the minute internal structures of the cell. The high specific heat of water enables it to absorb a great deal of heat without increasing very greatly in temperature and, conversely, to part with stored-up heat without falling very much in temperature. Sharp inequalities of temperature which might other- wise arise in living tissues are thus smoothed out by the "buffer action" of the prevailing solvent. It is of interest to consider the percentages of water which are contained in the various tissues of the animal body. The following are illustrative analyses cited after Hammarsten: 256 THE PROPERTIES OF PROTOPLASM Tissue. Percentage of water Fatty tissue 6-10 Bone (extremities and skull) , . . . . ... . . . 14-22 Bone (vertebra and ribs) , ..... ... . . 16-44 Tendon . . . .'.'. . . . 56-68 Brain, white substance . . ... . ; . . . .. . . 68-70 Muscular tissue 75-78 Thyroid gland ... . ...... . . . . . 77-82 Thymus ' . . . 81 Brain, gray substance 82-85 It will immediately be noted that the percentage of water is highest in those tissues which are undergoing the most rapid metabolic changes and which are called upon to function, in a chemical rather than a structural manner, most rapidly and frequently. The percentage of water is lower in adult than in embryonic -tissue, and decreases with advancing age of the tissues, and diminution of the speed of metab- olism. Living tissues which are exceptionally poor in water or which withstand dessication, such as seeds or bacterial spores, represent life latent, but arrested, only to be resumed in full vigor upon the readmis- sion of water. The force which impels the movement of water into or out of the the elements of living matter is the difference between the Osmotic Pressure of the fluids within the cell on the one hand, and the external medium which bathes the cell on the other. The manner in which this force may impel the migration of water will be evident if we consider the mechanism by which it originates. We may suppose that the molecules of a substance in solution are in a state of con- tinuous motion, as, indeed, their diffusibility shows that they must be. Let us consider the condition of affairs in a vessel filled with water (Fig. 9) and divided into two parts by a partition A-B, on the right-hand side of which we introduce such an amount of some diffus- ible substance, such as glucose, that there are ten molecules of glucose in the mixture for every ninety molecules of water. Evidently, on the left-hand side of it every molecule which collides with this parti- tion will be a water-molecule, but on the right-hand side every tenth molecule will be a sugar-molecule. If, now, the partition A-B is constructed of such material that it is porous to water, but impermeable for more bulky molecules such as those of sugar, it is evident that out of 100 molecules bombarding the partition from the left all will pass through into the right-hand chamber, while out of 100 molecules bombarding the partition from the right, only 90 will be able to penetrate into the left-hand chamber. In any given interval of time, therefore, an excess of water molecules will have entered into the right-hand chamber, and this excess will be directly proportionate to the concentration of sugar dissolved therein. Such a partition as that which we have described constitutes what is known as a Semipermeable Membrane, and membranes having the characteristic of permitting the passage of water but not of dissolved substances are very numerous. The one most frequently employed PROPERTIES CONFERRED BY CONSTITUENTS 257 for osmotic-pressure measurements is the membranous precipitate of Copper Ferrocyanide which is formed when a solution of copper sulphate comes into contact with a solution of potassium ferrocyanide. If the continuous entry of water into the right-hand chamber were permitted and the level of fluid did not rise so as to create a pressure, water would pass indefinitely from left to right until the sugar in the right-hand compartment was infinitely diluted. In this way no measurement of the attraction of the solution for water would be possible, since, in theory, if no frictional forces or pressures interfered with the free motion of water, every solution, concentrated or dilute, would attract an infinite volume of water. We may, however, measure the degree of attraction for water which is exerted by the dissolved substance by determining the pressure or temperature necessary to 100 H,0 90 H,0 10 SUGAR B FIG. 9 increase the force or frequency of the impacts on the right-hand side of the partition, until the greater speed of transit from right to left compensates for the greater volume of transit from left to right. If pressure be applied to the contents of the right-hand compartment, the force of the impacts of the molecules upon the partition is increased so that although only ninety water molecules collide with the right- hand side of the partition for every hundred which collide with the left-hand side, yet those colliding on the right do so more forcibly, and thus a greater proportion succeed in penetrating the membrane, until, when the pressure applied to the solution attains a certain magnitude the greater proportion of collisions leading to penetration of the membrane exactly balances the excess of the total number of collisions on the side which is bathed by pure water, 17 258 THE PROPERTIES OF PROTOPLASM The pressure which is required to exactly compensate the greater frequency of collisions on the side of the membrane which is bathed by pure water is termed the Osmotic Pressure of the solution. Its measurement may be rendered automatic by enclosing the solution in a vessel to the orifice of which a Manometer is attached, so that the entry of a very minute amount of water, into the vessel, insufficient to appreciably dilute the solution, causes a very considerable rise in the mercury column of the manometer, and a proportionately large increase of pressure. With practically negligible dilution of the solution, therefore, the necessary pressure is attained and cannot be exceeded, because the os notic pressure having once been attained, the rates of entry and exit of water into and oit of the vessel become equal and no further changes of pressure or composition can occur. Such a vessel, provided with a semipermeable membrane and a manometer, is termed an Osmometer. Since the pressures which are generated are usually very great, the walls of the vessel must be made of strong material, and the membrane, especially, must be constructed so as lot to break under the strain. These desiderata are attained by employing a vessel composed of earthenware, in the minute pores of which membranes are formed by filling the vessel with a solution of potassium ferrocyanide and immersing it in a solution of copper sulphate. The two reagents diffuse outward and inward, or may be induced to do so by electrolysis, until they meet at some point within the pores across which membranes of minute dia leter are formed. Such membranes withstand relatively enormous pressures, while a large continuous membrane would rupture under the strain of much smaller pressures. Another way of equalizing the rates of transposition of water across the membrane would be to raise the Temperature of the solution above that of the water upon the other side of the membrane. Increase in temperature results in proportionate increase of the mobility of the molecules, so that the collisions with the membrane would be proportionately more numerous per unit of time on the heated than on the unheated side. This procedure is not practicable with the membranes which we have hitherto been considering, because they would conduct heat from the one chamber to the other and the tem- peratures of the two compartments on either side of the membrane would soon be equalized. We may very easily employ this method, how- ever, if in the place of a thin solid membrane we employ a layer of air. Then, provided the dissolved substance is not volatile, i. e., soluble in air, we have in effect a semipermeable membrane which is a poor con- ductor of heat and which may be obtained of any thickness which we may desire. If two chambers or vessels, the one containing water, the other containing a solution, be both placed in a confined space or large vessel filled with air, water will, if the two liquids are at the same temperature, slowly distil over from the compart nent co itaining pure water into the compartment containing the solution, which thus becomes pro- PROPERTIES CONFERRED BY CONSTITUENTS 259 gressively more dilute. If unchecked by any balancing or opposing influence, this distillation will continue until the solution becomes infinitely dilute, i. e., practically equivalent to distilled water. In this case, therefore, as in the case of thin solid membranes, we can only measure the attraction of the solution for water by measuring the change in the condition of the solution requisite for its neutraliza- tion. This we may accomplish by heating the solution and thus increasing the mobility of the molecules which it contains, and so increasing the number of collisions per second of water-molecules with the supernatant layer of air. The temperature to which we must raise the solution in order to equalize the rates of distillation to and from the water and the solution is proportionate to the increase in the collisions per second which is requisite to produce this equaliza- tion, and this in turn must obviously be proportionate to the con- centration of the dissolved substance. Thus if the dissolved sub- stance constitutes one-tenth of the total molecules in the solution, we must raise the temperature of the solution sufficiently to increase the total collisions per second by one-tenth, in order to render the rate of distillation equal to that of pure water at the lower tempera- ture. If this rate of distillation is sufficient to cause ebullition, i. e., to render the pressure of water-vapor equal to that of the atmosphere, it is evident that the temperature required to attain it will be higher in the case of the solution than in the case of pure water. Hence, the Boiling-point of water is raised by dissolved substances, and that in proportion to their molecular concentration. There is yet another way in which we may equalize the rates of penetration of water from opposite sides of the membrane, and that is by cooling the water, and thus reducing the mobility of its molecules relatively to those of the solution. Now when a solution freezes, it is not the dissolved substance that freezes, but the solvent, in this case water. The dissolved substance, in fact, with certain intelligible exceptions, crystallizes out and becomes mechanically separated from the solvent when the latter freezes. In such a case the membrane is furnished by the surface separating the crystals of ice from the remainder of the solution. If, now, reverting to the diagram in Fig. 9, the water in the left-hand compartment be sufficiently cooled, relatively to the solution, water will pass over from the warm solution into the cool chamber of pure water, because of the greater mobility of the molecules in the warm solution. 1 Hence, in order to accom- plish the withdrawal of water from the dissolved substance which occurs at the freezing-point the water must be cooled to a temperature 1 Ultimately, however, the greater mobility of the molecules in the solution will fail to compensate for the progressively decreasing proportion of water molecules present in the solution, and the water compartment would have to be further cooled in order to continue withdrawal of water from the solution. This is the phenomenon of "under- cooling" which freezing salt solutions display. The correct freezing-point is that at which the first crystal of ice separates, and which is marked by a sudden slight rise of temperature due to the disengagement of the latent heat of fusion ol the ice. 260 THE .PROPERTIES OF PROTOPLASM below the freezing-point of pure water. Hence, the Freezing-point of water is lowered by 'dissolved substances, and that in proportion to their molecular concentration. The osmotic pressure of a solution of a diffusible substance may therefore be measured either directly, employing a semipermeable membrane, or indirectly, by measuring the elevation of the boiling- point or the lowering of the freezing-point. Conversely the molecular concentration of a dissolved substance may be estimated in the same ways. The osmotic pressure exerted by a molecular solution, that is, by one gram-molecule of substance dissolved in a liter of water is 22.4 atmospheres. The elevation of the boiling-point in the same solution is 0.54, while the depression of the freezing-point is 1.86. If, however, the dissolved substance undergoes Electrolytic Dissociation then each of the ions which it yields exerts osmotic pressure and affects the boiling- and freezing-points in the same way as a molecule, so that for a substance completely dissociated into two ions, such as sodium chloride in dilute solution, the osmotic pressure per gram- molecule of dissolved substance is double the above-mentioned figure, and the molecular elevation of the boiling-point and lowering of the freezing-point are similarly enhanced. If different solvents are employed the osmotic pressures obtained are the same as those obtained when water is used as a solvent, provided the molecular condition of the dissolved substance is the same in both solvents, but if it be ionized in one and not in the other, or forms double molecules in one and not in the other solvent, the pressures observed will, of course, differ from one another in a corresponding manner and degree. The magnitude of the effect upon the boiling- and freezing-points, although always proportionate in any one solvent to the molecular plus ionic concentration of the dissolved substance, differs with different solvents. The osmotic pressures of tissue-fluids and of fluids expressed from cells are usually estimated by the Cryoscopic Method or measurement of the lowering of the freezing-point of the solvent, in this case water. This measurement is much less tedious and less subject to interference by colloidal admixtures, etc., than the direct measurement of pressure in an osmometer. The elevation of the boiling-point is usually not applicable because of the extensive changes induced in these solutions by elevated temperatures, for example the coagulation of proteins and the transformation of bicarbonates into carbonates with the evolution of carbon dioxide. The former of these changes can be obviated, of course, by measuring the elevation of the boiling-point under reduced pressures when ebullition occurs at a correspondingly lower temperature, but the difficulty created by the evolution of carbon dioxide still remains. The following are illustrative measurements, obtained by Hamburger and others, of the lowering of the freezing-point in blood-sera* of various species of Mammalia: PROPERTIES CONFERRED BY CONSTITUENTS 261 Lowering of Species. freezing-point. Man . . 0.526 Ox 0.585 Horse . . 0.564 Pig 0.615 Rabbit . 0.592 Dog :...... 0.571 Cat. . - fc . .- -. . . . ^ . . . 0.638 Sheep 0.619 Echidna hystrix 0.600 From this table two remarkable facts are apparent: In the first place that the osmotic pressure of the blood of species of mammalia so diverse as man, herbivora, carnivora and the monotremes is extra- ordinarily constant, and in the second place that it has the magnitude of no less than some eight atmospheres, corresponding to the pressure exerted by a one-third molecular solution of a non-ionized substance such as sugar or urea, or a one-sixth molecular solution of sodium chloride. - The osmotic pressure of the blood-serum, as evaluated from the lowering of the freezing-point, rises slightly, but unmistakably, after the absorption of the products of digestion derived from the meal. The Lymph has usually a higher osmotic pressure than the blood, a fact which is attributed to the extraction of products of metabolic activity from the tissues somewhat more rapidly than they can be discharged from the lymph into the blood. Milk and Bile have the same osmotic pressure as blood, Saliva a lower pressure. Urine is in general much more concentrated in diffusible constituents than the blood or tissue-fluids and therefore displays a much greater lowering of the freezing-point, usually between 1.3 and 2.3. The blood-sera of Birds possess an osmotic pressure very similar to that of mammalian blood -sera. It is a curious fact, however, that the Eggs of birds have a distinctly lower osmotic pressure than that of the blood-serum of the birds that lay them, or of the blood-serum of the embryos that develop within them. This is strikingly shown by the following estimations of Atkins. Lowering of 1 Species. freezing-point. Fowl-blood . .- . 0.607 Fowl-egg 0.454 Duck-blood 0.574 Duck-egg 0.452 Goose-blood 0.552 Goose-egg '. . 0.420 During Incubation of the egg the osmotic pressure of its contents increases until it approximates to that of the blood. Since in so many anatomical particulars the Ontogeny of the individual represents an abbreviated outline of the Phylogeny of the species, Atkins has sug- gested that the low osmotic pressure of the egg-contents may indicate descent of the birds from ancestors in which the blood-serum was more dilute than it is in the birds of the present epoch. Since the birds 262 THE PROPERTIES OF PROTOPLASM are probably descended from ancient forms of Reptilia or from forms intermediate between the Reptilia and the Amphibia it is of interest to note that many of the Amphibia and some of the Reptilia which inhabit fresh water exhibit a low osmotic pressure of the blood-serum. The following are illustrative figures cited after Hober and Jona. Lowering of Species. freezing-point. Amphibia: Ranaescuknta . . . , . 0.465 Salamandra maculata 0.479 Reptilia: Emys europaia 0.474 Emydura macquariw 0.550 Thalassochelys caretta 0.610 It will be observed that the fresh-water turtle, Emys europaia, has blood of which the osmotic pressure approaches the amphibian type, the marine turtle, Thalassochelys caretta has the avian and mammalian type of osmotic pressure of the blood, while the tortoise, Emydura macquarice represents an intermediate pressure. The osmotic pressure of amphibian blood-serum closely approaches in magnitude the pres- sures obtaining in the eggs of birds. Among the various orders of Fishes in the Teleostomi or bony fishes, which are phylogenetically the most recent and highly developed forms, the osmotic pressure of the blood-serum approximates much more nearly to that of the blood of Mammalia, Aves and Reptilia than to the osmotic pressure of the ocean which the marine forms inhabit. In the phylogenetically older and less specialized forms, the Elasmobranchii or sharks, however, the osmotic pressure of the blood approximates that of sea-water, as the following figures, cited after Bottazzi, reveal: Lowering of Fluid. freezing-point. Sea-water 2.30 Elasmobranchii : Torpedo marmorata .- . 2.26 Mustelus vulgaris . . . . * . .-. . . 2.36 Trygon violacea 2.44 Marine teleostomi : Charax puntazzo 1.04 Cernagigas . 1.04 Crenilabrus pavo 0.75 Box salpa 0.84 Fresh- water teleostomi: Anguilla vulgaris 0.58-0.69 Barbus fluviatilis 0.475-0.558 Lcuciscus dobula 0.45 Perca fluviatilis 1 Cyprinus carpio [ n _ Tinea vulgaris ( Esox lucius In lower marine forms the tissue-fluids approximate still more closely in composition and concentration to the sea-water which these organisms inhabit. The following are results obtained by Bottazzi: OSMOTIC PRESSURE OF CELL CONTENTS 263 Lowering of freezing-point of Organism. tissue-fluids. Ccelenterata : Alcyonium palmatum 2.196 Echinodermata : Asteropecten aurantiacus 2.312 Holothuria tubulosa 2.315 Vermes: Sipunculus nudus 2.31 Crustacea: Majasquinado 2.36 H omarus vulgaris 2.29 Cephalopoda : Octopus macropus 2.24 With the enhanced specialization, therefore, which characterizes the higher and especially the vertebrate forms of life, independence of the external milieu has been acquired and the cells are bathed in a medium of relatively constant concentration and, as we shall see, of even more constant composition. THE OSMOTIC PRESSURE OF CELL-CONTENTS. The osmotic pressure of cell-contents can, of course, be determined indirectly by expressing the cell-sap and determining its freezing- point. In many cases, however, the measurement may be made in a very much more convenient manner by employing the method of Plasmolysis devised in 1884 by the Dutch botanist, de Vries. In many plants the protoplasm of the cells lies closely adherent to the cellulose cell-wall, and it is found if these cells be immersed in concentrated solutions of salts, sugars, urea or other diffusible sub- stances, that the protoplasm shrinks away from the supporting wall of cellulose, indicating that the protoplasm has diminished in volume. This loss of volume can only be due to the abstraction of water from the protoplasm, and since the agencies which accomplish this abstrac- tion of water are solutions of relatively high osmotic pressures, we infer that the external limiting membranes of the cells, within the cellulose cell-wall but bounding the exterior of the protoplasmic con- tents, is Semipermeable, admitting water but not admitting a variety of diffusible dissolved substances. If this interpretation be the correct one, then any solution having a higher osmotic pressure than the cell-fluids will cause plasmolysis, while the solutions which are of just the same osmotic pressure as the cellular fluid will fail to cause plasmolysis. The solutions which just fail to cause plasmolysis, or which are Isotonic with the cell- fluids, should therefore all be of the same molecular or molecular plus ionic concentration, independently of the nature of the dissolved substance, provided, only, that it is not able to penetrate the cell- membrane in measurable proportion within the period of time con- sumed by the shrinkage of the protoplasm. 264 THE PROPERTIES OF PROTOPLASM The following are results which were obtained by Over ton, employ- ins; the cells of spirogyra filaments: Isotonic concentration: Dissolved Molecular found, calculated, substance. weight. per cent. per cent. Cane-sugar . . 342 6.0 Mannitol 182 3.5 3.19 Glucose 180 3.3 3.15 Arabinose 150 2.7 2.63 Erythritol 122 2.14 Asparagin 132 2.5 GlycocoU 75 1.3 1.32 The tf calculated" values were computed as follows: The isotonic concentration of cane-sugar being 6 per cent, and its molecular weight 342, the concentration of an isotonic solution is evidently /$ = ^ molecular. A ^y molecular solution of glycocoll would contain |^- grams of glycocoll per liter, or 1.32 grams per hundred c.c. It will be seen that.the experimental and the calculated values are exceedingly close to one another and we may infer that, at all events so far as limited periods of time are concerned, the protoplasm of spirogyra is impermeable to the substances mentioned, although freely permeable to water. This method of estimating the isotonicity of solutions, however, is subject to several sources of error and uncertainty. In the first place we must take into consideration the fact that the protoplasmic limit- ing membrane must necessarily alter in form before we can perceive any solution to be Hypertonic, or in excess of the isotonic concentra- tion. Now the external limiting membranes of cells must undoubtedly possess some degree of Elasticity, in consequence of which they must themselves exert some pressure upon the cell-contents. The forces leading to shrinkage of the protoplasm are not solely osmotic there- fore, but to some slight extent elastic also, and we cannot positively estimate the proportion of the total force which this elasticity com- municates, since it will not improbably add a constant amount to each osmotic pressure investigated. Isotonic solutions are therefore isosmotic with one another, but not necessarily isosmotic with the cell-contents. In red blood-corpuscles this is probably the origin of the constant slight difference between the osmotic concentration of the contents of the corpuscles and the surrounding medium or plasma, amounting, according to Moore and Roaf, to a difference of freezing- point depression of 0.02 to 0.03 C., or an osmotic-pressure difference of from 0.24 to 0.36 of an atmosphere. -t In the second place, the Semipermeability of living cell-membranes is, of necessity, never absolute. This becomes obvious when we consider that the nutrition, and therefore, the maintenance and growth of cells depends upon their intake of substances dissolved in water. Unless a cell can be penetrated by the mineral or organic substances which constitute the components out of which protoplasm is built up, the progressive consumption of material and dissipation OSMOTIC PRESSURE OF CELL-CONTENTS 265 of energy by the cell must rapidly lead to its disintegration. Further- more, the solutions which Overton, in the results cited above, found to be isotonic with the cell-contents of spirogyra filaments exerted an osmotic pressure of some four and a half atmospheres, and the corresponding pressure in the cell-contents themselves, can only have been due to diffusible water-soluble substances which must therefore have penetrated the protoplasm at some period of its devel- opment. The semipermeability of cell-membranes is in fact, even in the most typical instances, apparent and not real. It. is purely a question of Relative Permeability, of the rapidity with which dissolved substances and water relatively penetrate the cell. In the case of Bacillus cholera, for example, the relativity of the semipermeability of cells can very clearly be seen, for these organisms, as well as certain other bacteria, are temporarily plasmolyzed by hypertonic salt solu- tions or sugar-solutions, but not at all by Glycerol solutions. Even the plasmolysis observed in salt- or sugar-solutions disappears in the course of an hour or two, because, after the lapse of this time, a suffi- cient proportion of the salt or sugar has penetrated the cells to restore isotonicity between the inner and outer fluids. Evidently, therefore, in the case of these cells water and glycerol penetrate the exterior limiting membrane almost instantaneously, salt and sugar more slowly. The disparity of the velocities of penetration for water and dissolved substances is greater in spirogyra filaments than in the above-mentioned species of bacteria, and this constitutes the origin of the apparent semipermeability of the protoplasm in spirogyra. It is a rather remarkable, and certainly a regrettable fact that physical chemists have hitherto paid so little attention to the investi- gation of the Time-relation of Osmosis. The comparative neglect of this and other fields of inquiry which would naturally suggest them- selves to the student of pure physics or mechanics, is undoubtedly attributable to the bias toward purely thermodynamical reasoning which has been communicated to the students of physical chemistry by the past generation of chemists. The thermodynamical relation- ships and equations contemplate only attained equilibria, not fluctuat- ing or kinetic phenomena. Hence, the relationship between the lapse of time and the degree of penetration of a 'membrane by various solvents or dissolved substances, which would seem to present a most obvious subject for inquiry, is as yet very imperfectly known. One would expect, however, that the quantity of penetration would be an exponential function of the time, and that this function would contain specific parameters or constants, characteristic for the membrane, the particular solvent employed, and the dissolved substance respec- tively. The evaluation of these parameters in the case of living cell- membranes would afford an accurate quantitative measure of Per- meability, for the estimation of which we must rely at present upon qualitative rather than upon quantitative data. In the plasmolytic method of estimating isotonic solutions we regard 266 THE PROPERTIES OF PROTOPLASM as isotonic those solutions which are just insufficiently Hypertonic to cause withdrawal of water from the cell-contents. The effects of Hypotonic solutions or solutions which are more dilute than the cell- content are more readily studied in cells which possess no rigid support- ing framework, such as the exterior cellulose wall of plant-cells. The Red Blood-corpuscles were first employed by Hamburger for this purpose. If these cells are suspended in sufficiently hypotonic solu- tions, the excessive penetration of water into the cells results in their rupture by the internal pressure which results, and hemoglobin is set free, tingeing the supernatant fluid red. The technique, therefore, consists in suspending the corpuscles in solutions of varying concen- tr'ation and allowing them to settle to the bottom of the tube. A solution which is just sufficiently hypotonic to burst some of the corpuscles will be tinged with hemoglobin and the corpuscles are then said to have undergone Hemolysis. The degree of hypotonicity required to rupture red blood-corpuscles is apparently the same for a variety of dissolved substances, so that the solutions are found to be isotonic with one another, as the following data show: Limiting concentration which causes Substance. Molecular weight. hemolysis, per cent. NaCl ,58.5 0.585 CHsCOOK .....;.... 98.1 1.04 KNO 3 . . . ....... . .101.1 1.00 NaBr . . . ... . , 102.9 1.02 Nal 149.9 1.55 KI . . . . . . . .... . 166.0 1.65 Solutions which cause hemolysis, although isotonic with one another are, of course, by no means isotonic with the fluid contents of the corpuscles, for the bursting of the cells indicates not a slight but a very considerable excess of pressure within them. Solutions insufficiently hypotonic to cause actual rupture of the cells will nevertheless cause them to swell through absorption of water, while slightly hypertonic solutions will, on the contrary, cause shrinkage of the cells through the withdrawal of water, just as in the plasmolysis of plant-cells. This is the foundation of the Hematocrit method of measuring isotonicity, devised by Hedin and Koeppe. Blood-corpuscles, freed from serum by washing them with isotonic salt solution, are suspended in measured amounts of various solutions to be investigated, and the mixtures are placed in specially constructed centrifuge-tubes of very narrow bore and provided with fine graduations. The tubes are then centrifuged and the heights of the columns of corpuscles compared in the various tubes. If the corpuscles have swollen they will occupy a larger volume in the tube, if they have lost water they will occupy a smaller volume than the corpuscles which are immersed in strictly isotonic salt solu- tions. From the lowering of the freezing-point we know that blood- serum is isotonic with NaCl or f sugar solutions, and it is experi- mentally found that in the majority of instances salt solutions which OSMOTIC PRESSURE OF CELL CONTENTS 267 slightly exceed this concentration cause shrinkage of the corpuscles, while solutions which are less concentrated than blood-serum cause swelling of the corpuscles. Assuming the corpuscles in normal serum to be withstanding no pressure or, at the most, a very slight one, it is of some interest to calculate from the degree of hypotonicity the pressure which is required to rupture the corpuscles so as to discharge hemoglobin into the solu- tion. The solutions which are just sufficiently concentrated to prevent rupture are, as we have seen, isotonic with a one-tenth molecular sodium chloride solution or, which comes in terms of osmotic pressure to the same thing, a one-fifth molecular solution of sugar. When neither swollen nor shrunken these cells are isotonic with a one third molecular solution of sugar. The degree of hypotonicity required to rupture the cells therefore, corresponds to the pressure exerted by a 3 i = A molecular solution of sugar, i. e., to a pressure of about three atmospheres. From the data which we have cited the surface of a red blood-cor- puscle would appear to afford an example of a strictly semipermeable membrane. Here again, however, semipermeability is relative and not absolute. Not only water can enter the cells with ease but also other substances with varying difficulty. An ingenious method of illustrating this fact is that which has been devised by Hedin. A measured amount of the substance for which the permeability of the corpuscles is to be tested is dissolved in defibrinated blood, i. e., in a mixture of serum and corpuscles. The serum of this blood will be found to freeze at a lower temperature than untreated serum, because a certain proportion of an additional diffusible substance is contained in it. The depression of the freezing-point of this serum may be designated "a." Now to an equal volume of serum which does not contain any corpuscles an equal amount of the same substance is added. This serum will also freeze at a lower temperature than normal serum, and the depression of the freezing-point which it exhibits may be designated "b." Now, it is evident that if the substance which was added to the defibrinated blood penetrated the corpuscles and dissolved in them to the same extent as in an equal volume of serum, the concentrations of the substance in the two samples of serum would be equal to one another, and we would have a = b. If the blood corpuscles in the defibrinated blood took up less of the dissolved substance than an equal volume of serum, then the substance would be present in greater concentration in the first sample of serum than in the second, and we would have a > b or jj > 1. If, on the other hand, the blood-corpuscles took up more of the dissolved substance than an equal volume of serum then we would have a < b or ^ < 1 . The results of this method show that the salts of the alkalies and alkaline earths and the amino-acids and sugars penetrate the corpuscles with great difficulty. Ammonium Salts and Urea, however, pass into the 268 THE PROPERTIES OF PROTOPLASM corpuscles readily. Among the alcohols there is an interesting relation- ship between the number of hydroxyl-groups which they c6ntain and the readiness with which they penetrate the corpuscles. The hexa- tomic and pentatomic alcohols hardly penetrate the corpuscles at all. Erythritol, which is a tetra-atomic alcohol arid Glycerol which is tria- tomic penetrate slowly. Ethylene Glycol, which is a diatomic alcohol, penetrates the cells rather rapidly, while the Monatomic Alchols divide themselves immediately in equal proportion between the corpuscles and the serum. Ether, esters, aldehyde and acetone, on the other hand, are preferentially absorbed by the corpuscles, so that they become more concentrated in the corpuscles than in the serum which bathes them. Of course only those substances which fail to enter the cells quickly can cause shrinkage of the corpuscles in hypertonic solutions. THE COMPOSITION OF THE MINERAL CONSTITUENTS OF TISSUE FLUIDS. It was first pointed out by Ringer in 1882 that the relative propor- tions of the mineral constituents in the blood-sera of different mam- mals are most remarkably constant and, furthermore, that notwith- standing the fact that potassium and calcium salts are present in blood- serum only in minute proportion relatively to the sodium salts, yet their presence in the established proportion is actually essential to the proper performance of their functions by the tissues, a very slight alteration in the mineral composition of the fluid bathing them being very deleterious. On the basis of numerous analyses of the ash of blood-sera, the following .composition was established by Locke as the most suitable circulating fluid for mammalian tissues: NaCl, 0.9 per cent.; KC1, 0.042 per cent.; CaCl 2 , 0.024 per cent. To this mixture a small propor- tion (0.01 to 0.03 per cent.) of sodium bicarbonate is generally added to neutralize the acids which are produced by tissue-activities and a little glucose (0.1 per cent.) has been shown to prolong the life of excised tissues which are kept for prolonged periods in this artificial circulating fluid. The glucose is consumed by the tissue and probably serves as a nutrient. When the glucose is omitted this mixture is usually desig- nated Ringer's Solution. For amphibian tissues a slightly more dilute solution is employed. The solution originally recommended by Ringer was a 0.6 per cent, solution of sodium chloride saturated with calcium phosphate to which 0.03 per cent, of potassium chloride was added. A suitable fluid may also be prepared by simply adding to Locke's Solution one-third of its volume of distilled water. Loeb has pointed out that the proportions of the various salts in Ringer's and Locke's solutions correspond approximately to the ratios: 100 molecules of NaCl to 2 molecules COMPOSITION OF THE MINERAL CONSTITUENTS 269 of KC1 to 2 molecules of CaCl 2 , the total concentration being one-sixth molecular.' Not only, however, are the mineral constituents of mammalian serum constant in composition, but even in the blood of fishes we find that substantially the same relative proportions obtain. The following analyses are cited after Macallum, the percentage-concentTSition of sodium being taken as 100 and the percentages of the remaining metals reduced to the same units: Species. Na K Ca Mg Dogfish (A canthias vulgaris) .100 4.6 2.7 2.5 Cod (Gadus callarias) ... 100 9.5 3.9 1.4 Pollock (Pollachius mrens) . . 100 4.3 3.1 1.5 Dog 100 6.9 2.5 0.8 Mammal (average) .... 100 6.7 2.6 0.8 Man 100 6.1 2.7 0.9 The remarkable uniformity of composition which is thus displayed by the blood-sera of such diverse organisms, suggests that it is deter- mined by some common cause, more especially since a slight alteration of the normal mineral composition of blood-sera causes profound disturbance of the functions of the tissues. The interesting suggestion has been put forward by Macallum that the mineral composition of vertebrate blood-sera represents the composition of the sea-water at the time when the early ancestors of the present vertebrate forms first acquired an organ, namely the kidney, of which the function is to maintain constancy of composition in the body-fluids. In the lower marine forms of the present day which do not possess any corresponding excretory organ, the composition and concentration of the body-fluids is practically identical with that of the sea-water in which they live, but in mammals the tissue-fluids are not only more dilute than present- day sea-water, but they differ from it in containing a much smaller proportion of one mineral constituent, namely Magnesium. The fol- lowing figures are cited after Macallum, the percental-concentration of sodium, as before, being taken as 100 and the percentages of the remaining metals reduced to the same standard. Fluid. Na K Ca Mg Ocean-water 100 3.6 3.9 12.1 Tissue-fluid of a jellyfish (Aurelia flavidula) 100 5.2 4.1 11.4 Blood-serum of a dog ... 100 6.9 2.5 0.8 The correspondence of the three sets of figures, excepting in regard to magnesium, is certainly striking and the oceanic origin of these widely- found ratios appears very probable. Among the crustaceans the more primitive forms, such as Limulus possess a blood-serum which is practically of the same composition as sea-water. In more highly developed forms such as Homarus an 1 The actual ratios in Locke's solution are 100 NaCl : 3.6 KC1 : 1.4 CaCl?, In sea- water the ratios are: 100 NaCl : 2.2 KCJ : 1.5 CaCl?, 270 THE PROPERTIES OF PROTOPLASM approach toward the vertebrate composition of the serum is already indicated, as the following figures reveal: Fluid. Na K Ca Mg Ocean-water 100 3.6 3.9 12.1 Serum of Limulus polyphemus .100 5.6 4.1 11.2 Serum of Homarus americanus .100 3.7 4.9 1.7 According to Macallum the development of a kidney in the proto- vertebrate forms from which vertebrates have arisen, fixed the com- position of the tissue-fluids of the vertebrata for all time, since the primitive kidney was adapted to the concentration and proportions of the mineral constituents of the ocean of that period. In the early Cambrian or pre-Cambrian period at which the ancestral forms of the vertebrates arose, the sea-water must have been very much more dilute than it is at present day, because sodium chloride is constantly accumu- lating, since it is not deposited in important amounts in the marine geological formations. Calcium and potassium are deposited from sea- water in the form of limestone and minerals such as glauconite at about the same rate as that at which they are carried into the sea by rivers. Magnesium, however, is increasing in the sea-water not only absolutely but also relatively to the sodium, the rate of deposition being much slower than the rate of addition. It is quite probable, therefore, that the sea-water of the early Cambrian epoch was not only much more dilute than the sea-water of our day, but also contained both absolutely and relatively much less magnesium. The blood-serum of mammals therefore resembles a diluted sea- water with the exception that its magnesium content is both absolutely and relatively much lower than the magnesium content of the sea-water of our own day. Just as the homoiothermal animals have acquired a large measure of independence of the temperature of their environment, so, and at an earlier stage of evolution, the vertebrates have acquired a large measure of independence of their osmotic environment, they are "homoiosmotic," while the more elementary forms are "poikilos- motic" and the cells of which they are composed are exposed to all the disadvantages of an irregularly fluctuating milieu. At a still earlier stage of evolution the multicellular organisms acquired, as we shall see, more or less efficient means of maintaining constancy of the reaction or hydrogen ion concentration of their tissue-fluids. Each of these successive stages marked an additional degree of emancipation from the fortuitous inequalities of an unstable environment and a step toward the self-creation of an equable "internal environment/' suit- able for the maximum furtherance of vital activities. The mechanism by which this environmental stability is brought about is similar in each of the three cases and consists in a balance between income and output so adjusted that the dissipating agencies (excretory activity of the kidneys, radiation of heat from the surface of the body, and release of carbon dioxide from the lungs, respectively) NEUTRALITY OF THE TISSUES AND TISSUE-FLUIDS 271 discharge their functions under the stimulus of a definite positive or negative pressure, acting like so many dams, to maintain the reservoir of mineral constituents, heat, or bases at a certain height while the inflow and outflow are equalized so that the height of the reservoir does not progressively increase or decrease. We have in fact in each case a number of balanced activities in dynamic equilibrium, a type of mechanism which is repeatedly reduplicated in life-phenomena. Notwithstanding the fact that the mineral composition of mam- malian blood-sera differs appreciably from that of sea-water only in total concentration and in the relative content of a single constituent, Magnesium, yet this latter difference renders sea-water, even when diluted to isotonicity with blood-serum, far from a physiologically neutral fluid for mammalian tissues. It has been shown by Burnett that sterilized sea-water, rendered isotonic with blood-serum by dilution, causes Glycosuria, considerable amounts of glucose appearing in the urine when the sea-water is injected into the circulation of rabbits. The same effect is brought about by Locke's or Ringer's solutions, if magnesium is added to them in the proportion in which it is present in sea-water. Hence diluted and sterilized sea-water cannot be employed for surgical purposes as a substitute for Locke's or Ringer's solution. THE NEUTRALITY OF THE TISSUES AND TISSUE-FLUIDS. The statements concerning the alkalinity of the blood which are to be found in the physiological and medical literature of the last and early part of this century are totally unreliable since they were based upon the erroneous belief that it is possible to ascertain the reaction of such a fluid as the blood by titration. The method of titration merely informs us of the quantity of bases which are present either uncombined or else combined with weak acids such as carbonic acid, which are dis- placed from their compounds by the stronger acids used in titration. If all of the bases are present in the free, uncombined form then, in dilute solutions at all events, the true alkalinity or hydroxyl ion con- centration may be fairly accurately estimated to be equivalent to the amount of acid required for neutralization. But this is not at all the case if. the bases are partially or wholly combined with weak acids, because in that event the addition of the acid employed in titration displaces the weak acid which, when uncombined, by reason of its slight dissociability, ceases to affect materially the reaction of the fluid, and the condition which we are seeking to measure alters con- tinuously throughout the titration. Thus it is possible to estimate all of the sodium in a solution of sodium bicarbonate by direct titration with sulphuric acid, using Methyl Orange as an indicator, because the carbon dioxide which is displaced by the sulphuric acid, is so slightly dissociated in comparison with the acid used for titration that its con- tribution to the final reaction of the mixture is negligible. Yet a 272 THE PROPERTIES OF PROTOPLASM solution of sodium bicarbonate is far from possessing the alkalinity of a solution of free sodium hydroxide of the same concentration, although so far as the results of titratiori reveal there is no distinction between them. The blood and other tissue-fluids contain a large proportion of the sodium salts of weak acids, namely carbonic acid, phosphoric acid and proteins. When blood-serum is titrated with hydrochloric or sulphuric NEUTRALITY OF THE TISSUES AND TISSUE-FLUIDS 273 acid, using methyl orange as an indicator, by the time the red color appears these compounds have been successively decomposed, and, in fact, some proportion of the acid employed in the titration has actually entered into combination with the proteins which are now acting as bases instead of acting, as they do in normal blood, as weak acids. The Titratable Alkalinity of the blood, therefore, bears no relationship to its actual alkalinity or Hydroxyl ion Concentration. It does, how- ever, bear some relationship, as we shall see to the power of the blood to maintain its neutrality, in other words to the "Alkali-reserve" of blood. FIG. 11. Modified Cottrell hydrogen electrode. (After Schmidt.) In order to ascertain the actual reaction of a complex mixture of weak and strong acids and bases such as the blood or other tissue- fluids, therefore, a method of measurement must be employed which is static and not dynamic, i.e., which leaves the state of the* blood unal- tered in respect to the balance of acids and bases which it contains. For this purpose no method is better adapted for obtaining accurate results than the electrometric or Potentiometric Method. The principle of this method has already been explained upon page 154. For the degree of accuracy usually required in biochemical or physiological researches the apparatus employed by Hoagland (1) and illustrated on page 272 (Fig. 10) is undoubtedly the simplest and most convenient. For solutions not containing volatile acids, the Cottrell form of elec- trode as modified by C. L. A. Schmidt is the best (Fig. 11), but when the fluid to be investigated contains carbon dioxide which would be 18 274 THE PROPERTIES OF PROTOPLASM blown out by a continuous stream of gas, the electrod e must be enclosed in a gas-tight vessel containing hydrogen and the fluid to be investi- gated and the vessel must be shaken to secure continuous contact of the electrode with hydrogen so as to maintain its saturation (Fig. 12) . Certain special precautions must be taken, when potentiometrically measuring the reaction of fluids containing proteins, especially that of bringing the hydrogen electrode to equilibrium with neutral distilled water before immersing it in the protein solution, for otherwise the acid reaction of the platinum due to the great excess of hydrogen ions which it contains will precipitate many proteins in a film upon its surface. Foaming, which is often troubesome in protein solutions, may be prevented by addition of a few drops of octyl alcohol, or of a mixture of amyl alcohol and kerosine, or of isoamyl isovalerate. FIG. 12. "Shaking" hydrogen electrode. (After Clark.) The potentiometric method was first employed for the estimation of the reaction of blood by Hoeber. The alkalinity of the blood which was indicated by his earliest measurements was excessive, owing to the fact that the stream of hydrogen employed to saturate the electrode blew out the carbon dioxide which in circulating blood stands in equilibrium with the bicarbonates, and contributes materially to the maintenance of neutrality. Later and more accurate measurements by Hoeber and many others are unanimous in establishing the fact that the normal reaction of the blood is so faintly alkaline as to approximate very closely to neutrality. Thus at absolute neutrality, as in neutral distilled water, the hydrogen and hydroxyl ions are equal in concentra- tion, namely 0.8 x 10~ 7 normal. The actual hydroxyl concentration in the blood is only about double this, namely 1.6 x 10~ 7 or less than one five-millionth normal at the CO 2 -pressures prevailing in the circulating blood (0.028 to 0.054 atmosphere). NEUTRALITY OF THE TISSUES AND TISSUE-FLUIDS 275 Another method which has been extensively employed in the investi- gation of the reactions of blood-serum and of other tissue-fluids is the Indicator-method of Friedenthal which has been especially applied to these investigations by Sorensen. This method consists in adding to the fluid under investigation a number of different indicators known to display color-changes, at differing hydrogen or hydroxyl ion concen- trations. The same indicators are also added to a series of mixtures of monosodium and disodium phosphate, of which the former is acid in reaction and the latter alkaline. The hydrogen ion concentration of all possible mixtures of these salts has been determined, and that mixture which yields most nearly the same tints with indicators as the unknown fluid evidently corresponds to it in hydrogen ion concentra- tion. This method is not applicable to a highly colored fluid such as whole blood since the tints of indicators are not accurately appreciable in such a fluid. Furthermore it is to be noted that the indicators most suitable for this purpose are precisely those which are least desirable for the ordinary purposes of direct titration, because the best indicator for titration is that which displays a sharp change from one tint to another at a certain reaction, whereas the best indicator for the indirect method of titration just described is evidently one which offers a large number of appreciable changes of shade or tint within a limited range of hydrogen ion concentrations. The most suitable indicator for the purposes of indirect titration within the range of reactions commonly met with in tissue-fluids, is Phenol Sulphonphthalein. Finally it should be carefully noted that the choice of indicators is limited to those which do not react chemically with the proteins or other substances commonly present in tissue-fluids. A variety of dyes which "are commonly employed as indicators in direct titration are unsuitable for our purpose because they interact with proteins and the compounds which are formed do not change color at the hydrogen ion concentration at which the free dye changes color, or even may display totally different colors from those which the free dye exhibits. By these various methods it has been ascertained that not only is the blood of all vertebrates very nearly neutral in reaction, but almost all of the tissue-fluids are also approximately neutral. Thus the Pan- creatic Juice, the most alkaline of body-fluids, contains 5 x 10~ 9 H+, corresponding to an alkalinity of 13 x 10~ 7 OH~ or a little over one millionth normal. Hitherto, according to Friedenthal, no animal fluid has been found which contains less than 10~ 10 H + , that is, more than about 10Q 6 000 normal OH~. Now the neutrality of the blood is maintained with extraordinary exactitude despite the fact that a large proportion of the products of metabolism are acid in reaction and are washed out of the tissues in which they are formed, into the blood. The products of muscular activity include carbon dioxide, lactic acid and acid phosphates, and the muscular exertion which is involved, for example, in climbing a steep hill involves the expenditure of a very considerable number of 276 THE PROPERTIES OF PROTOPLASM foot-pounds of energy, and the oxidation of a correspondingly large quantity of carbohydrate material, of which the carbon is converted ultimately into carbon dioxide, which is carried to the lungs through the mediation of the blood. Yet while this large production of acid products may cause some slight distress of breathing in the unaccus- tomed individual, it barely perceptibly modifies the reaction of the blood. The intravenous injection of large quantities of acid produces an altogether disproportionately small effect upon the alkalinity of the blood. In Diabetes the faulty oxidation of fats produces a quantity of non-volatile acids which cannot be discharged as carbon dioxide is discharged, through the respiratory epithelium of the lungs, and yet in many cases of advanced diabetes the reaction of the blood is only very slightly affected so that even in diabetic coma the acidity of the blood is only raised to 1 x 10~ 7 normal H + , a reaction which would be communicated to a hundred liters of neutral distilled water by the addition of a single drop of normal acid solution. The mechanism whereby this extraordinary stability of reaction is attained is a dynamic equilibrium which involves a variety of coordi- nated factors. Thus the kidneys assist in removing excess of acids by excreting a predominance of acid salts and of non-volatile acids. The lungs are, however, the most important organs of acid-elimination, since they contribute to the reduction of the hydrogen ion concentra- tion of the blood by permitting the escape of carbon dioxide. On the other hand the tissues themselves can contribute to the neutralization of injurious excess of hydrogen ions by arresting the formation of urea from protein nitrogen at the intermediate stage of ammonia, the ammonium salts of the excessive acids being excreted as such in the urine. Hence, in Acidosis such as that encountered in diabetes and in many toxemias, an unusual quantity of Ammonia appears in the urine. The prime agent in accomplishing the regulation of the reaction of the tissues and tissue-fluids is, however, the blood itself. This may very readily be perceived by comparing the relative powers of blood and of distilled water or sodium chloride solution to neutralize acids. If two indicators be chosen which change color at differing hydrogen ion concentrations, and distilled water and blood-serum respectively be neutralized first to one, and then to the other indicator, the difference between the two /iters will be extremely small in the case of distilled water and of very considerable magnitude in the case of the blood- serum. It can be shown in fact, that provided the carbon dioxide tension be maintained at the levels which prevail in circulating blood, one hundred volumes of blood of normal reaction can neutralize no less than 125 volumes of ^ hydrochloric acid before attaining the hydro- gen ion concentration of advanced acidosis, namely l.OOx 10~ 7 at 38. This would be equivalent, in a man whose circulation contains five liters of blood, to the neutralization of over six liters of or six hun- NEUTRALITY OF THE TISSUES AND TISSUE-FLUIDS 277 dred cubic centimeters of ^ acid. In the body, it must be remembered, this remarkable neutralizing-power of the blood is assisted by the added ventilation of carbon dioxide from the lungs, which occurs in conse- quence of the Dyspnea or rapid breathing which results from a slight decrease of the alkalinity of the blood, and by the excretion of acid salts by the kidneys and by the production of ammonia from the tissues. The origin of the neutralizing-power of the blood is threefold : in the first place the Bicarbonates of the blood are capable of neutralizing large quantities of acid without any great change in the hydrogen ion concentration by undergoing the reactions : NaHCOs + HA = NaA + H 2 CO 3 H 2 CO 3 = H 2 O + CO 2 Thus if the acid HA is strongly dissociated, the effect of these trans- formations is to replace it by the exceedingly weakly dissociated car- bonic acid or by the neutral gas carbon dioxide. In a similar manner the Phosphates of the blood contribute to maintain neutrality by under- going the reaction : Na 2 HPO 4 + HA = NaH 2 PO 4 + NaA whereby the strongly dissociated acid HA is replaced by the faintly acid salt, monosodium phosphate. Finally the Protein Salts in the blood also assist in the preservation of neutrality by entering into reactions of the type: Na Protein + HA = H Protein + NaA the strong acid being in this instance replaced by practically neutral uncombined protein. Of these three agencies the bicarbonates are quantitatively much the most important. This arises from their abundance in plasma and also from the fact that the dissociation-constant of carbonic acid, or proportion of hydrogen ions to undissociated acid in the reaction of dissociation : H 2 CO 3 ^ H+ + HCO-3 is very nearly equal to the hydrogen ion concentration in distilled water at absolute neutrality (0.8 x 10~ 7 normal). Now L. J. Henderson has shown that the rate of change in the alkalinity or acidity of a solution of an acid when alkalies or acids are added to it is a minimum when the dissociation-constant of the acid is of this magnitude. He illustrates this principle by the following table, showing the amount of tenth normal alkali required to secure a definite but arbitrarily chosen change in alkalinity when added to equal amounts of the undermentioned acids : Dissociation-constant. Cubic centimeters of Acid. X10~ 7 alkali required. Phenol 0.0013 0.01 Boric acid 0.017 0.08 Hydrogen sulphide 0.57 1.10 Monosodium phosphate 2.0 1 . 00 Carbonic acid 3.0 0.72 Picolinic acid 18.0 0.10 Acetic acid .180.0 0.03 278 THE PROPERTIES OF PROTOPLASM The ability of sodium bicarbonate, in equilibrium with carbonic acid, to maintain the neutrality of its solutions is strikingly illustrated by Henderson in the following way : " Suppose, for example, a solution of 100 liters containing one kilogram of sodium' bicarbonate in equilibrium with an atmosphere containing one gram of carbon dioxide per liter. Let hydrochloric acid be added in successive small portions to the solution. Further, let the solution be constantly stirred and shaken, and let the experiment be conducted slowly, so that there shall always be equilibrium between the carbonic acid in the solution and in the atmosphere. Further, let the temperature be such that the absorption-coefficient of carbon dioxide shall be 1.000. Then the successive states of the solution will be approximately as recorded in the following table. HCl added, grams. Ratio of H 2 CO3 to NaHCOs. H + normal. OH normal. Relative acidity. Relative alkalinity. . 2. 27 to 11.90 0.000000057 0.000000176 0.57 1.76 10 . 2. 27 to 11.50 0.000000059 0.000000170 0.59 1.70 50 . 2. 27 to 10.00 0.000000068 0.000000147 0.68 1.47 100 . 2. 27 to 8.20 0.000000083 0.000000120 0.83 1.20 150 . 2. 27 to 6.30 0.000000108 0.000000093 1.08 0.93 ' 200 . 2. 27 to 4.40 0.000000154 0.000000065 1.54 0.65 250 . 2. 27 to 2.60 0.00000026 0.000000039 2.60 0.39 300 . 2. 27 to 0.68 0.0000010 0.000000010 10. 0.10 310 . 2.27to 0.31 0.0000022 0.0000000045 22. 0.045 318 . ex 0.00026 0.00000000039 260. 0.0039 320 . . 0.00045 0.00000000022 450. 0.0022 330 . . 0. 0027 0.000000000037 2700. 0.00037 "From the beginning of the experiment until almost 250 grams of hydrochloric acid have been added, neither alkalinity nor acidity is double in intensity the values which obtain in a perfectly neutral solution." "Such close approach to neutrality can be attained with pure water only after elaborate and very difficult purification, yet in the presence of carbonic acid it is the natural condition." In laboratory-glassware a mixture of disodium and monosodium Phosphates would perhaps be almost as efficient as sodium bicarbonate in preserving neutrality. In the body, however, they are not so efficient as the bicarbonates because in the first place they are not nearly so abundant and in the second place the elimination of the acid phosphates which are formed in the neutralization of acids has to take place by the relatively slow and roundabout channel of the kidneys, while the elimination of carbon dioxide takes place rapidly through ventilation from the lungs. Direct determinations by the potentiometric method have shown that the proteins contribute just about one-fifth of the neutraliz ing- power of the blood. In the tissues their proportional importance in maintaining neutrality is probably greater, because they are present therein in higher concentration than they are in the blood. NEUTRALITY OF THE TISSUES AND TISSUE-FLUIDS 279 Solutions such as those of sodium bicarbonate, disodium phosphate or sodium proteinate which conserve the neutrality of the water in which they are dissolved are very frequently designated "Buffer- solutions" from the resemblance of their action obliterating rapid changes of hydrogen concentration to the action of a buffer on a vehicle in obliterating dangerously sudden changes of velocity of motion. Buffer-solutions are frequently employed now, and must necessarily be more and more widely employed, wherever stable conditions of environment are requisite, as in bacterial cultures, cultures of living tissue in vitro, aquarium-media for marine or fresh-water organisms, and artificial circulating media. An estimation of the very greatest importance in all disease-condi- tions or metabolic disturbances which involve Acidosis is that of the Alkali-reserve or neutraliz ing-power of the blood. When large quanti- ties of "fixed" i.e., non- volatile acids are thrown into the blood the sodium with which they combine is rendered unavailable for neutraliz- ing other portions of acid or for binding carbon dioxide. The alkali- reserve in such cases is diminished and the ability of the blood to maintain its neutrality is proportionately impaired. A low alkali- reserve is therefore, in general, a relatively hazardous condition. Various methods have been proposed for measuring the alkali-reserve, the majority depending upon the fact that as the sodium bicarbonate of the blood has been diminished and the uncombined carbon dioxide stands in almost constant proportion to it, the carbon dioxide obtain- able from the blood by a standard procedure is diminished. The method suggested by Van Slyke, and now employed very widely, consists in taking a sample of blood from a vein in the forearm and introducing it into a vessel filled with the alveolar air of the patient obtained by breathing and rebreathing into the vessel several times. The blood is then shaken up with the alveolar air to bring it into equi- librium with the carbon dioxide contained therein and a measured sample, without loss of carbon dioxide, is introduced into a special form of gas-burette (Fig. 13) and acidified with sulphuric acid to decompose the bicarbonates. The chamber containing the sample is now evacuated by means of a column of mercury and the gas which is evolved is measured at atmospheric temperature and pressure. An alternative and perhaps preferable method which is, however, somewhat less simple to manipulate, consists in directly analyzing the carbon dioxide content of Alveolar Air obtained by rebreathing into a closed vessel. When the alkali-reserve is low, the carbon-dioxide content of the blood being diminished, the carbon-dioxide output through the lungs and the partial pressure of carbon dioxide in the alveolar air are correspondingly diminished. Another feasible method of measuring the alkali-reserve, or, which comes to the same thing, the Neutralizing-power of tissue-fluids is that which has been employed by Marshall in the analysis of Saliva. The 280 THE PROPERTIES OF PROTOPLASM Petition 1 \rnrn. bore Position 3 Is 6ocm below position FIG. 13. Van Slyke's apparatus for the determination of carbon dioxide in blood. (After Van Slyke.) NEUTRALITY OF THE TISSUES AND TISSUE-FLUIDS 281 various samples of saliva having been brought to a common reaction on the alkaline side of absolute neutrality (neutrality to phenolphtha- lein), the quantity of acid is estimated, by direct titration, which is necessary to bring the reaction of the fluid to an arbitrarily chosen reaction on the acid side of neutrality (neutrality to paranitrophenol) . The measurement is in fact analogous to that employed by Henderson in estimating the power of different acids to maintain the neutrality of their solutions. Of course, to obtain physiologically interpretable results with blood-serum it would be necessary to carry out the titra- tions under a standard partial pressure of carbon dioxide, for example in a vessel filled with alveolar air. The choice of indicators is limited when the fluid under investigation is even faintly tinged with color. For example the faint yellowish tinge of blood-serum interferes with the sharpness of the end-point with paranitrophenol. We thus see that by a variety of interlocking mechanisms, consisting in every instance of dynamic equilibria, the tissue-fluids of the higher animals, which are to their individual cells the external media in which they live, are kept extraordinarily constant in concentration, composi- tion of mineral constituents, and hydrogen ion concentration. The very great susceptibility of most of the chemical reactions which are involved in life-phenomena to slight changes of reaction, may very readily be seen to involve relative stability of reaction as a requisite to the orderly performance of life-processes. It is in fact an almost universal rule, in the words of Loeb, that " life-phenomena occur in a neutral liquid. " The ocean which is the original home of life, is, thanks to the presence of bicarbonates and phosphates, a "buffer"- solution and nearly neutral in reaction despite the life which swarms therein. According to Palitzsch the extreme variation in the hydrogen ion concentration of the ocean is from 1.1 x 10~ 8 N to 0.45 x 10~ 8 N H + , corresponding to an exceedingly faint alkalinity of the order of that found in the blood of mammals. In a very few instances only does life subsist in a medium which deviates far from neutrality. When secreting gastric juice, in the absence of neutralizing substances, the cells of the gastric mucosa are bathed on the side toward the lumen of the stomach by a fluid which may attain an acidity or hydrogen ion concentration due to hydrochloric acid of no less than one hundredth normal, or ten thousand times the acidity which would correspond to the alkalinity of pancreatic juice. The "salivary glands" of certain carnivorous molluscs, which probably correspond in function to the gastric glands in mammalia, similarly secrete an acid juice in which the high hydrogen ion concentration is attributable to Sulphuric Acid. With such rare exceptions, exhibited only by highly specialized and adapted cells, the immediate environment in which living matter subsists is extremely invariable in certain physical characteristics, and this invariability which is essential to the normal occurrence of life-phenomena, is brought about through the interplay of unique 282 THE PROPERTIES OF PROTOPLASM physical and chemical properties which are possessed by water and carbon dioxide. Even the additional stability of the environment of life which is brought about by the maintenance of constant temperature in the homoiothermal animals, is dependent upon the unique specific heat of water. The fitness of our environment for life is therefore essentially dependent upon these substances. As Henderson has pointed out, it is not that living matter has become adapted in an evolutionary sense to this medium, although specific organs concerned in the maintenance of the stability of the environment in higher organisms, such as the kidney, may have been subject to evolutionary adaptation. For the environment or the conditions from which it inevitably arose long antedated life itself, and the earliest forms of life must have been fitted to this environment no less exactly than the later. A direct chemical interrelationship between life phenomena and the particular type of environment in which they occur is thus indicated. It is somewhat idle to speculate whether or not life could subsist in some quite different environment with some other element such as silicon or boron as a base instead of carbon. Such "life," if it could correctly be so called, lies of necessity outside our experience. But the absolute dependence of life as we know it upon water in the liquid form and carbon dioxide in the gaseous form, renders the temperature limits between which life can subsist excessively narrow in comparison with the vast range of temperatures found in the various portions of our universe. Of the 6500 degrees which separate the temperature of interstellar space from that of the surface of the sun, only 65 or one per cent, of the total range is suitable for the occurrence of life-pheno- mena. In view of this exceedingly narrow margin upon which life precariously depends, the probability of its presence in any other of the bodies in our solar system must be regarded as exceedingly small. Concerning the possibility of life in other suns or planets which may be associated with them, we are of course in complete ignorance, but Arrhenius has put forward the interesting hypothesis that life may be transmissable, in the latent form which is embodied in bacterial spores, from one part of the universe to another in association with cosmic dust. Bacterial Spores have been experimentally shown to be exceed- ingly resistant to desiccation and low temperatures, retaining their ability to give rise to functionally active protoplasm so soon as they encounter a favorable environment. The computations of Arrhenius show that the known properties of certain bacterial spores are not inconsistent with the view that they might survive a journey through space, impelled by light-pressure, from one solar system to another. If this view be correct, then the existence of life in any part of the universe might sow the whole with seeds ready to develop at any mo- moment at which the environment of a particular cosmic body becomes suitable for the maintenance of the processes of functionally active life. NEUTRALITY OF THE TISSUES AND TISSUE-FLUIDS 283 REFERENCES. GENERAL: Hamburger: Osmotische Druck und lonenlehre. Wiesbaden, 1907. Hoeber: Physikalische Chemie der Zelle und der Gewebe: Leipzig. Fourth edition. Loeb, J.: Studies in General Physiology. Chicago, 1905. Loeb, J.: The Dynamics of Living Matter. New York, 1906. Philip: Physical Chemistry, its Bearing on Biology and Medicine. London,- 1914. Michaelis: Die Wasserstoffionen Konzentration. Berlin, 1914. OSMOTIC PRESSURE OF TISSUE-FLUIDS: Fano and Bottazzi: Arch. Ital. di Biol., 1896, 26, p. 45. Bottazzi and Ducceschi: Ibid., 1896, 26, p. 161. Bottazzi: Ibid., 1897, 28, p. 61. Arch, di Fisiol., 1906, 3, pp. 416 and 547. Bottazzi and Gabrieli: Arch. Int. de Physiol., 1905-6, 3, p. 156. Bottazzi: Ergeb. der Physiol., 1908, 7, p. 161. Atkins: Biochem. Jour., 1909, 4, p. 480. Jona: Ibid., 1912, 6, p. 130. OSMOTIC PRESSURE OF CELL-CONTENTS: Hedin: Skand. Arch. f. Physiol., 1895, 5, pp. 207, 238, 377. Pfliiger's Arch., 1897, 68, p. 229; 1898, 70, p. 525. Koeppe: Arch. f. Anat. u. Physiol., Abt., 1895, p. 154. COMPOSITION OF THE MINERAL CONSTITUENTS OF TISSUE-FLUIDS: Macallum: The Ancient Factors in the Relations between the Blood-plasma and the Kidneys, Trans, of the Coll. of Phys. of Philadelphia, 1917 NEUTRALITY OF THE TISSUES AND TISSUE-FLUIDS: Hoeber: Pfliiger's Arch., 1900, 81, p. 522; 1903, 99, p. 572. Friedcnthal: Zeit. f. allg. Physiol., 1902, 1, p. 56; 1904, 4, p. 44. Farkas: Arch. f. Anat. und Physiol., Abt. Supp., 1903, p. 517; Pfliiger's Arch., 1903, 98, p. 551. Fraenkel: Ibid., 1903, 96, p. 601. v. Szily: Ibid., 1906, 115, pp. 72 and 82. Robertson: Jour. Biol. Chem., 1909-10, 7, p. 351. Hasselbalch: Biochem. Zeit., 1910, 30, p. 317; 1913, 49, p. 451. Hasselbalch and Tundsgaard: Ibid., 1912, 38, p. 77. Henderson: The Fitness of the Environment, New York, 1913. METHOD OF ESTIMATING THE REACTION OF TISSUE-FLUIDS: Schmidt: University of California, Physiology Pub., 1909, 3, p. 101. Sorensen: Ergeb. d. Physiol., 1912, 12, p. 393. Sharp and Hoagland: Jour. Agr. Res., 1916, 7, p. 123. Clark andLubs: Jour. Bact., 1917, 2, pp. I, 109, 191. Schmidt and Hoagland: Table of PH, H + and OH~ values Corresponding to Electro- motive Forces Determined in Hydrogen Electrode Measurements, University of California Publications, Physiology, 1919, 5, p. 23 (consult for literatxire on gas-chain). Baker and Van Slyke: 1918, 35, p. 137. METHODS OF ESTIMATING THE ALKALI-RESERVE: Henderson: The Excretion of Acid in Health and Disease, Harvey Lectures, 1 Oth Ser., Philadelphia, 1914-15, p. 132. Levy and Rowntree: Arch. Int. Med., 1916, 17, p. 525. Sellards: The Principles of Acidosis and Clinical Methods for its Study. Cam- bridge, Mass., 1917. Van Slyke: Jour. Biol. Chem., 1917, 30, pp. 289, 347. CHAPTER XIII. PROPERTIES CONFERRED BY THE COLLOIDAL CON- STITUENTS: STRUCTURE AND CONSISTENCY. THE EMULSION-STRUCTURE OF PROTOPLASM. One of the most important aspects of the relationship of the Lipoids to the properties and behavior of protoplasm is that arising out of the marked effect upon the tension of protoplasmic surfaces which the lipoids and their decomposition-products are capable of bringing about. The Surface-tension of the interface between water and gas, or an immiscible fluid or solid, is very markedly reduced by Oils, Fatty Acids or Soaps, and this fact contributes in the first place to the deter- mination of the distribution of these substances in the cell, and in the second place to the stability of the emulsified substances in living cells, which, despite their immiscibility in water, remain suspended in the form of stable emulsions within the material composing the protoplasm. The distribution of soluble fatty materials in the cell, such as the Lecithins must be considerably influenced by the extent and variety of the surfaces which are presented by the sponge- or foam-like structure of protoplasm. The reason for this is that all those substances which reduce superficial tensions also tend, if possible, to become concen- trated upon any surfaces presented to them. This is very strikingly shown, for example, in a classical experiment adduced by J. J. Thomson. If a deeply colored solution of Potassium Permanganate be passed through a long column of well washed and finely ground quartz-sand, the first few drops of fluid which percolate through the column will be found to be colorless, the whole of the permanganate in this first quantum having been abstracted from the solution by the surfaces over which it has passed, not because of any chemical interaction between the sand and the reagent, but in consequence of the reduction of the tension of the water-sand interface by the permanganate. Similarly, if aqueous solutions of Saponins or of Bile-salts be shaken up with petroleum-oils, the dissolved substance will be found to have become concentrated at the surface of the oil-drops, and in the foam which forms when saponin solutions are shaken in air, the saponin is more concentrated than it is in the body of the liquid. The mechanism of this retention of dissolved substances by sur- faces is as follows: In the accompanying diagram (Fig. 14) of a spherical droplet partially enclosed by a layer of molecules which coat it and separate it excepting at the gap A from direct contact with the surrounding medium, if the enveloping molecules reduce the EMULSION-STRUCTURE OF PROTOPLASM 285 tension of the interface between the drop and the medium in which it is suspended, it is evident that the tension of the exposed gap in the surface will be greater than the tension of the covered portions of the surface. The two portions of the surface will be pulling unequally, therefore, and unbalanced excess of tension will exist at the gap in the sense indicated by the arrows, and the tendency of this tension in the case of a gap of molecular dimensions will obviously be to draw together the edges of the enveloping film and reduce the tension of all parts of the surface to a uniform value. Any molecules of such a substance coming into contact with the surface will therefore tend to be held or "trapped" there, and since, in the course of the fortuitous motions of the dissolved molecules, a very large number must repeatedly come into contact with any surfaces exposed to a solution, the accumulation of adhering molecules will continue until the droplet becomes covered by a layer of such thickness that the molecular attraction between the underlying molecules of the drop and those of the surrounding medium becomes inappreciable owing to the distance through which it has to be exerted. This will occur when the thickness of the film is of the order of that of a soap-bubble just before it bursts, namely about one two millionth of a millimeter. A FIG. 14. Illustrating the tendency of a lipoidal layer at the interface of two aqueous phases to repair itself when broken. The extent to which the Surface-tension of water is reduced by lipoidal substances and soaps may be inferred from the following results reported by Lord Rayleigh. The measurements refer to an air-water interface. Dynes per linear c.m. Tension of pure water 1 74 Tension of greasy water 33 Tension of water saturated with olive oil 41 Tension of water saturated with sodium oleate 25 The trace of Olive Oil which dissolves in water, therefore, reduces the tension of the air-water interface to one-half its normal magnitude. The lipoids in the cell are present partly in soluble forms, such as ^The tension of the air- water interface is usually considered to be 81 dynes per square centimeter which is the estimate of Quincke. The estimate of Lord Rayleigh who employed very refined method of measurement is more probably correct. In any case, all of the estimates having been made by the same method, they are comparable with one another. 286 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS the Lecithins, partly in a very finely emulsified form, the individual particles being of ultramicroscopic dimensions, and partly in a coarsely emulsified form such as that found in fatty connective tissues. The presence of a large proportion of ultramicroscopically divided fat is shown by the high fat-content of many tissues in which microscopical examination after appropriate staining fails to reveal the presence of visible fat-globules. Under certain conditions, especially in Phosphorus- poisoning and in Anaphylactic Shock, the ultramicroscopic particles in certain tissues coalesce to form coarse emulsions and the particular tissues affected, as for example the liver in phosphorus-poisoning, are then easily seen to be heavily infiltrated with fat. Direct analysis, has shown that in such cases the fat-content of the tissues is neverthe- less normal, in other words the normal liver-cell contains just as much fat as the liver-cell which has undergone fatty degeneration in conse- quence of phosphorus-poisoning, but in the normal cell the Emulsifica- tion of the fat is so thorough that the greater part of the fat is present in particles too small to be visible under the microscope. The soluble lipoids and the soaps and other substances which reduce the tension of an oil-water interface are probably in large proportion concentrated at the extensive surfaces which arise from this subdivision. The Emulsification of fats in water is greatly facilitated by the presence in the water of a substance which reduces the interfacial tension, provided that at the same time the substance forms a viscous or sparingly soluble coating over the oil-droplets which retards their coalescence when they come fortuitously into contact with one another. We have already had occasion to dwell upon the importance of soaps and of the bile-salts in bringing about the emulsification of the fats in the diet prior to their hydrolysis by the digestive enzymes. When olive oil is shaken up with pure water little or no emulsification occurs. Even when the mixture has been very thoroughly shaken, the oil and water separate completely within a comparatively brief period. If, however, a little sodium carbonate or hydroxide be added to the water in order to form soap with the trace of fatty acid which oil contains, the effect of shaking the mixture is now very different. A milky or creamy emulsion is formed with comparatively little expendi- ture of mechanical effort in shaking, and no separation of the two fluids will occur even after long intervals of time. The emulsifying action of alkalies is also strikingly illustrated by floating drops of Olive Oil upon distilled water and one per cent, sodium carbonate solution respectively. In the latter case the oil-droplet spreads out, fluctuations of superficial tension at the edges of the drop cause deformations, and result in a species of "fraying" of the edges, minute particles of the oil breaking off to form a milky emulsion which gradually spreads through the solution. In the Emulsions of oil in water which are thus formed the spherical droplets of oil are surrounded and completely enveloped by the water. The power of a given quantity of water to surround oil must evidently EMULSION-STRUCTURE OF PROTOPLASM 287 be limited, however, for no matter how tightly packed the particles of oil may be, the thickness of the layer of water between them cannot be of less than molecular dimensions at its thinnest, and must of course be much greater in the interstices of the emulsion. In other words a limited quantity of water cannot emulsify an unlimited quantity of oil and, as a matter of fact, when a given quantity of oil is shaken with varying proportions of alkaline water, if the volume of water is below a certain critical fraction of the volume of the oil, the character of the emulsion which is obtained is altered altogether, and we now have emulsions of Water in Oil. A convenient method of symbolically representing these differing types of emulsions is to enclose the Internal Phase of the emulsion in brackets. Thus an emulsion of oil in water would be designated: water (oil) while an emulsion of water in oil would be distinguished by the symbol : (water) oil When the proportion of water to oil is in the neighborhood of the critical ratio, complex intermediate forms of emulsion may be encoun- tered, such as emulsions of oil in water emulsified in oil, thus : ((oil) water) oil It is, in fact, highly probable that the majority of emulsions of water in oil are really of this more complex type. The character of an emulsion obtained by shaking together olive oil and alkaline water may very readily be ascertained without micro- scopical examination by the simple device of sprinkling upon the surface of the emulsion a little finely powdered Sudan III or Scarlet R. These dyes are soluble in oil, but insoluble in water. Hence if they are sprinkled upon the surface of an emulsion of oil in water, the dye simply dissolves in and stains the drops of oil with which it comes into actual contact, leaving the remainder of the emulsion unstained. If, however, the emulsion is one in which water is the internal phase and oil the external, the dye dissolves in the interstitial oil and spreads over the surface of the emulsion. The following are illustrative results obtained by shaking together olive oil and water- at an approximately uniform rate of shaking, and in the presence of a fixed proportion of alkali : Components of emulsion. Oil Water 5N.NaOH c.c. c.c. c.c. Character of the emulsion obtained. (Water) oil; fluid, yellow. (Water) oil; fluid, yellow. (Water) oil ; fluid, yellow. (Water) oil; fluid, creamy. (Water) oil; fluid, creamy. Water (oil) ; white, very viscous. 98 1 96 3 92 7 91 8 90 9 89 10 Water (oil) ; white, very viscous. The critical ratio was in this instance: ^ oil 90.5 288 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS The value of the critical ratio varies with different samples of oil, because of their varying fatty-acid content. It also varies with the proportion of alkali employed, since if the quantity of soap be insuffi- cient to surround all the droplets with a layer of molecular thickness the stable emulsification of the whole of a large excess of oil becomes an impossibility, and the critical ratio is increased. It will be observed that upon passing the critical ratio the char- acteristics of the emulsion change very markedly. Instead of the yellow, fluid emulsions obtained while water is the internal phase, creamy and more viscous emulsions result when water is the external phase. In the neighborhood of the critical ratio the viscosity of the water (oil) emulsions is very greatly enhanced, and emulsions of an almost butter-like consistency may be obtained. This probably arises from the fact that when the water in the emulsion is just, and only just sufficient to surround all of the oil-droplets, any deformation whatever of the tightly-packed oil-droplets must increase the size of the interstices between them; but this can only be accomplished by a complete disrup- tion and inversion of the emulsion, since the water is already stretched to the utmost limit of its covering-power. The viscosity, or resist- ance to deformation of these emulsions, therefore, represents the force required to invert their structure. Not only the lipoid constituents of cells, but also the Proteins tend to form films at the surfaces of suspended droplets, and thus facilitate the formation of emulsions. If Chloroform be shaken up with pure distilled water no emulsion arises; the two liquids separating com- pletely after a very brief interval. If, however, a protein be added to the water the chloroform, instead of separating out in the form of a continuous layer, separates out in small discrete droplets which, if numerous, form a milky layer at the bottom of the vessel; by trans- mitted light, however, they appear perfectly transparent. These droplets are extraordinarily stable and do not coalesce, however long they may stand in contact with one another. They may be repeatedly washed in water until all traces of protein have disappeared from the wash-fluid, and they still remain perfectly stable and distinct from one another. They may be shaken up in chloroform itself or treated with dilute sodium hydroxide solution without impairing their form or stability. If, however, they be heated to nearly the boiling-point of chloroform under a layer of water the droplets burst and coalesce, forming a homogeneous layer of chloroform. If treated with alcohol they immediately dissolve leaving a fine membranous precipitate of protein floating in the water. Thus if we shake up chloroform with about twice its volume of a one per cent, solution of Protamine Sulphate or a one per cent, solution of Gelatin, and, after allowing the droplets to settle, pour off the supernatant fluid and repeatedly wash the drop- lets with water, then if we suspend these droplets in a small amount of water and add to the water an equal volume of Alcohol and gently shake the test-tube, the droplets which are thus stirred up into the EMULSION-STRUCTURE OF PROTOPLASM 289 ' alcohol-water layer can be seen to swell up rapidly and burst, and the fine membranes which surrounded them can then be seen falling down through the alcohol-water. If we now add several volumes of alcohol and shake up the liquid, the chloroform droplets all disappear and what we now have is a clear, homogeneous solution, in which innumerable minute membranes can be clearly seen floating. The phenomena of Relative Semipermeability may also be illustrated by these droplets. Substances which are soluble in water and also in chloroform penetrate the membranes, and if, like Alcohol, Ether or Ethyl Acetate they chance to be more soluble in chloroform than in water, the chloroform in the droplets may take up so much of the sub- stance that they swell to the extent of rupturing their enveloping membranes. If, however, the substances in which the droplets are immersed are sufficiently insoluble in water they fail to penetrate the membranes and then the droplets may be " plasmolyzed," that is, the chloroform may be extracted from them leaving the enveloping membranes shrunken and empty. This occurs when the droplets are suspended in Toluol, Xylol or Carbon Bisulphide. Fat emulsions which contain protein tend to form films at surfaces with which they come in contact, consisting of a more concentrated emulsion, both in respect to fat and in respect to protein, than that which constitutes the body of the liquid. This is very well illustrated by the film which forms on the surface of Milk when it is heated. The heating of the milk renders the Calcium Caseinate which it contains somewhat less soluble, and the concentrated layer of calcium caseinate and fat particles which forms at the surface becomes, at temperatures above 45, sufficiently viscous to assume the consistency of a semi-solid film, which, owing to its high viscosity, does not readily pass back into solution upon cooling. A pure solution of calcium caseinate becomes markedly opalescent on heating to 45 but does not form a sufficiently viscous film at its surface to be mechanically separable from the underlying liquid. A living cell consists essentially of a more or less finely emulsified suspension of fat-like substances in a semi-gelatinous solution of pro- tein. The film which forms at the surface of warm milk may be regarded as an extreme illustration of the type of surface-layer which we may therefore expect to exist at the periphery of living cells, namely an emulsion of fat and protein, more concentrated and, therefore, more viscous than the emulsion which constitutes the underlying protoplasm. The emulsion-structure of the superficial layer in cells enables us to account for a very widespread property of living cells which would otherwise be almost inexplicable, namely the property of One-sided Permeability. This phenomenon is very well illustrated by the follow- ing experiment of Overton's: If tadpoles are immersed in a five or six per cent, solution of cane-sugar or a 0.6 per cent, solution of sodium chloride they are unaffected either in size or in any other notable 19 290 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS respect. If, however, they are immersed in solutions which are hypertonic to these, for example in eight per cent, cane-sugar or 0.8 per cent, sodium chloride, they lose a quantity of water and in twenty- four hours they are found to have shrunk decidedly in volume. Evi- dently, then, the sugar or salt cannot enter the limiting membranes of the cells of the skin while water can pass through them freely in the direction tissues -> external medium. One might imagine, therefore, that the epithelium of a tadpole resembles an ordinary semipermeable membrane, permitting the passage of water but not of dissolved substances. If this were really the case, then on immersing the tad- poles in solutions which are hypotonic to 0.6 sodium chloride we should expect them to take up water and to increase in volume just as much as they decrease in volume in hypertonic solutions. This does not occur, however, and tadpoles immersed in hypotonic solutions do not take up water to any greater extent than from isotonic solutions. We can only infer, therefore, that the superficial epithelium of the tadpole permits the passage of water from within outward, but not in the reverse direction; that this membrane is permeable to water in the direction tissues -> external medium, but not in the direction external medium -> tissues. The property of one-sided permeability is displayed by many living membranes, but not by all. A very striking contrast is shown in this respect by the Pavement Epithelium which lines the peritoneal cavity, on the one hand, and the Columnar Epithelium which lines the lumen of the small intestine, on the other. Thus Heidenhain introduced 50 c.c. of a three per cent, solution of Glucose into the peritoneal cavity of a dog, and at the same time 44 c.c. of the same solution into an isolated loop of the intestine. After ninety minutes the quantity and composi- tion of the residual fluid in the peritoneal cavity were as follows : Quantity of fluid. Glucose. Sodium chloride. 19.5 c.c. 1 . per cent. . 55 per cent. while in twenty-five minutes the composition of the residual fluid in the loop of intestine was as follows : Quantity of fluid. Glucose. Sodium chloride. 19 . c.c. 3 . 8 per cent. . 04 per cent. From the peritoneal cavity both water and glucose had issued into the tissue-fluids, the glucose even more rapidly than the water, while sodium chloride, which was absent from the fluid originally introduced, had diffused from the tissues into the peritoneal cavity. The peri- toneal epithelium, therefore, behaved like a membrane of parchment, permitting the passage of dissolved substances in either direction in proportion to their relative concentrations on the two sides of the membrane. From the intestinal loop, both water and glucose had issued into the tissue-fluids, water somewhat more rapidly than glucose. But prac- tically no sodium chloride had diffused into the intestinal fluid from the EMULSION-STRUCTURE OF PROTOPLASM 291 tissue-fluids. Evidently the intestinal epithelium permits the passage of certain dissolved substances into the tissue-fluids behind it, but not the migration of dissolved substances in the reverse direction. The maintenance of one-sided permeability in tissues is dependent upon the maintenance of the unimpaired structure of the cells. Thus the phenomenon of one-sided permeability is nowhere more clearly illustrated than it is in the kidneys, where the dissolved constituents of Urine are constantly excreted against a high pressure, the tissues of the kidney being much more permeable for dissolved substances in the direction blood - urine than in the direction urine - blood. If, how- ever, the epithelium of the renal tubules is injured by perfusion with solutions of certain substances, for example Sodium Fluoride, it loses this power and comes to resemble much more closely a membrane of parchment. This is very clearly illustrated by the following experi- ment by Bottazzi. One kidney in a dog was injured by perfusion with sodium fluoride solution. The ureters of the two kidneys were then separately catheterized and the freezing-points of the samples of urine collected from the two kidneys were determined from time to time. The following were illustrative results : Depression of Depression of Urine c.c.: freezing-point. freezing-point Time. .. . of blood. Normal. Injured. Normal. Injured. 2.30 to 3.00 3.30 to 4.00 4.50 to 5.20 5. 30 to 6.00 6.00 to 6.30 9.30 to 10.00 8.00 to 8.30 9. 12 1.616 0.979 0.572 14. 20 1.118 0.294 14. 22 0.584 0.240 10. 22 0.570 0.224 12. 20 0.572 0.212 0.560 4. 9 1.002 0.206 2.5 6 1.304 0.302 0.569 It is evident that the phenomenon of one-sided permeability must be dependent upon a heterogeneous structure of the membrane which displays it. The phenomenon is not, and could not be displayed by structureless membranes, or by membranes having a uniform structure in the direction of penetration, i. e., perpendicularly to their surface. For instance, consider a membrane formed of successive columns, of two different materials, one of which permits the passage of substances soluble in water while the other does not. Then if the arrangement of these two components were th,at displayed in Fig. 15 substances soluble in water could penetrate the unshaded channels just as easily from below as from above the membrane. But if the membrane were curved, so as to bring the columns of impenetrable material closer together on the under than on the upper surface, or if they were pyramidal in shape so as to achieve the same end, so that the arrange- ment would be that displayed in Fig. 16 then it is evident that the penetrable area on the under surface of the membrane would be a much smaller proportion of the whole area than on the upper surface of the membrane, so that substances penetrating from above would do so with comparative ease, while substances issuing from below the membrane would do so with difficulty. 292 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS Now the lipoid constituents of the cell may be assumed to be gener- ally impenetrable by substances which are insoluble in fats and oils, so that these must seek entry to the cell through the interstitial spaces between the lipoid constituents of the superficial membrane of the cell. If, therefore, these intersitital spaces filled with a solution or gel of protein, were so constructed as to be narrower at one end than at the other, the superficial membrane of the cell would evidently be more readily permeable in one direction than in the opposite. The most usual spatial arrangement of the various structures or constituents of a cell is that of Radial Symmetry. The primitive arrangement of strictly radial symmetry so frequently displayed in spherical cells becomes modified or distorted in those cells, such as the majority of epithelial cells, which, through mutual compression or for other reasons, have assumed a columnar, stratified or flattened outline. In such cases the radial arrangement of structures may be confined to the sides or margins of the cell, and differ in character in the protoplasm underlying the various facets of the cell. FIG. 15 FIG. 16 FIGS. 15 and 16. These figures illustrate the effect of curvature of the surface of a radially dispersed emulsion in producing funnel-shaped interstitial pores between the radiating columns of lipoid globules. In Fig. 15 both surfaces of the cortical layer being plane, the diameter of the interstitial orifices are the same at the exterior and interior surfaces. In Fig. 16 the cortical layer being curved, the interstitial orifices are narrower upon the un der than upon the upper surface of the cortical layer. A radial arrangement of the ultramicroscopic fat-granules of the cell would obviously lead to the formation upon the surface and in the subjacent protoplasm of minute Funnel-shaped Pores, of which the interstitial openings would be permeable to substances soluble in water, while the walls, being composed of fat-granules, would be impermeable or with difficulty permeable by such substances. The interstitial openings at the margin distal from the center, from which the fat- granules radiate, would be relatively large, while at points lying nearer to the center of radiation, that is, in general, deeper within the cell, the diameter of the pores would be very considerably contracted. Substances which are soluble in water might evidently penetrate such a cell with relative ease, since a relatively large proportion of the exterior cell-surface would consist of the water-soluble phase of the emulsion, but they would issue from the interior of the cell with relative difficulty, since a relatively large proportion of the area which they would have to traverse to find an outlet would consist of the lipoidal phase. If the modifications of radial symmetry which are so characteristic of Epithelial Cells should result in the confinement of this structure to one surface or facet of the cell, it is obviously possible that one-sided permeability of a tissue composed of such cells might EMULSION-STRUCTURE OF PROTOPLASM 293 be the consequence. This may be seen by reference to the diagram in Fig. 16. It should be borne in mind that the existence of funnel-shaped pores in the surface of a cell or in a membrane would only give rise to one- sided permeability provided the diameter of the pore at the constricted end were comparable with the mean free path of the penetrating mole- cules. Were the least diameter of the pores less than the mean free path of the pentrating molecule, then the membrane would be a strictly semipermeable membrane for this type of molecule. Were the least diameter of the pores on the contrary, very large in comparison with the mean free path of the molecule concerned, then the membrane would be freely permeable by this molecule in either direction. Thus it is readily conceivable that membranes of this type might exhibit One-sided Permeability for certain substances dissolved in water, Absolute Permeability for others, and Semipermeability for yet other molecules. On the other hand, if the above sketch represents truly the structure of the superficial layer of cells, substances which are soluble in fats would enter the cell through the radiating columns of lipoidal material. Now the phenomenon of one-sided permeability has not as yet been observed to be displayed toward substances which are soluble in lipoids. Indeed, in general, the penetrability of cells by substances which are soluble in lipoids is very much greater than their penetra- bility by other substances, no matter how soluble they may be in water. This fact is very strikingly illustrated by the experiments of Overton and Meyer, who measured the minimal concentrations of various Narcotics dissolved in water which would induce narcosis in tadpoles, the narcosis being evidenced by cessation of movement. The same narcotics were dissolved in water and the water shaken up in Olive Oil and the relative solubilities of the narcotics in water and in oil estimated by the distribution of the narcotic between the two solvents. The following were illustrative results obtained with various Alcohols : Solubility in water. Narcotic. molecules per liter. Solubility in oil. Methyl alcohol . . . . 0.52-0.62 Solubility in water = OC solublein50 parts of oil. Ethyl alcohol .... 0.27-0.31 30 : 1 Propyl alcohol .... 0.11 8:1 Butyl alcohol .... 0.038 Soluble in 12 parts of solubility in water; oil = OC . Caprylic alcohol . . . 0.0004 Soluble in 2000 parts of solubility in water; oil = (X . The following results were obtained with other narcotics: ' Solubility in oil. Critical concentration. Solubility in water. Narcotic. . ------ - -- - - At 3. At 30. At 3. At 30. Salicylamide .... Vwoo i/ 600 22.232 14.002 Benzamide ..... l /5oo Yaoo 0.672 0.437 Monacetin ..... Vo l /io 0.099 0.066 Ethyl alcohol ..*../ V 0-026 0.047 Chloral hydrate . . . VBO V*o 0.053 0.236 Acetone ...... J /3 VT 0.146 0.235 294 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS Hence if tadpoles, anesthetized at 30 by a 2 f solution of Chloral Hydrate be cooled they recover their mobility, on warming they are again anesthetized, and at 30 the solubility of chloral hydrate in olive oil is much greater than it is at lower temperatures. While the inference drawn by Overton and Meyer from these experi- ments, that only those substances which are soluble in Lipoids can penetrate the cell, obviously cannot be substantiated, for otherwise neither water, inorganic salts nor amino-acids could ever gain entry into protoplasm, yet it is very manifest from these and many other experi- ments of a like nature that substances which are soluble in lipoids do enter living cells with exceptional ease. We may probably infer with safety that the lipoidal elements in the superficial membranes of cells constitute a large proportion of the surface, and the interstices a rela- tively small proportion, so that substances which are insoluble in lipoids enter living cells with comparative difficulty FIG. 17. Illustrating the increase in the diameter of the interstitial spaces which results from increase in the diameter of the fat-droplets in an emulsion. Any reagent or condition which affects the State of Aggregation of the fat-globules in the limiting membrane of cells must necessarily affect the diameter of the interstices between them. In general those conditions involving the formation of large aggregates would increase the permeability of tissue by enlarging the diameter of the radiating fat-droplets and, therefore, that of the interstitial spaces (Fig. 17). This is very strikingly illustrated by the eggs of certain marine forms such as the sea-urchin which, when exposed to the action of fat-solvents become permeable to water which they take up from the surrounding sea-water. The water thus absorbed accumulates in a layer just under the superficial membrane of the cell, lifting it off the underlying pro- toplasm and forming the "Fertilization Membrane" which is normally the effect of a cytolytic agent carried into the egg-cell by the head of the spermatozoon. TJie permeability of the surface of the cell is also increased for inorganic salts, for McClendon has shown that the Electrical Conductivity of a suspension of sea-urchin eggs is increased by fertilization while Osterhout has shown that an increase in the electrical conductivity of living tissues is indicative of increased per- meability of the surface of the cell for inorganic salts. Since the lipoidal droplets in cells are suspended in a gelatinous or VISCOSITY OF PROTOPLASM 295 semi-liquid solution of Protein we may also assume that any condition tending to alter the consistency of the interstitial protein solution would deform the structure of the emulsion. Coagulating Agents especially might be expected to reduce the interstitial gel to discrete granules or separate flocculi, thus removing the obstacle to coalescence of the fat globules and the consequent coarsening of structure and widening of interstices. Corresponding to this conception we find that simple heating of a piece of frog's skin renders it freely permeable to water in either direction, instead of only in one. The effect of coagulating agents' upon permeability may also be strikingly illustrated in the following way: If paramcecia be washed free from culture medium with pure distilled water and suspended in a solution of Methyl Green (free from methyl violet), the protoplasm of the infusorians takes on a faint greenish tinge, but the large pseudo-nucleus remains white and unstained. After removing the excess of methyl green by washing the organisms in distilled water, a little Cupric Chloride may now be added to the water. Immediately the nucleus becomes stained a deep green, indicating that the impenetrability of the nuclear membrane for the dye prevents it from being stained in the normal cell, but after the action of this protein coagulant, which kills the organisms, the permea- bility of the nuclear membrane is so enhanced that the dye can readily enter and even attain a greater concentration therein than it does in the cytoplasm. THE VISCOSITY OF PROTOPLASM. The major part of the high degree of Viscosity which protoplasm displays is attributable to the Protein which it contains. The viscosity of a protein solution increases very rapidly indeed with its concentra- tion, so rapidly, in fact, that earlier observers were inclined to the belief that the viscosity changed suddenly at definite critical con- centrations instead of changing evenly and with regularity as it does in solutions of crystalloids. Later observations have shown us, how- ever, that the viscosity of protein solutions increases with the concen- tration in accordance with the usual formula ~ = A n , where 77 is the viscosity of the solutiorr, 170 that of the solvent, n the concentration of the solution and A a constant, the numerical value of which depends upon the nature of the dissolved substance, and upon the temperature. The following are results obtained by Sackur, employing Sodium Caseinate : n (in equivalents - (15 C.). logio A. of sodium) . % 0.01830 1.870 14.8 0.01370 1.581 14.5 0.00915 1.363 14.3 0.00547 1.202 14.6 0.00458 , 1.165 14.5 296 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS A remarkable feature of these results is the extraordinarily high value of A, involving a very rapid increase of viscosity with increasing concentration. For the majority of crystalloids the value of A is not greatly in excess of unity, while for sodium caseinate it is of the Order of 10 14 . This fact alone would lead us to suspect that the mechanism which produces the viscosity of these solutions is different in nature from that which produces the viscosity of solutions of crystalloids. The viscosity of a protein solution is also very greatly increased by lonization, the viscosity of protein solutions being at a minimum when ionic protein is absent, i. e., when the protein is uncombined with acids or bases. Indeed a very little consideration suffices to show that the viscosity of a protein solution is of a very different type from the viscosity, for example, of solutions of Sugar or Glycerol in water. Apart from the extraordinary magnitude of A, the type of viscosity exhibited by solutions of proteins differs from the viscosity of a glycerol-water mixture in that it affords no hindrance, or very slight hindrance, to the motion of ions and of crystalloidal molecules. The velocities with which various crystalloids diffuse through Gelatin jellies are remarkably close to the diffusion-velocities of the same substances in distilled water. The jelly causes a very slight retardation of diffusion but the hindrance to molecular movement is disproportionately small in comparison with the enormous viscosity of the jellies. It has repeatedly been shown that the specific mobilities of the majority of inorganic ions is the same in gelatin or agar jellies as it is in distilled water. In fact, if allowance be made for the diminution of the cross-section of the conducting field which is occasioned by the presence of gelatin molecules we find that the electrical conductivities of inorganic salt solutions in gelatin jellies are only very slightly less than those of equally concentrated solutions in pure water, implying that the ions of the electrolyte move as freely in the insterstices between the protein molecules as they would move in distilled water. This is true even when the ions are protein ions, for the dependence of the Electrical Conductivity of protein solutions upon their dilution is of a perfectly normal character, resembling the dependence of the conduc- tivity of a solution of a crystalloid upon dilution, although, in the range of concentrations employed, the viscosity of the protein solution increases with its concentration very greatly, while that of a salt solution, for example, increases almost imperceptibly. On the other hand the intimate dependence of the conductivities of solutions of electrolytes upon the ordinary types of viscosity has been commented upon, and quantitatively estimated by a host of observers. Viscosities, very much less than those of the most dilute Jellies, if caused by such substances as sugar or glycerol, profoundly diminish the conductive power of electrolytes. Not only inorganic, but also protein ions are very greatly hindered in their mobilities by the type of viscousness which alcohol-water or glycerol-water mixtures VISCOSITY OF PROTOPLASM 297 exhibit. In fact, whereas doubling the viscosity of a solution of Sodium Caseinate by the addition of protein does not measurably affect its conductivity, doubling its viscosity by the addition of forty per cent, of alcohol reduces the mobility of the caseinate ions to one-half, and the conductivity of the solution to a still smaller proportion. In estimating the influence of viscosity upon the mobilities of protein ions we can entirely disregard that portion of the viscosity of the solution which, although comparable in magnitude with the viscosity of the solvent, is attributable to the protein itself. There are thus two kinds of viscosity which may be displayed by solutions, the one which impedes the motion of molecules or ions, and the other which does not hinder the motion of such small particles, although it does very greatly impede the passage of the fluid through a narrow tube or the rate of oscillation of a rotating disc suspended within the fluid. The former type of viscosity is displayed by solutions of inorganic substances and the simpler organic substances, the latter type of viscosity by solutions of the proteins. The customary method of measuring viscosity, such as the measure- ment of the time taken by a given volume of fluid to pass, under the force of gravity, through a specified length of a narrow tube, all involve deformation of the fluid, whereas the estimation of viscosity which depends upon the diffusion of molecules or ions through it, does not require any displacement of the particles of the solvent in which the diffusion is occurring. Deformation is especially resisted by protein solutions, but internal molecular motions are not impeded. This fact strongly suggests the existence of a Structure within solutions of the proteins. It appears very probable that the molecules of protein in solution are loosely connected with one another so as to form a mesh- work or three-dimensional net throughout the body of the solution. Such a net, which, in two-dimensional section, we may picture as some- thing analogous to a tennis-net with microscopic or ultramicroscopic meshes, would offer no hindrance to the passage through it of a quickly- moving body which is much smaller than its meshes, but to any force involving deformation of its structure, for instance to a force tending to drag it through a small tube, it would offer a very considerable resistance. In measuring the resistance which a protein solution offers to passage through a capillary tube, we are not measuring true viscosity or internal friction between adjacent molecules, therefore, but the resistance of the structure of the solution to deformation. A common method of measuring the viscosity of fluid consists in suspending a disc within the fluid and causing it to oscillate, the decrease of the rate of oscillation being a measure of the viscosity. When this method is applied to protein solutions, however, it is found that the decrease in the rate of oscillation of the disc is abnormally rapid, but if the liquid be slightly shaken or the disc taken out and replaced, the decrease in the rate of oscillation becomes normal again for a brief period. Evidently the protein network adheres to the disc, 298 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS so that, in the course of time, the motion of the disc is not merely opposed by the friction of immediately adjacent molecules, but by the inertia of all of the protein molecules of the fluid which are attached indirectly, through a continuous meshwork, to the oscillating disc. JELLIES AND GELATINIZATION. The structure which confers upon protein solutions their peculiar type of Viscosity leads in many cases when the solutions are sufficiently concentrated to their acquiring certain of the properties of solids. Such solutions are what we term Jellies, and they resemble solids in presenting pronounced resistance to deformation which, however, yields to the slightest force if its action be sufficiently prolonged. Where forces of an instantaneous character are concerned, therefore, the jellies are solids, but where forces of prolonged action are concerned they are fluids. The distinction between a solid and a jelly is, in fact, largely a matter of degree. A solid will flow under a sufficiently great pressure applied for a relatively brief period of time, but a sharp impact affects it as it affects an elastic solid, causing oscillation and recoil, but not deformation. Intermediate states of matter are afforded by such materials as Sealing-wax which, even at ordinary temperatures, will flow under slight pressure applied for very prolonged periods, but which under even considerable forces acting sufficiently suddenly exhibits all the brittleness of a solid. Under certain conditions, when the meshes are sufficiently coarse, various jellies or "gels" clearly display a network or spongy structure. If an insoluble gel, such as White of Egg coagulated by fixatives, the gel of Colloidion produced by the action of chloroform upon an ether solution, common black India-rubber, or the hydrogel of Silica be examined under high magnification they can all be seen to possess a fine sponge-like structure. When, for example, a thirteen per cent, solution of egg-white is fixed with sublimate, sections are found to show a sponge- structure, or, what corresponds to a sponge in two dimensions, a network-structure. W. B. Hardy, who has especially investigated this gel, failed to obtain with acid or basic dyes any staining of the substance within the meshes of the net, and pressure applied to the gel resulted in the squeezing of fluid out of its interstices. The structure of the gel is, therefore, that of an open sponge-work of solid, containing fluid within its meshes. Direct experimentation with A gar jellies has shown that in a gel containing one r>er cent, of agar, the solid framework is a solution of water in agar, while the fluid in the interstices is a dilute solution of agar in water. Upon heating the solution the two components become miscible in each other and we obtain what appears to be a homogeneous solution. Upon the basis of these facts Hardy draws a far-reaching analogy between the jellies which liquefy when heated, and gel when cooled, and the system Phenol-water, which, if it contains more than 71 per cent, or less than JELLIES AND GELATINIZATION 299 76 per cent, of phenol, separates, at temperatures below 80 C., into two phases, the one a solution of phenol in water, the other a solution of water in phenol. According to the view developed by Hardy the two cases differ only in the fact that upon separation of the two phases in the agar-water system the system retains a structure, while in the phenol-water system no structure is retained and the components separate into two clearly demarcated layers. Essentially, the dif- ference between the two systems consists in this : that when the phenol- water system separates into two phases, the phases become separated by the minimal possible surface, namely a plane; while when the agar- water system separates into two phases they remain in contact over an area far larger than the minimum. In the latter case it would seem that the surface-tension at the interface of the two phases is very low, so that the force leading to the diminution of surface is small. The resistance to the diminution of the interface is also very large because of the high viscosity of the gel. The manner in which the structure of a gel is built up can be readily observed in the ternary mixture, alcohol, gelatin and water. If 13.5 grams of Gelatin are mixed with 50 c.c. of water and 50 c.c. of absolute alcohol, a mixture is formed which is optically homogeneous at 17 to 20 C., but which separates into two phases at temperatures below this. Hardy thus describes the sequence of events on cooling this mixture below the temperature of gelation: "As the temperature falls below the limit a clouding occurs which I find to be due to the appear- ance of fluid droplets which gradually increase in size until they measure 3 /z.ju. On cooling further, these fluid droplets become solid and they begin to adhere to one another. The framework is therefore an open structure which holds the fluid phase in its inter- stices." "When once formed the phases have considerable stability. If the droplets are composed of a solid solution one may, by the addi- tion of water, cause them to increase to relatively vast dimensions without their being destroyed; as they increase in size their refractive index approximates more and more to that of the external phase until they are finally lost sight of. The addition of alcohol, however, once more brings them into view and causes them to shrink. Owing to this stability, once a configuration has been established, one has to far overstep the conditions of its formation in order to destroy it. This would account for the remarkable hysteresis observed in reversible gels. . . . When water is added to a ternary mixture so as to considerably swell the droplets, the system is unstable, and the two phases mix at once when it is mechanically agitated." In jellies of this type which are dilute with respect to the colloid constituent, therefore, the structure is that of an open sponge-work, the meshes being filled with water or a water-rich solution of the substance forming the gel while the framework of the sponge consists of anastomosing threads composed of linearly arranged globules of the water-poor phase. In such gels, therefore, the surface of the water- 300 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS rich phase is concave; in other words the water-proof phase is the Internal Phase of the gel, and the water-rich material constitutes the External Phase of the gel. If, however, to a ternary mixture of gelatin, alcohol and water which forms such a gel as that described above, more gelatin be added, the character of the gel changes entirely and its structure becomes inverted. The water-poor phase becomes con- cave and the water-rich phase, instead of being, as formerly, concave, becomes convex to it. On cooling such a mixture to a temperature below that at which it forms an optically homogeneous solution, droplets separate out which are poor in gelatin, while the interstitial portion of the system, which is rich in gelatin, solidifies. Thus the gel comes to possess a Honeycomb-structure the droplets being poor in gelatin and rich in water. This is very clearly shown by the following analyses made by Hardy : TEMPERATURE OF THE MIXTURE, 15 C. (EQUAL PARTS OF WATER AND ALCOHOL). Per cent, gelatin in mixture. 67 Per cent, gelatin in droplets (internal phase) . 17.0 Per cent, gelatin in interstices (external phase). 2.0 13 5 . . . . . . 18.0 5 5 36.5 , 8.5 40.0 From these analyses it is also clear that the two phases in a protein gel are not of constant composition, but may, under different conditions of total concentration, etc., vary widely in their relative and absolute gelatin and water-content. This system differs, therefore, from the system phenol-water, not only in the extent of the surface which separates the phase, but also in the variability of the composition of its phases, in this respect resembling rather the system hydrated silica-water. The reason for this inversion of structure which occurs in concen- trated gelatin Jellies is the same as that which is the origin of the inversion of structure in olive-oil-water Emulsions when the proportion of oil to water is increased beyond a certain limit. The spreading- or covering-power of water is not unlimited and therefore the amount of oil or gelatin which it can surround is correspondingly restricted. The question has been raised whether the jelly which is formed by gelatin dissolved in water (instead of alcohol-water mixtures) really possesses a structure analogous to that observed by Hardy in ternary systems. It has been urged that this structure is an artefact arising out of partial Coagulation of the protein, since it is not directly visible in binary systems. The action of coagulants such as alcohol or sublimate upon jellies which already possess a structure of this type, however, is not to otherwise alter, but merely to coarsen their structure. This is due to loss of water on the part of the colloid-rich droplets with a consequent diminution of the volume of the colloid-rich phase and an increase in the volume of the more fluid interstices. This can be shown, not only JELLIES AND GELATINIZATION 301 by direct observation, but also by the relative ease with which water can be expressed from the jelly before and after "fixation." From Poiseuilles' Law for the outflow of liquid from capillary tubes, it follows that the pressure required to express the fluid must vary approximately as the inverse fourth power of the diameter of the meshes, although, of course, the variable viscosity of the expressed fluid will be a factor introducing departures from this simple law. Now a hydrogel con- taining 13 per cent, of Gelatin at a temperature of 15 C. will endure a pressure of 400 pounds to the square inch without expression of water; after fixation with formalin or corrosive sublimate, however, the fluid can be expressed from the gel like water from a sponge, with simple hand-pressure. Since more complete coagulation does not alter the type of structure possessed by jellies of partially coagulated protein, but merely coarsens it, it is a fair inference that jellies which have undergone no measure of coagulation also possess the type of structure outlined by Hardy, but that owing to its fineness the details of this structure are not visible. The existence of a structure in jellies formed by the solution of gelatin in water is also objectively demonstrated by the observation of Liese- gang, that when silver nitrate diffuses into gelatin which is impregnated with potassium bichromate, the precipitation of insoluble Silver Bichromate does not occur indifferently in all parts of the area of diffu- sion, but in concentric circles. It has also been shown by Rohonyi that when thin films of gelatin are frozen the ice-crystals are formed in concentric rings. It is difficult to clearly conceive any mechanism which would permit this in a perfectly homogeneous medium. The theory that crystallization is inhibited by the gelatin until a certain degree of supersaturation is attained might account for failure of precipitation or crystallization at certain points, but, provided the jelly were strictly homogeneous and structureless, it fails to account for its appearance at other points. The experiments of Hardy show that on adding water to the system alcohol-water-gelatin the gelatin-rich phase progressively imbibes water until it passes by a series of insensible transitions into a Solution of gelatin. We have seen that solutions of protein show evidence, in the peculiar type of resistance to deformation which they display, of possessing a structure which is most easily conceived as a spongework of protein molecules with intercommunicating meshes filled with water. The Structure of the solution is therefore that of an attenuated jelly and there is no distinction of kind, but only of degree, between a protein solution and a protein jelly. As a matter of fact, if the Viscosity of a solution of gelatin sufficiently concentrated to gelatinize at room- temperature be measured at intervals while it is cooling, no sharp change of viscosity is found to occur at gelation, the viscosity of the solution just prior to that point being so great as to afford clear indica- tion of the forthcoming semi-solidification. The structure of protoplasm, therefore, consisting as it does of an 302 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS emulsion of lipoids suspended in a solution or jelly of protein must be very complex. Essentially it is an emulsion enclosed within another emulsion and many diversities of structure and arrangement may evidently exist. One would anticipate that the architecture of such a complex system would be profoundly affected by any factors affecting the solubility of the proteins, and therefore their affinity for water. A relative alteration of volume of the water-poor and water-rich phases of the protein emulsion must necessarily disturb all the space-relations of the enclosed fat-emulsion, and these displacements acting at the Surface of the cell would be equivalent to opening or shutting so many doors for the entry of water-soluble substances into the cell. The striking effects of various inorganic substances upon the Permeability of cells upon which we shall dwell in the succeeding chapter, probably originate in changes of the affinity of the cell-proteins for water with consequent dilatations or contraction of the constituent phases of the protein jelly and enlargement or constriction of the interstitial spaces between the lipoidal elements of the superficies of the cell. THE OSMOTIC PRESSURE OF PROTEIN SOLUTIONS. It was formerly believed that proteins in solution exerted, in common with other colloids, either no osmotic pressure at all, or a pressure of immeasurably small extent. More recent investigations have shown, however, that the difference in this as in other respects between the colloids and the "typical" crystalloids is merely a quantitative differ- ence which is directly attributable to and deducible from the relatively enormous size of their molecules. Thus a one per cent, solution of Glucose contains y-g- gram-molecules of glucose per liter and exerts an osmotic pressure of nearly one and a quarter atmospheres, but a one per cent, solution of Hemoglobin, which has a molecular weight of sixteen thousand, only contains y^ro gram-molecules of protein per liter and, therefore, may be expected only to exert an osmotic pressure of 0.014 of an atmosphere. The direct determination of the osmotic pressure of protein solutions is a task fraught with immense difficulties, on account of the difficulty of preparing ideally pure proteins. The investigations of Graham, the originator of the distinction between crystalloids and colloids, appeared to indicate that colloids in general exert a high osmotic pressure. Subsequent investigators, however, attributed these results to an admixture of crystalloids, which, as the above numerical compari- son shows, might be expected to exert a disproportionate effect upon the pressures exhibited. Starling endeavored to measure directly the osmotic pressure of the proteins in blood-serum by using for his Osmom- eter a membrane permeable to salts but impermeable to proteins, and this method has, since then, been employed in all accurate work upon the subject, since, as Reid has pointed out, it is the only method of procedure which is applicable to the problem. Such a membrane is to the colloids what an ideally semipermeable membrane is to all OSMOTIC PRESSURE OF PROTEIN SOLUTIONS 303 dissolved substances, inclusive of the colloids. We have no assurance that any given protein preparation is totally free from impurities which may influence the direct measurement of osmotic pressure; it is, there- fore, essential to employ a membrane which is permeable to such im- purities and thus, if time be allowed for the system to come to equi- librium, differentiates between protein and non-protein constituents of the solution under investigation. For this purpose Reid employs a membrane of vegetable parchment, which, as he has shown, is per- meable even to nucleic acid, although it is impermeable to the proteins which he employed in his investigations. By extremely prolonged purification Reid has succeeded in obtaining preparations of Egg- albumin which exhibit no measurable osmotic pressure when examined by this method. In subsequent investigations, however, he obtained osmotic pressures, due to dissolved Hemoglobin of perfectly constant value and such as to indicate a molecular weight of about 48,000. Barcroft and Hill have, however, demonstrated by thermodynamical methods that in solutions containing hemoglobin prepared by less prolonged dialysis the molecular weight of this substance is close to 16,669 which is the figure calculated from the content of Iron, assuming each molecule of hemoglobin to contain only one atom of iron. Roaf, employing the differential osmotic method just described, finds that the molecular weight of hemoglobin, dissolved in distilled water, is about 32,000, while in sodium carbonate solutions it is 16,000. These results appear to show that when protein is uncombined with acids or bases it is polymerized, and so exerts a considerably smaller pressure than protein salts. The extremely important discovery has been made by R. S. Lillie, that the osmotic pressure which is exerted by proteins (determined differentially as described) varies very pronouncedly with the nature of the inorganic acids bases or salts which their solutions contain. The following are illustrative results, obtained when dilute acids or alkalies are employed as solvents: 1.5 PER CENT. GELATIN IN DILUTE HC1 SOLUTIONS. Osmotic pressure of Solve Water W /3100 H -/2050 W /1550 W /1025 TO /770 m /620 /412 the protein in nt. mm. Hg . - 'i , 82 21 .. 68 12.3 ' ' a . . . - . + ... . V . 17 9 *.....,..... 26 5 . 32 4 34.9 39.3 1.5 PER CENT. GELATIN IN DILUTE KOH SOLUTIONS. Osmotic pressure of the protein in Solvent. mm. Hg Water 7.9 "YsiooKOH 14.1 m /62o " 23.7 m /412 " 25.1 m /no " .... ? ... r .. ? ..,. 29. Q 304 PROPERTIES CONFERRED BY COLLOIDAL CONSTITUENTS In Lillie's words, " In the presence of either acid or alkali the osmotic pressure of the gelatin thus shows a marked increase, which, within the above range of concentrations, exhibits a certain proportionality to the quantity of acid or alkali added. For equivalent concentrations acid produces a somewhat greater increase than alkali. The change in osmotic properties is to be attributed to a finer subdivision of the col- loidal particles and a consequent increase in the, surface of intersec- tion between colloidal particles and medium." The osmotic pressure of gelatin and of egg-albumin is unaffected by the addition of non-electro- lytes, such as cane-sugar, glucose, glycerol and urea, but is considerably affected by the addition of Inorganic Salts, being depressed thereby. The decrease of the osmotic pressure exerted by the protein depends upon the nature of both the anion and the cation of the added salt. The depression increases in the order: Alkali metals < alkaline earths < heavy metals (for cations); and CNS tissues, but only in the opposite direction. The only hypertonic solutions employed, in which osmotic phenomena might have played a role, were those of the chlorides of the alkalies and ammonium. It was first discovered by Biedermann in 1880 that solutions of certain Sodium Salts cause skeletal muscles which may be immersed in them to enter into more or less rhythmic contractions, reminiscent of those of heart-muscles in normal serum or in Ringer's solution. He also pointed out that since these contractions continue to take place in the presence of Curare, which paralyzes the neuromuscular junctions, the stimulus which evokes them must originate in the muscular tissues themselves, i. e., the contractions are Myogenic. This phenomenon was reinvestigated by Loeb, who found the following minimal con- centrations of the various sodium salts just sufficed to evoke the semi- rhythmic contractions in frogs' muscles, the solutions being rendered isotonic with amphibian serum by the addition of sugar or urea. Minimal effective Substance. concentration. Sodium chloride . ... . ... . m /ie Sodium bromide . . . . ... . ;'"'.. . . . . . m /u to m /s2 Sodium iodide . . . . .' . . . '. , . r . . m /32 Sodium carbonate . . . . . m /ie to m /32 Sodium sulphate . . . . . m / 32 Sodium acetate . . . -. w / 32 to m /64 Sodium fluoride TO / 64 to OT / 3 6 Sodium formate m / 80 to w /ieo Sodium oxalate TO / 26 o to m /3oo Sodium phosphate . TO /i 2 8 to m /266 Sodium citrate "/ 2 oo It will be observed that the most efficient stimulators in this series are the salts of sodium combined with an acid (oxalic, phosphoric, citric, hydrofluoric) which precipitates Calcium from its solutions. DISTURBANCE OF THE INORGANIC ENVIRONMENT 313 It is a very significant fact, therefore, that the addition of traces of soluble calcium salts to any of these solutions, whether containing a calcium-precipitating acid or not, results in prompt suppression of the contractions. Evidently the calcium is not required in these cases to supply a nutrient to the muscular tissues, but to antagonize an action of an excess of Sodium which results in abnormality of function. The simultaneous action of an excess of sodium ions and a calcium- precipitating anion is more effective than excess of sodium alone, because the calcium already present in the tissues partially antagonizes the excess of sodium in the environment. These facts led Loeb to emphasise the importance of the ^| ratio in living tissues and in their environment. Any pronounced increase in this ratio leads to hyperirritability of nervous and muscular tissues and, in fact, as Loeb has pointed out, it is only the calcium in our blood and tissue-fluids which prevents all our skeletal muscles from beating rhythmically like the heart. The fact that the heart continues to beat rhythmically in the presence of the calcium in the blood, although the skeletal muscles cannot do so, draws our attention to the very important fact that the effect of the inorganic environment differs in different types of living tissues. This fact is very strikingly illustrated by the effects of salt solutions upon different parts of the swimming-bells of jellyfish. These bells, in normal sea-water, are almost constantly contracting in a rhythmic manner and it is by the rhythmic impetus created by the expelled water that the jellyfish propels itself through the water. It was first pointed out by the English Biologist Romanes that when the swimming-bell of the jellyfish Gonionemus is deprived by section of its margin, the center of the bell will no longer beat in sea-water, while the margin continues as before to beat rhythmically. Since the margin contains all the nervous ganglia of the bell Romanes concluded that the beats of the swimming-bell were initiated and regulated by these nervous tissues. Loeb, however, found that if the centers, with the margin excised, be placed in pure sodium chloride or sodium bromide solutions which are isotonic with sea-water, they will beat rhythmically just as the intact bell does in sea-water. The experiment really indicates, therefore, that the optimal salt-mixture for rhythmic excitation differs in the nervous and the contractile 'tissues of the bell. Another experiment which illustrates in a very striking manner the differing effects of the inorganic environment upon tissues of diverse function is the following: When the last abdominal segment of a recently-killed fly 1 is torn out with a pair of forceps a length of intestine is usually extracted from the abdomen. The muscular tissue in the intestines of the Insecta, unlike ours, is striated. If this be wetted with M/6 sodium chloride solution and examined under a 1 The species actually employed was the large Australian "bluebottle," Callophora rillosa. 314 PROPERTIES OF THE COLLOIDAL CONSTITUENTS microscope, rhythmic contractions will be seen travelling in rapid succession from the upper to the lower end of the intestine. On touching the intestine at about the middle point with a finely pointed camePs-hair brush wetted with M/6 calcium chloride solution the rhythmic contractions in the affected area are immediately suppressed, but on following a wave of contraction with the eye as it enters this- area and disappears, it can be seen to re-issue below the affected area at the moment when it would have appeared, had the contraction actually traversed that section of the intestine. Evidently, while the contractile function has been suppressed, the conductive functions of the tissues are still unimpaired. If, however, the middle part of the intestine is touched with potassium chloride solution instead of calcium chloride solution, both conduction and contraction are sup- pressed and rhythmic contractions remain confined to the region above the affected area, the region below remaining quiescent. Conduction and contraction are therefore very diversely affected by these inorganic salts. EFFECTS OF REMOVAL OF CALCIUM FROM THE TISSUES AND TISSUE-FLUID. We have seen that an increase in the Q-* ratio in excised muscular tissues leads to hyperirritability of the tissues and that the ratio may be increased in either of two ways, namely by increasing the concen- tration of the Sodium Ions in the environment or by decreasing the Calcium Ions by employing a salt of which the acid component either precipitates calcium or forms a sparingly dissociated compound with it. The effects of injection of calcium precipitants such as citrates, oxalates, fluorides, tartrates, oleates and other soaps, etc., are very widespread and fundamental. They are traceable to muscular, ner- vous and glandular tissues. In small doses whether taken by mouth or injected intravenously, they act as Cathartics, inducing enhanced peristalsis and the evacuation of fluid f eces. In larger do ses we obtain, in addition to purgation, peripheral twitchings, i. e., irregular involun- tary contractions of the muscles of the extremities. An effect upon the renal epithelium is also evidenced by a marked Diuresis, or abnormal volume and dilution of the urine. In still larger doses a very curious complex of symptoms is elicited. Shortly after the injection of massive doses of sodium citrate subcutaneously in rabbits, peripheral twitch- ings occur which are rapidly succeeded by convulsive movements and marked disturbances of equilibrium. The forelegs are stiffly extended and continually shuffling forward with a motion resembling an effort to maintain equilibrium upon a slippery or moving surface. The head is thrown back and the jaws are continuously chewing. Not infre- quently the animals throw themselves into backward somersaults. Exactly the same effects, without the peripheral twitchings, purgation or diuresis, are obtained if, instead of administering massive doses to REMOVAL OF CALCIUM FROM THE TISSUES 315 the whole animal, minute doses are applied to the White matter of the Cerebellum, by direct injection below the gray matter of the surface. The convulsive effects of large doses are therefore attributable to excitation of the white matter of the cerebellum. Chronic ingestion or injection of calcium precipitants leads to partial solution of the bones which become thin and soft, a condition not infrequently met with in sheep that have been feeding for some time upon plants of the Oxalis group. The effects of calcium precipitants in the order in which they occur are therefore: 1. Purgation. 2. Peripheral muscular twitchings and diuresis, 3. Cerebellar excitation. 4. Partial solution of the bones. A measure of Tolerance to the first three groups of effects is developed after repeated administration. The variety of sensitiveness and response of differing tissues to modifications of the inorganic environment is again displayed in these effects. The very striking instance of the varying sensibility of differ- ent cells to this type of environmental disturbance is afforded by the complete insensitiveness of the Gray Matter of the central nervous system to calcium precipitants. We have noted above that the cere- bellar effects of calcium precipitants are only elicited when the salt reaches the white matter of the cerebellum, and Maxwell has shown that solutions of the various calcium precipitants are without effect when placed upon the surface of the motor-area of the cerebrum, but immediately induce convulsions when they penetrate by diffusion or injection to the underlying white fibers. The origin of the purgation by the Saline Cathartics has been the subject of much and prolonged discussion. The earliest suggestion was that made by Poiseuille and Liebig, to the effect that the action of these cathartics was a purely osmotic one; the excess of salt within the intestinal cavity withdrawing water from the tissues and tissue- fluids, while the tension of the intestinal musculature caused by this collection of fluid within the lumen of the intestine led to rapid expul- sion of the contents. The great French Physiologist, Claude Bernard, however, showed that the intravenous injection of sulphates caused purgation, although the osmotic effect in this instance should be the reverse of that imagined by Poiseuille and Liebig, and water should be withdrawn from the intestine into the circulation. To meet this objection a modification of the osmotic theory was subsequently brought forward by Wagner and Schmiedeberg, who suggested that the saline cathartics modify the Permeability of the intestinal epithe- lium, in such a manner that the absorption of water from the intestine is hindered and the fluidity of the contents and distention of the mus- cular walls, which ensues from the accumulation of unabsorbed fluids, leads to the rapid evacuation of fluid feces. The discovery by Loeb that those salts which increase the -^ ratio, and especially those which precipitate calcium, induce hyperirri- tability in muscular tissues, at once threw a new light upon the action of the saline cathartics. A large proportion of these cathartics are 316 PROPERTIES OF THE COLLOIDAL CONSTITUENTS sodium or magnesium salts of acids such as sulphuric, carbonic, phos- phoric, citric or tartaric acids which form insoluble or sparingly dis- sociated compounds with calcium, and their action in stimulating the muscles of the intestine may be regarded simply as an instance of a general effect upon contractile tissues. Barium Chloride, which stands in the peculiar position of not being a calcium precipitant, and never- theless being a powerful stimulant of muscular tissues, is also a very drastic purgative. The inorganic reagents which induce contractions in excised skeletal muscles, therefore, cause purgation when adminis- tered by mouth or injected intravenously. Continuing and extending the above-cited investigations of Claude Bernard, J. B. Macallum showed that if 10 c.c. doses of M/6 sodium citrate, sulphate or tartrate be administered subcutaneously to rabbits, followed ten minutes later by 5 c.c., and ten minutes after that by 5 c.c. more, very pronounced purgation follows. Purgation may also be induced by perfusion of these solutions into the bloodvessels supplying a loop of intestine, or even by painting the solution upon the peritoneal surface of the intestine. By whatever avenue the salt reaches the muscular tissue, therefore, contractions are induced. This disposes of the original osmotic theory. The theory of Wagner and Schmiedeberg, that the fluidity of the feces induced by these salts is due to the non-absorption of water from the intestine, was shown to be unnecessary by the discovery by Macallum that part, at least, of the fluidity of the feces is attributable to the active secretion of fluid from the mucous glands of the intestine into its lumen. Thus, a loop of intestine about 30 centimeters long in a rabbit was thoroughly cleaned out by compression and the ends tied. From time to time before and after the administration of a cathartic salt the loop was opened and the content of fluid determined. The following is an illustrative result: Loop contained at the beginning . . . . . . . . . . 5.0 c.c. Fluid secreted in the first ten minutes 0.2 " Fluid secreted in the second ten minutes 0.5 " 2 c.c. of m / barium chloride injected subcutaneously. Fluid secreted in the first ten minutes after injection . . . . 4.0 c.c. Fluid secreted in the second ten minutes after injection . . . 3.4 " Fluid secreted in the third ten minutes after injection . . . . 3.0" the loop after the administration of the barium chloride, also under- went powerful contractions. Even when the saline cathartic is administered by mouth, the operative portion of it is that which reaches the intestine through the medium of the circulation, so that even in this instance an osmotic effect of the salt is excluded. This has been very strikingly shown by the experiments of Hertz, Cook and Schlesinger, conducted in Guy's Hospital in London. These observers employed human subjects for their experiments, following the passage of the cathartic down the intestine by simultaneously administering bismuth oxychloride and following the shadow cast by this substance on an .r-ray plate. Sepa- REMOVAL OF CALCIUM FROM THE TISSUES 317 rate experiments upon a patient with an iliac fistula showed that the cathartic and the bismuth oxychloride travelled down the intestine together, i. e., the cathartic did not reach any point of the intestine in appreciable advance of the shadow cast upon the plate. Three persons received two ounces each of bismuth oxychloride i n half a pint of cold water at 8 A.M. Breakfast was given at 8.30. Cecal sounds were heard and a shadow appeared in the cecum four hours after the meal. Two days later the same persons received a Seidlitz powder with the same mixture. The shadow appeared in the cecum at the usual time, namely, four hours after the meal, but while normal feces were passed before breakfast, fluid stools, due to the cathartic, were passed at 9.15, 9.40 and 9.45 respectively, no less than three hours before the first trace of bismuth or of the saline cathartic reached the cecum by the way of direct passage through the intestine. The same conclusion was reached by chemical analysis of the feces, a sulphate being in this instance employed as the cathartic salt: Per cent. Per cent. Feces. of water. Total SO4 of SO4. First day normal 80.9 0.037 0.045 Second day normal (10.15) . . . 80.0 0.016 0.032 Second day watery (11.25) . . . 91.1 0.091 0.041 Third day normal 77.3 0.270 0.220 Thus the watery feces evacuated in response to the cathartic con- tained very little more sulphate than the normal feces of the pre- ceding day, while the normal feces of the day following the purgation contained less than the normal percentage of water, and a great excess of sulphates. Were either Liebig's or Wagner and Schmiedeberg's hypothesis the correct interpretation of the facts, we would expect these feces to be very fluid, whereas the experiment shows that the sulphate that remains unabsorbed is actually much less efficient in promoting peristalsis than the proportion which circulates in the blood- stream. That an excess of sulphates were actually circulating in the blood-stream while purgation was taking place is evidenced by the fact that the urine collected between 8 A.M. and 4 P.M. on the second day contained 0.624 grammes more S0 4 than the urine collected during the same period on the previous day. It must be admitted that the purgative action of the saline cathar- tics is not to be entirely accounted for by the precipitation of the calcium in the tissues, since Barium and Magnesium, irrespective of whether they are combined with calcium precipitating acids or not, will induce purgation. The possibility must be borne in mind, how- ever, although we as yet possess no direct evidence which bears upon it, that barium and magnesium, being related divalent metals, may possibly displace calcium from certain compounds in the protoplasm of the tisues affected, and in this connection it is perhaps significant that the urinary output of calcium runs parallel to the output of 318 PROPERTIES OF THE COLLOIDAL CONSTITUENTS magnesium. Furthermore both the cathartic and the anesthetic actions of magnesium salts are antagonized and annulled by the administration of calcium salts. At all events barium salts share with the calcium precipitants the common property of inducing hyperirri- tability in muscular tissues, while the exceptional sensitiveness of the intestinal musculature to magnesium may be perhaps regarded as affording another instance of the diverse susceptibility of the various types of tissue-cells to the influence of changes in the inorganic environ- ment. That the intestinal musculature is not the only tissue which is profoundly affected by magnesium salts is shown by the fact that the introduction of a considerable excess of magnesium chloride into the blood-stream induces Glycosuria in rabbits. THE MUTUALLY ANTAGONISTIC ACTION OF SALTS, AND PHYSIO- LOGICALLY BALANCED SOLUTIONS. We have already seen that a small proportion of calcium inhibits the action of sodium salts in inducing rhythmic pulsations of many contractile tissues. This is, however, merely a particular instance of a very general phenomenon, as the investigations of Loeb in animal physiology and of Osterhout in plant-physiology have most abundantly demonstrated. For example, the fertilized eggs of the marine fish Fundulus will develop normally in distilled water. Inorganic salts are therefore not necessary for their nutrition. They will also develop normally, of course, in sea-water in which the various saline constituents other than bicarbonates and phosphates are present in approximately the following concentration and proportion (Van t'Hoff's solution) : 5/8m sodium chloride 1000 parts by volume 5/8m magnesium chloride 78 " " 5/8m magnesium sulphate 38 " " 5/8m potassium chloride . . . . 22 " " 5/8m calcium chloride t 10 " " In | m sodium chloride solution, without the admixture of the other salts, however, the eggs will not live for more than twelve hours, despite the fact that this solution is isotonic with sea-water. Evidently sodium chloride is definitely toxic for these organisms. In the following mixtures: 96 c.c. of 5/8m NaCl + 4 c.c. of 5/8m MgCl 2 96 c.c. of 5/8m NaCl + 4 c.c. of S/Sm.KCl 96 c.c. of 5/8m NaCl + 4 c.c. of 5/8m CaCl 2 they will live only for about twenty-four hours or even less. When the eggs are placed in the following solutions: 96 c. c. of 5/8m NaCl + 2 c.c. of 5/8m MgCh + 2 c.c. of 5/8m CaCl 2 96 c.c. of 5/8m NaCl + 2 c. c. of 5/8m MgCl 2 + 2 c.c. of 5/8m KC1 96 c.c. of 5/8m NaCl + 2 c.c. of 5/8m CaCl 2 + 2 c.c. of 5/8m KC1 MUTUALLY ANTAGONISTIC ACTION OF SALTS 319 the eggs live for less than thirty days in the first two solutions, but in the third, which corresponds in composition with a concentrated Ringer's Solution, the eggs live for an indefinite period and develop normally. Evidently the toxic properties of sodium chloride are neutralized or antagonized by admixture with a small proportion of other inorganic salts. When the correct mixture is obtained the solu- tion is devoid of toxicity and we speak of it as a "Physiologically Balanced" salt solution. Evidently Ringer's solution and sea-water are physiologically balanced solutions in so far as the tissues of Fundulus are concerned. Sodium chloride is not peculiar in exerting a toxic effect in pure solution. In fact it may be said that any salt without admixture with other salts is more or less toxic for living protoplasm. This is very clearly demonstrated by the following among very many experi- ments of this character which we owe to Loeb. Antagonism between sodium chloride and zinc sulphate (Fundulus). Percentage of - eggs which Solution. develop: lOOc.c. H 2 O ................. 49 lOOc.c. H 2 O + 8 c.c. l/32m ZnSO 4 .......... 100 c.c. 8/8m NaCl + 8 c.c. l/32m ZnSO 4 ........ . 1 100 c.c. 7/8m NaCl + 8 c.c. l/32m ZnSO 4 ......... 6 100 c.c. 6/8m NaCl + 8 c.c. l/32m ZnSO 4 ......... 8 100 c.c. 5/8m NaCl + 8 c.c. l/32m ZnSO 4 ......... 29 100 c.c. 4/8m NaCl + 8 c.c. l/32m ZnSO 4 ......... 34 100 c.c. 3/8m NaCl + 8 c.c. l/32m ZnSO 4 ......... 37 lOOc.c. 2/8m NaCl + 8 c.c. l/32m ZnSO 4 ......... 38 Evidently a very dilute solution of zinc sulphate is highly toxic for Fundulus eggs. Sodium chloride in excess is also very toxic. A definite admixture of these two toxic salts may be found, however, which is almost devoid of toxicity. In the above experiment we have an instance of antagonism between a monovalent metal and a divalent metal. Antagonism may also be displayed between two monovalent metals or between two divalent metals. The following is an illustrative example: Antagonism between magnesium chloride and strontium chloride (Fundulus) . Percentage of eggs which Solution. develop: 100 c.c. 5/8m MgCl 2 ............. 100 c.c. 5/8m MgCl 2 + 1 c.c. of 5/8m SrCl 2 ..... ' . 25 lOOc.c. 5/8m MgCl 2 + 2 c.c. of 5/8m SrCl 2 ...... 22 100 c.c. 5/8m MgCl 2 + 3 c.c. of 5/8m SrCl 2 ...... 9 lOOc.c. 5/8m MgCl 2 + 4 c.c. of 5/8m SrCl 2 ...... lOOc.c. 5/8m MgCl 2 + 5 c.c. of 5/8m SrCl 2 ........ Similar antagonism was found to subsist between lithium chloride and zinc sulphate, potassium chloride and zinc sulphate, ammonium chloride and zinc sulphate, sodium acetate and lead acetate, sodium chloride and manganese chloride, sodium chloride and cobalt chloride, 320 PROPERTIES OF THE COLLOIDAL CONSTITUENTS sodium chloride and lead acetate, sodium chloride and aluminium sul- phate, sodium chloride and chromium sulphate, potassium chloride and calcium nitrate and, in fact, some measure of mutual antagonism is usually but not invariably found to subsist between every pair of inor- ganic salts. Very striking examples of the mutual antagonism of inorganic salts are afforded by the experiments of Osterhout upon plant-tissues. The following shows the aggregate length of roots produced after sixty days by wheat-seeds allowed to germinate in various salt solutions of 2^ molecular concentration. Aggregate length Solution. of roots mm. Sodium chloride . . . ' . . " . .'. . . . 59 Potassium chloride . . ., . . ''. 68 Magnesium chloride .-.:'.. 7 Calcium chloride 70 1000NaCl+ 10CaCl 2 . . . . ; 254 lOOONaCl + 22KC1 + 10CaCl 2 324 1000 NaCl + 78MgCl 2 + 10CaCl 2 377 1000 NaCl + 78MgCl 2 + 38MgSO 4 + 22KC1 + 10CaCl 2 . . . . . 360 Distilled water 740 Since the roots exhibit a maximum growth in distilled water the various salts are evidently not required for the nutrition of the plants. The individual salts in pure solution are all highly toxic as compared with distilled water, but mixtures of the salts in proportions approxi- mating to those found in sea-water permit very extensive growth of roots to occur. The following data show the percentage-increase in the length of the thallus which develops from the seeds of Equisetum in various salt solutions of T f ^ molecular concentration. Percentage increase in length of thallus Solution. after 50 days. Sodium chloride . . . Potassium chloride Magnesium chloride Calcium chloride 700 lOOONaCl + 22KC1 lOOONaCl + 78MgCl 2 40 lOOONaCl + 78MgCi 2 + 22KC1 40 lOOONaCl + 10CaCl 2 980 lOOONaCl + 22KCI + 10CaCl 2 . 1500 lOOONaCl + 78MgCl 2 + 10CaCl 2 1760 lOOONaCl + 78MgCl 2 + 38MgSO 4 + 22KC1 + 10CaCl 2 ... 1500 Distilled water 1760 In certain instances the toxicity of such a universally distributed substance as sodium chloride may be extremely great. Thus Osterhout found specimens of Vaucheria which were killed within a few days by so small a concentration as y^^ sodium chloride, although the running water in which these algse were growing contained no less than twelve times this concentration of sodium chloride. In the brook, however, the toxicity of the sodium chloride was completely annulled by the MUTUAL ANTAGONISM OF INORGANIC SALTS 321 traces of other salts, magnesium, potassium and calcium chlorides which the water contained. Similar phenomena of antagonism have been observed by C. B. Lipman in culture-media containing bacteria. In certain cases, how- ever, no mutual antagonism was observed, as in the case of magnesium and calcium salts acting upon Bacillus subtilis. Furthermore, although the toxicities of potassium chloride and calcium chloride for this, and probably for other am nonifying bacteria, are mutually diminished by their admixture, this is not the case for sodium and calcium chlorides, a mixture of these two salts being more toxic for all proportions of cal- cium than sodium chloride alone. These exceptional phenomena appear to differentiate the ammonifying bacteria very sharply from other types of living tissue. The mutually antagonistic toxicity of inorganic salts is therefore a phenomenon which is universally displayed whatever type of proto- plasm we employ. In certain types, as those afforded by the ammoni- fying bacteria, certain antagonisms may fail to be exhibited, but other pairs of salts, again, will clearly annul each other's toxicity. We may infer therefore, that the toxicity of pure salts for protoplasm is a universal property, and that in the majority of instances a mixture of any two salts is less toxic than either of the components alone. It is certainly not an accident that for all the forms of life which have been investigated, the most nearly innocuous mixtures correspond in composition either to sea-water (Van t'Hoff's solution) or to Ringer's solution. In these mixtures of five and three salts respectively the annulment of toxicity is far more complete than in any binary mixture. Sea-water and Ringer's solution are therefore, Physiologically Balanced Solutions, but for certain of the higher animals, for example in the mammals of which the tissues are adapted to an environment having the composition of Ringer's solution, sea-water, as it is composed today, is no longer a physiologically balanced solution. The determination of the physiological balance depends, therefore, upon the properties of the protoplasm upon which the salts are acting and not upon any peculiar properties of the salt-mixture in question, such as double salt- formation, etc. THE ORIGIN OF THE MUTUAL ANTAGONISM OF INORGANIC SALTS. Since the mutual antagonism of salts originates in a property of protoplasm rather than in any physical peculiarity of the salt-mixtures, we are led to infer that the phenomenon must probably be due to chemical interactions between the constituents of the salt-mixture and some constituents of the cells. Now antagonism, as we have seen, may be displayed between almost any pair of metal ions, but it may also be displayed between different pairs of acid radicals. More- over the toxicity of both acids and bases may be partially annulled by 21 322 PROPERTIES OF THE COLLOIDAL CONSTITUENTS suitable neutral salts. It is clear, therefore, that any constituent of the cell which is responsible for these phenomena must be capable of entering into combination with both acids and bases and with both the acid and the basic radicals of salts. The Proteins are the only abundant constituents of the cell which have been demonstrated to possess these properties, and it has therefore been inferred by Loeb and is now very generally assumed, that the toxicity of sodium salts, for example, is attributable to the formation of sodium proteinates which, if present in too great a proportion in the cell, confer upon the protoplasm properties which are incompatible with the maintenance of normal function. The toxicity of calcium salts is regarded as being attributable in like manner to the undue predominance of calcium proteinates in the cell. An admixture of several types of protein salts is requisite to confer upon the protoplasm of the cell the exact complex of qualities essential to the maximal furtherance of its vital activities. Much light has been thrown upon this question by two very striking series of investigations, namely the Flotation Experiments of Loeb and the Conductivity Experiments of Osterhout. The eggs of the marine fish Fundulus which were employed in the earlier experiments cited above have a specific gravity which is considerably in excess of that of sea-water. They will float in a --- molecular solution of sodium chloride, while they sink in a V~ molecular solution. The experiments consisted in placing the eggs in solutions exceeding --f- molecular in concentration, which is, of course, considerably hypertonic to the contents of the eggs, and observing how long they will float in such solutions. The withdrawal of water from the eggs is manifested not only by shrinkage of volume, but by a coincident increase in specific gravity which results finally in the eggs acquiring a higher specific gravity than the medium so that they sink in it. Continued flotation in hypertonic solutions is therefore evidence of impermeability of the superficies of the cell for water. It is found that if the eggs are placed in a 3 molecular solution of Sodium Chloride they will float, but as a rule not longer than three hours. After that they sink to the bottom of the test-tube while the loss of water which has led to their sinking is evidenced by collapse of the egg-membrane, and shrinkage of the yolk-sac. When the eggs are placed in a -/ molecular solution of Calcium Chloride they float at first, but they sink in about half an hour. If, however, the eggs are placed in a mixture of 50 c.c. of 3 molecular sodium chloride and 2 c.c. of --f- molecular calcium chloride, they will float for three days or more at the surface of the solution, the eggs shrink but little or not at all, and the embryos continue to live. In a mixture of 50 c.c. of 2J m. NaCl + 1 c.c. of 2J m. KC1 + 0.75 c.c. of 2J m. CaCl 2 some of the eggs will con- tinue to float for as long as ten days, while in a 2J m. solution of pure sodium chloride they do not float for more than a few hours. These phenomena admit of only one explanation, namely, that in normal sea-water the superficies of the Fundulus egg is practically MUTUAL ANTAGONISM OF INORGANIC SALTS 323 impermeable to water, but that in a physiologically unbalanced salt solution this natural impermeability is lost, and hence, if the solution is at the same time hypertonic, water diffuses out of the egg and the resultant increase in specific gravity causes it to sink. The same solu- tions which cause this loss of water are also toxic for the developing embryos. Hence the toxicity of unbalanced solutions is associated with an increased permeability of the cells. The same conclusion has been reached by Osterhout in quite another way. This observer has employed the electrical conductivity of plant- tissues as a measure of their permeability for ions, that is of the resist- ance which the surfaces of the cells offer to the transport of ions across them. Discs about 13 m.m. in diameter were cut from the fronds of marine'algse (Laminaria), the average thickness of a frond being about 0.5 m.m. One or two hundred of these discs were then packed together like a roll of coins, into a solid cylinder of from 50 m.m. to 100 m.m. in length. They were held in place by glass rods so arranged as to make a hollow cylinder which closely fitted over the outside of the solid cyl- inder of tissue. The spaces between the rods allowed free access of various salt solutions to the living tissue. At each end of the cylinder of tissue was placed a platinum electrode which could be pressed firmly by means of a screw against the opposite ends of the cylinder. The conductivity of the cylinder was estimated in the usual way. The surface in and out of which ions were forced by the current, amounted to from 26,000 to 53,000 square centimeters; an increase in the con- ductivity of the cylinder implied decreased resistance to the passage of ions across the surfaces of the tissue, i. e., an increased permeability for electrolytes, while a decrease in the conductivity of the cylinder implied, on the contrary, decreased permeability of the cells. It will be observed that the permeability measured by Osterhout was per- meability for dissolved electrolytes, while that measured by Loeb was permeability for water. On transferring the cylinder of Laminaria from sea-water to Sodium Chloride solution of the same temperature and conductivity (0.52 molecular), the resistance fell from the initial value of 1100 ohms in sea-water to 890 ohms in ten minutes. In fifteen minutes it had fallen to 780 ohms, after sixty minutes to 420 ohms, and thereafter continued to fall steadily until it reached a constant minimal value of 320 ohms, which was found to be the resistance of a column of sea-water of the same length and diameter. In other words, in pure sodium chloride solution the cell-surfaces in Laminaria increase in permeability until finally they interpose no resistance at all to the transference of ions across them. A very striking contrast to this result is obtained if a similar column of tissue be transferred from sea-water to a solution of Calcium Chloride having the same conductivity as sea-water. In this case the resistance of the tissue initially rises, very often from the initial sea-water value of 1100 ohms to 1750 ohms in the first fifteen minutes. The resistance 324 PROPERTIES OF THE COLLOIDAL CONSTITUENTS remains stationary at this level for some hours, and then slowly sinks until it finally reaches the level of 320 ohms, which represents zero resistance on the part of the surfaces within the tissue. The permea- bility of the cell-surfaces in calcium chloride solutions, therefore, at first decreases and later increases. A mixture of 1000 c.c. of molecular sodium chloride + 15 c.c. of molecular calcium chloride was then diluted until it had the same conductivity as sea-water. A similar column of Laminaria tissue, when placed in this mixture, neither gained nor lost resistance, and had the same conductivity after twenty-four hours as it normally has in sea- water. The antagonistic action of calcium chloride upon the toxicity of sodium chloride is therefore seen to depend upon the maintenance of the normal permeability of the cells. In general it was found that while the salts of monovalent cations such as sodium, potassium, csesium, rubidium, lithium and ammo- nium increase the permeability of the tissue from the beginning, the salts of divalent cations, such as magnesium, calcium, barium, stron- tium, manganese, cobalt, iron, nickel, zinc, cadmium and tin agree in bringing about an initial decrease of conductivity followed by a relatively gradual increase. The initial decrease is, however, very slight in the case of magnesium. Acids resemble the divalent cations in causing an initial decrease followed by an increase of permeability, but both the increase and the decrease are much more rapid than they are in solutions of neutral salts of divalent cations. Alkalies resemble the salts of the monovalent cations in causing an increase of permeability from the first. If the increase in permeability does not exceed a certain limit, the return of the tissue to normal sea-water results in the restoration of normal permeability and the tissue is not permanently injured. If, however, the increase of permeability exceeds this limit then the normal permeability is not recoverable and the attainment of absolute permeability, i. e., zero resistance of the surfaces of the cells to the passage of electrolytes across them, indicates death of the tissue. The toxic action of pure salts is therefore seen to originate in the irreparable impairment of the normal resistance which the surface of the cell opposes to the penetration or exit of water and inorganic salts. Having regard to the fact that the Proteins of the cell are the only abundant constituents which are capable of entering into combination with all of these diverse substances we may assume that the alterations of Permeability which attend immersion of living tissue in abnormal inorganic environments are due to alterations in the physical consist- ency of the interstitial protein solution or jelly which holds the lipoidal elements in suspension. Alterations in the consistency of the Inter- stitial Protein Medium, and especially alteration of the texture of the spongework of which it is composed, must necessarily modify the spacing of the superficial lipoidal elements, and by widening or narrow- ing the interstitial pores, increase or decrease the permeability of the MUTUAL ANTAGONISM OF INORGANIC SALTS 325 cell for water and for substances which are soluble in water, but insoluble in fats. Of course the alteration of the texture of the interstitial protein jelly which ensues when cells are immersed in abnormal inorganic media may be expected, not only to affect the permeability of the cells, but also a variety of other properties of the cells, and in this way to affect a variety of their functions. Thus, as Loeb has pointed out, the effects of diverse salt solutions, and especially those of calcium precipitants upon the phenomena of motility, are not solely and directly to be attributed to changes in the permeability of the superficies of the contractile elements. Indeed it would be manifestly unreasonable on a priori grounds to make such an assumption. The permeability of the cells having been affected, however, the salts which penetrate them induce further changes which modify their performance of func- tion. This is very clearly indicated by the following experiments in which Loeb sought to ascertain whether the ratio of ^ or of jjjg~$~C which is requisite for the maintenance of life is the same as that required for the maintenance of motility. The eggs of Fundulus were immersed in solutions of sodium chloride of varying concentration, and the con- centration of calcium chloride which had to be added to each sodium chloride solution to permit fifty per cent, of embryos to form was determined. It was found that if the concentration of sodium chloride varies in the ratio 1:2:3 the requisite additions of calcium chloride vary in the proportion 0.3 : 1.3 : 3.2. In other words, if we double the concentration of sodium chloride we must quadruple the amount of calcium chloride, and if we triple the concentration of sodium chloride we must add about ten times as much calcium chloride. To permit normal development and therefore, presumably, to maintain normal Permeability calcium chloride must be added almost in the ratio of the square of the concentration of the sodium chloride. Now when we turn to the proportion of calcium necessary for the maintenance of unimpaired Motility we find a very different relation- ship obtaining. For this investigation the newly hatched larva? of a barnacle (Balanus eburneus) were employed. These larvae are incessant swimmers, and they rise to the surface of the water. They are able to live in sea-water varying in concentration from Vie m to 6 /s m. When the larvae are put into a pure solution of NaCl+KCl in the proportions in which these two salts exist in sea-water, they will all fall to the bottom of the vessel which contains them. They are unable to swim, although they may live for a number of hours in such a solution. If one salt with a bivalent cation be added, for example CaCl 2 or Srds in sufficient quantity, they will rise to the surface but they cannot stay there very long. If, however, enough of a mixture of CaCl 2 +MgCl 2 is added, in the proportions in which calcium and magnesium are present in sea-water, the larvae will rise to the surface and remain there, constantly swimming. Various concentrations of the Na+K mixture were employed and the concentration of bivalent cations, Mg+Ca, required to preserve motil- 326 PROPERTIES OF THE COLLOIDAL CONSTITUENTS ity in each solution was determined. The results showed that the ratio ^g *^ was constant over a wide diversity of concentrations. In other words the concentration of bivalent ions necessary to preserve motility varied directly as the concentration of monovalent ions and not as the square, as in the case of permeability. While motility, therefore, is affected by changes in permeability, the effects upon motility involve changes which are not identical with those which underlie the alterations of permeability. The permeability of the surface of the cell for substances dissolved in water is presumably determined by the diameter of the interstitial pores filled with protein jelly which comprise the spaces between the lipoidal elements of the superficial emulsion. We have seen that permeability is affected by reagents which presumably affect the solu- bility or state of aggregation of the protein constituents of the cell. We should expect, however, to find the permeability of the surface of the cell also affected by lipoid-solvents, especially if these enter into the lipoidal droplets and increase their diameter. FIG. 18. Showing successive effects of increasing diameter of the oil-droplets in an emulsion upon the size of the interstitial spaces. As the droplets increase in size until they touch each other the area of the interstitial spaces diminishes. Further increase in the diameter of the oil-droplets increases the sectional area of the interstitial spaces. According to the measurements of Osterhout, the various lipoid- solvents, in particular, ether, chloroform, chloral hydrate and alcohol which are also Anesthetics, exert two effects upon protoplasm: The one consists in a decrease of permeability which is reversible, i. e., disappears after removal of the anesthetic. The other effect, which requires large dosages, in an increase of permeability which is found to be irreversible. Since anesthesia is reversible we may presume it to be associated with the former of these effects while the ultimate toxic or lethal effects of these drugs may be referred to the irreversible in- crease of permeability. The absorption of these substances by the lipoidal elements of the superficial emulsion with consequent increase in the volume of the lipoidal droplets might lead either to decreased or increased perme- ability for substances which are soluble in water. Provided the lipoidal droplets are not, in the normal superficies of the egg, in physical con- tact with one another, the interstitial spaces between the droplets will ORIGIN OF ACID SECRETIONS 327 be reduced in diameter by the swelling of the droplets. As soon as the droplets come to touch one another, however, any further increase in their diameter will push their peripheries further apart and increase the diameter of the interstitial pores. This will be clear from the accompanying diagram depicting the three conditions indicated (Fig. 18). It can readily be seen, therefore, how a lipoid-solvent may, in small doses, decrease the permeability of cells for water-soluble substances and, in larger doses, increase it. THE ORIGIN OF ACID SECRETIONS. It has always been, until within very recent years, a fact exceedingly puzzling to physiologists that certain secretory glands, particularly the glands of the gastric mucosa and the "salivary" glands of car- nivorous molluscs, elaborate a strongly acid secretion from an alkaline medium, namely blood or other tissue-fluids. The alkalinity of the medium was, of course, greatly overestimated by the earlier observers. On the other hand, however, the results of the most exact measure- ments show that the blood and tissue-fluids are on the alkaline side of neutrality, while the acidity of gastric juice, of which the components must in the long run have been derived from the blood, is comparable with that of a hundredth-molecular solution of hydrochloric acid. The first hypothesis which was advanced in explanation of this phenomenon is usually but erroneously attributed to the German biological chemist, Maly, who published it in 1874. It actually originated with an American, E. N. Horsford whose account of this hypothesis is contained in an article contributed to the Proceedings of the Royal Society of London in 1869. He observed that if a mixture of neutral or even weakly alkaline salts, such as the Phosphates, be enclosed within a parchment-membrane and allowed to diffuse through it into distilled water, the water outside the membrane becomes acid in reaction, while that within the membrane becomes correspondingly more alkaline. This phenomenon is due to the fact that the diffusion- velocity of acids is more rapid than that of the alkaline salts which are formed within the .dialyzer. From the alkaline blood, containing chlorides and phosphates, therefore, the acid hydrochloric juice was supposed to arise in an analogous manner. The difficulty which con- fronts this hypothesis is, however, that it proves too much, since by parity of reasoning all the secretions of the tissues should be acid in reaction, whereas, as a matter of fact, the majority of the secretions resemble the blood in reaction or else, as in the case of the pancreatic juice, are actually more alkaline than the blood. Moreover the effects observed in the dialysis of salt mixtures are too small in magnitude to account for the relatively high acidity of gastric juice. An alternative hypothesis advanced by Koeppe is even more difficult of acceptance. This investigator supposes that the gastric mucosa is permeable to sodium ions but not for chlorine ions. As sodium ions in the food 328 PROPERTIES OF THE COLLOIDAL CONSTITUENTS leave the stomach and penetrate the tissues an equivalent number of hydrogen ions migrate from the tissues into the lumen of the stomach and there combine with the chlorine ions to form Hydrochloric Acid. In the first place the assumption of the differential permeability of the stomach-wall for sodium and hydrogen ions is purely gratuitous and has no foundation in direct observation, and in the second place the theory would require the presence of food in the stomach before acid gastric juice could be secreted, whereas, as Pavlov has shown, the secretion of acid gastric juice may be excited reflexly without the presence of any foodstuffs in the stomach. T. B. Osborne has, however, drawn attention to a mechanism where- by an acid fluid may be elaborated through the intermediation of Proteins. When Edestin is dissolved in sodium chloride solutions and then precipitated by passing in a stream of carbon dioxide, it is found that the precipitate contains an excess of combined hydrochloric acid, while, on the other hand, an equivalent mass of sodium carbonate or bicarbonate has been formed in the fluid and may be estimated by titration with methyl orange. When, in other words, the excess of sodium hydroxide is neutralized by carbon dioxide, this protein com- pound of sodium chloride breaks up, setting free sodium hydroxide and retaining hydrochloric acid in combination. A precisely similar phenomenon occurs when Red Blood-corpuscles are repeatedly washed with isotonic salt solution until the washings become perfectly neutral and are then suspended in neutral sodium chloride solution and treated with a stream of carbon dioxide. The external fluid becomes alkaline and the blood-corpuscles become richer in chlorine (Giirber). In this way hydrochloric acid is brought into combination with a non-diffusible base, and may be subsequently separated from it by hydrolytic dis- sociation, followed by the diffusion of the hydrochloric acid into the surrounding medium, or in the particular instance under consideration, into the gastric juice. We may infer, therefore, that the secretion of an acid juice depends upon the existence in the secreting cells of a protein which is capable of decomposing sodium chloride in the presence of carbon dioxide. The appearance of the free hydrochloric acid in the secretion being attributable to the colloidal, indiffusible character of the protein base. THE SELECTIVE ACTION OF TISSUES AND THE " OLIGODYNAMIC " ACTIONS OF HEAVY METALS. It is a universal phenomenon in living tissues that despite the fact that the exact composition of the inorganic milieu is so definitely related to their welfare and can depart so little from normality without induc- ing disturbances of permeability, yet the relative proportions of the various inorganic constituents of the protoplasm do not conform at all to -.the proportions subsisting in the medium which they inhabit. SELECTIVE ACTION OF TISSUES 329 Thus the Red Blood-corpuscles and the Skeletal Muscles, although bathed by fluids which contain a marked excess of sodium over potas- sium salts, nevertheless, in themselves, contain a very marked excess of potassium over sodium salts. Again, although in fresh-water streams the relative content of potassium is often extremely low, the plants which live in them are capable of storing up a comparatively large amount of potassium in their tissues. One of the most extreme instances of this selection by living tissues of components in disproportion to their abundance in the surrounding medium is that afforded by the presence of Iodine in considerable amounts in the tissues of the Thyroid Gland in mammals and in the tissues of Marine Algae. Iodine is present in normal blood only in undetectable traces and in sea-water in extra- ordinarily small amounts. If we place within a dialyzer an excess of diffusible potassium salts over diffusible sodium salts and dialyze against a solution containing excess of diffusible sodium salts, the proportions of sodium to potassium within and without the dialyzer sooner or later readjust themselves, approaching equality. Now the surface of the living cell, although, perhaps, sparingly permeable to water-soluble substances is neverthe- less not absolutely impermeable to them, and in the course of time if the inorganic constituents of the cell are present therein wholly in diffusible forms, the concentrations of the various inorganic components within and without the cell must ultimately attain equality. Even the One-sided Permeability of the cell-surface would not alter the proportions of the various constituents from those prevailing in the external medium, although their total concentration would, in conse- quence of this, be constantly maintained at a somewhat higher level than that prevailing in the external medium. Hence this phenomenon admits, as Loeb has pointed out, of only one- explanation, namely that the inorganic constituents of a tissue which are found therein in excess of the proportion in which they occur in the fluids which bathe it, must exist within the tissue in the form of non-dissociated and non- diffusible compounds. "If a tissue utilizes one kind of metal in this way, for example K, while another metal, for example Na, is chiefly used for the formation of dissociable compounds with Na as the free ion, the consequence will be that the ashes of the tissue contain K and Na in altogether different proportions from those in which they are contained in the surrounding solution. I think we may take it for granted that, at least, potassium forms a non-dissociable constituent of the protoplasm of a number of tissues of animals and plants' ' (Loeb.) The proteins are the only abundant constituents of protoplasm which possesses the amphoteric property necessary for simultaneous combination with acid and basic radicals. We have seen, furthermore, that the compounds of proteins with inorganic bases, acids and salts, do not yield any inorganic ions to the solution; they are non-dis- sociable compounds in so far as the inorganic component is concerned. 330 PROPERTIES OF THE COLLOIDAL CONSTITUENTS It is to the protein compounds in the main that we must look, therefore, for the origin of the selective ability of tissues. Many of the Heavy Metal Salts, such as those of mercury, silver, lead or copper are highly toxic for living organisms in extraordinarily high dilutions. Even water distilled from a metallic still, or collected in a metallic condenser may be extremely toxic to many forms of life. This phenomenon appeared so impressive to the botanist Nageli that he invented a special phrase " oligodynamic action" to describe it. The phenomenon is not so surprising as it might appear, however, when we recollect that heavy metal ions are protein precipitants and especially tend to form insoluble and non-dissociated compounds with proteins. The effect of this is to reduce the concentration of heavy metal ions in any region containing protein, and if the protein is surrounded by a medium which still contains free metal ions these will diffuse in to take the place of those precipitated or rendered non- dissociable. These in turn will be removed from the solution and so the process will go on until, although the original concentration of metal ions in the external medium may have been very small, in the end the concentration of combined metal in a cell may be considerably greater and quite sufficient to constitute a lethal dosage. As W. A. Osborne has shown, this sequence of events may be directly observed by plac- ing a protein solution inside a parchment-dialyzer and immersing the dialyzer in an exceedingly dilute solution of mercuric chloride. The mercury quickly attains a higher concentration within the dialyzer than without, because as rapidly as it enters it is bound, and the osmotic gradient remains positive from the medium without to the protein solution within the dialyzer. THE BIOLOGICAL INDIVIDUALITY OF TISSUES AND TISSUE- FLUIDS. In discussing the various compounds which the Proteins are capable of forming we had occasion in Chapter VIII to dwell upon the existence and the properties of the compounds of proteins with other proteins and especially upon the demonstration afforded by the investigations of Hardy, that the Serum-globulin which is separable from blood- serum by dilution and acidification is not present as such in the untreated serum but in the form of a complex, probably arising out of the union of several protein molecules. The presence of these protein complexes in the tissues and tissue- fluids affords a simple and readily intelligible explanation of what would otherwise constitute an exceedingly puzzling fact, namely, the Biological Individuality of the various tissues and tissue-fluids. The individual proteins which are found in the tissue-fluids of tolerably nearly related animals, for example in the tissue-fluids of the various species of mammalia, appear, on analysis, to be either identical or BIOLOGICAL INDIVIDUALITY OF TISSUES 331 very nearly identical with one another. Thus the casein of human milk has been shown by Abderhalden to be chemically identical with the casein of goat's milk, in so far as the relative yields of the various amino-acids enable us to judge. Similarly the serum-albumins and globulins of goose-blood are identical with those of horse-blood, and the investigations of Abderhalden together with more recent analyses by Gortner and Wuertz have shown that within the closest approxi- mation attainable by present methods of analysis the amino-acid yields from the fibrins of ox-blood, horse-blood, sheeps' blood and the blood of swine, are all identical. Yet when the blood or blood- serum of any species of animal is injected into the circulation of another species it is treated as a foreign intrusion, and results in the appear- ance of specific "antibodies" such as the Hemolysins or the Precipitins which react with the blood of the species injected, but with no other. Thus if a rabbit be injected repeatedly with human blood- serum, the serum of this rabbit acquires the abnormal property of causing a precipitate to form when it is mixed with human serum. It makes no difference to the result what human being may furnish the serum, but if we employ sera from other and unrelated mammals we obtain little if any precipitate after mixing with the serum of the immunized rabbit. With the sera of related species some precipitate will be obtained, but it is not so abundant as that which is yielded by human serum. The relationship of man to the primates was thus established upon a quantative basis by Nuttal, to whom the following measurements are due: Anti-human serum mixed with : Blood of : Amount of precipit ate. Percentage. Man 0.031 100 Chimpanzee 0.040 130 1 Gorilla 0.021 64 Ourang 0.013 42 Dog 0.001 3 Cat 0.001 3 Tiger 0.0005 2 Ox 0.003 10 Sheep 0.003 10 Guinea-pig . . 0.000 Rabbit ..; 0.000 Kangaroo (Macropus bennetti) . 000 In a similar manner, if a rabbit be immunized against the serum of some other vertebrate than man, the serum of the rabbit so treated will develop a precipitin for that species and its near relatives, and not for other vertebrates. The blood-serum of each species, and in fact the tissues and tissue-fluids in general of each species are so many separate Antigens, producing in immunized animals antibodies which may in certain cases be related to one another but which are clearly not in 1 The estimate of the quantity of precipitate yielded by chimpanzee-serum was much too high, because, as occasionally happens, the precipitate did not settle properly and its true value could not be estimated. 332 PROPERTIES OF THE COLLOIDAL CONSTITUENTS any case identical with one another. Now as far as our experience ex- tends, all antigenic substances are proteins. All attempts to demonstrate antigenic properties in substances unrelated to proteins have resulted in failure and in particular the investigations of Fitzgerald and Leathes, have shown that the Lipoids are non-antigenic. Yet, as we have seen the individual proteins which may be isolated from the tissue-fluids are identical in widely differing species. If, however, the individual proteins which are separable from blood- serum by chemical procedures are present wholly or in part in the unaltered serum in the form of complexes of several proteins united together, we can readily understand how different sera come to contain differing antigens. Two protein-complexes of this type might well be built up out of identical units, and yet differ fundamentally, owing to differences in the combining proportions, and consequently in the mode of linkage of these units. Just as a wide and conceivably infinite variety of proteins may be built up out of differing permutations and combinations of eighteen or nineteen amino-acids, so an infinite variety of protein complexes might be built up by the union in varying proportions and arrangements of the comparatively limited number of different proteins which are individually separable from a tissue-fluid. In pursuance of this idea Gay and Robertson and C L. A. Schmidt have investigated the antigenic properties of several compound pro- teins. If compound proteins differ in their biological specificity from their constituents, then a Compound Protein should represent a new Antigen giving rise to antibodies for itself, as distinguished from the antibodies for its constituents. Unfortunately a formidable technical difficulty stands in the way of clearly recognizing the presence of anti- bodies which are specific for the compound protein. This is the diffi- culty which is constituted by the fact that any protein which is capable of being split by hydrolysis into moieties which are still proteins (in the sense that they are antigenic) gives rise on injection into animals to antibodies not only for itself but also for these split-products. Analogously, a compound protein gives rise to antibodies for its con- stituent parts, and it is only possible to distinguish between these, which would appear in the blood of immunized animals after injection of the separate constituents, and any antibodies which may be formed for the compound as a whole, in those doubtless exceptional instances in which the antibody for the compound reacts with a constituent which is not normally antigenic. The above-mentioned observers have therefore investigated, from this point of view, certain compound proteins in which one constituent is non-antigenic, such as Protamine Caseinate, of which the protamine constituent is non-antigenic and toxic, while the casein constituent is antigenic and non-toxic, and Globin Caseinate of which the globin con- stituent is toxic and non-antigenic. Protamine caseinate displays no antigenic characteristics which BIOLOGICAL INDIVIDUALITY OF TISSUES 333 enable it to be distinguished from casein. It is non-toxic, but whether this lack of toxicity is attributable to the masking of the toxic proper- ties of protamine by its combination with casein, or to the smallness of the proportion of protamine contained in the compound, has not yet been definitely established. It gives rise to antibodies for Casein by virtue of its casein-content, just as casein gives rise to antibodies for its infraprotein split-product Paranuclein, but it does not give rise to any antibody which will react with its protamine constituent. Globin caseinate, however, differs very markedly in its antigenic behavior from either of its constituents. In the first place it is non-toxic, and the failure to exhibit toxicity can hardly be attributable to dilution of the globin constituent by admixture with casein since globin caseinate contains 66 per cent, of globin (see Chapter VIII). Still more striking is the fact that it yields antibodies which react (i. e., display Alexin- fixation) not only with the casein constituent of the compound but also with the globin constituent. It would appear evident, therefore, that injection of globin caseinate into animals gives rise to an antibody which does not appear in response to separate injections of its con- stituents. We have in this case therefore an instance created in labora- tory glassware of what we have assumed to occur in tissue-fluids, namely the formation of a protein complex which differs from other proteins, even from those out of which it is itself built up, in the anti- bodies to which it gives rise when it is injected into animals. REFERENCES. GENERAL: Loeb: Studies in General Physiology, Chicago, 1905. The Dynamics of Living Matter, New York, 1906 The Mechanistic Conception of Life, Chicago, 1912. The Organism as a Whole, New York, 1916. Robertson: Ergeb. der Physiol., 1910, 10, p. 216 (consult for literature). EFFECTS OF REMOVAL OF CALCIUM: Macallum: On the Mechanism of the Physiological Action of the Cathartics, University of California Pubs., Physiology, 1906. Meltzer and Auer: Am. Jour. Physiol., 1905, 14, p, 366; 1908, 21, p. 400. Merckx: Arch. Int. Pharmaco-dynamie, 1906, 16, p. 301. Bancroft: Pfliiger's Arch., 1908, 122, p. 616. Hertz, Cook and Schlesinger: Proc. Roy. Soc. Med., London, 1908, 2, p. 23. Robertson and Burnett: Jour. Pharm. Exp. Ther., 1911-12, 3, p. 635. ANTAGONISTIC ACTION OF SALTS AND BALANCED SOLUTIONS: Loeb: Am. Jour. Physiol., 1899-1900, 3, p. 434. Proc. Nat. Ac. Sc., Washington, U. S. A., 1915, 1, p. 473. Jour. Biol. Chem., 1915, 23, p. 423. Osterhout: Bot. Gazette, 1906, 42, p. 127; 1907, 44, p. 259; 1908, 45, p. 117; 1908, 46, p. 53; 1913, 55, p. 446; 1914, 58, pp. 178, 272, 367; 1915, 59, pp. 242, 317; 1915, 60, p. 228. University of California Publications, Botany, 1907, 2, p 317; Jour. Biol. Chem., 1905-06, 1, p. 363; 1914, 19, pp. 335, 493, 517; 1918, 34, p. 363. Zeit. Physikal. Chem., 1909, 70, p. 408. Science N. S., 1911, 34, p. 187; 1912, 35, pp. Ill and 112; 1912, 36, pp. 350 and 637; 1913, 37, p. Ill; 1914, 40, p. 214; 1916, 44, pp. 318, 395; 1917, 45, p. 97. The Plant World, 1913, 16, p. 129. Jahrb. wiss Botan., 1914, 54, p. 645. Proc. Am. Phil. Soc., 1916, 55, p. 533. Lillie: Am. Jour. Physiol., 1912, 30, p. 1. Lipman: Bot. Gazette, 1909, 48, p. 105; 1910, 49, pp. 41 and 207. Centr. Bakt. Par. und Infek.: 1912, 32, p. 58; 1912, 33, p. 305; 1912, 35, p. 647. Waynick: Univ. California Pubs., Agric. Sc. , 1918, 3, p. 135. 334 PROPERTIES OF THE COLLOIDAL CONSTITUENTS ORIGIN or ACID SECRETIONS: Osborne: Am. Jour. Physiol., 1901, 5, p. 180. BIOLOGICAL INDIVIDUALITY: Nuttall: Blood Immunity and Blood Relationship. London, 1904. Robertson: Univ. California Pubs., Physiology, 1911, 4, p. 25. The Physical Chem- istry of the Proteins, New York, 1918. Gay and Robertson: Jour. Exp. Med., 1912, 16, pp. 470 and 479; 1913, 17, p. 535. ' Schmidt, C. L. A.: Univ. California Pubs., Pathology, 1916, 2, p. 157. Gartner and Wuertz: Jour. Am. Chem. Soc., 1917; 39, p. 2239. PAET III. THE CHEMICAL CORRELATION OF THE TISSUES. CHAPTER XV. THE VEHICLES OF CHEMICAL CORRELATION; BLOOD AND LYMPH. THE COMPOSITION OF THE BLOOD. The distributing agents which accomplish the transportation of substances from one part of the body to another are the Blood and Lymph. Through their intermediation oxygen and the products arising from the digestion of the foodstuffs are carried to the tissues, the waste- products which result from their activity are carried from the tissues to the excretory organs, and an exchange of products between diverse and widely separated tissues is also rendered possible. Among this latter class of materials there are included a number of substances which, arising in one tissue or group of tissues, stimulate other and distant tissues to correlated activity. These substances are collectively designated Hormones, or chemical messengers (from op/mco, I arouse, or excite). The blood consists of a suspension of cellular elements, the red cor- puscles or Erythrocytes and the white corpuscles or Leukocytes in a pale, straw-colored or almost colorless fluid, the Plasma. Of the two types of corpuscles the erythrocytes are much more abundant than the leukocytes, the normal average number of erythrocytes in man lying between five and six million per cubic millimeter of blood, while the leukocytes vary in number between 7000 and 15,000 per cubic milli- meter. In other species the number of formed elements per cubic millimeter of the blood may te higher or lower than in man. Thus in the mouse the normal erythrocyte-count lies between ten and twelve million per cubic millimeter. When the blood is shed from the vessels it forms within a few minutes a gelatinous clot, which is due to the separation from the plasma of an insoluble protein Fibrin. On standing, the clot shrinks or undergoes Syneresis, expressing a colorless or very pale yellowish fluid, rich in 336 VEHICLES OF CHEMICAL CORRELATION protein and containing in fact all of the constituents of the plasma with the exception of the formed elements and the protein Fibrinogen, from which the fibrin arose. This fluid is termed the Serum, and it may be obtained in greater abundance and more rapidly by removing the fibrin from freshly shed blood by whipping it with glass rods or by shaking it up with beads. The fibrin adheres in long strings to the rods or beads and may be removed with them from the fluid which is now termed Defibrinated Blood. From this the corpuscles, red and white, may be removed by centrifugalization, the supernatant fluid consisting of serum. The relative volumes of the plasma and corpuscles may be deter- mined in several ways of which the most accurate is probably the method devised by Hoppe-Seyler, which suffers from the disadvantage, however, of being somewhat lengthy and tedious. Defibrinated blood is employed for the estimation, the removal of fibrin from the whole blood introducing only a very slight error which, if desired, may be separately estimated. Three determinations are made, namely: (a) The total protein including hemoglobin in 1000 grams of whole blood, (b) The total protein, including hemoglobin, in the blood- corpuscles derived from 1000 grams of blood by centrifugalization followed by repeated washing with isotonic salt solution, until the washings are free from protein, (c) The total proteins in 1000 grams of serum free from corpuscles. The difference between (a) and (6) yields the proteins in the serum contained in 1000 grams of blood, so that the ratio ~ yields the proportion of 1000 grams which is constituted by the serum in that weight of whole blood. For example, in an actual estimation, the total protein in a kilogram of blood amounted to 172.9 grams, while the corpuscles from this amount of blood contained 124.0 grams of protein. The serum in a kilo- gram of blood, therefore, contained 172.9 124.0 = 48.9 grams of protein. One kilogram of serum, however, contained 72.5 grams of protein. Therefore the serum in a kilogram of blood comprised HT ths of a kilogram or 674.5 grams. The Serum (or plasma as it is termed before the fibrin is removed from the blood) therefore forms about two-thirds of the whole blood and the corpuscles one-third. This proportion is, however, subject to very wide variations. The blood- count itself, i. e., the number of corpuscles contained in a cubic milli- meter of whole blood, is variable and in conditions of Anemia may fall to one-half the normal value. Then the volume occupied by the indi- vidual corpuscles varies with the osmotic pressure of the serum, hyper- tonicity involving shrinkage and hypotonicity involving dilation of the corpuscles. The Electrical Conductivity of the whole blood compared with that of the serum derived from it may also be employed, as Stewart has shown, for the determination of the relative volumes of the corpuscles and serum. The results yielded by this and by other methods are in substantial agreement with those furnished by the method of Hoppe- Seyler. COMPOSITION OF THE BLOOD 337 In addition to the red and white blood corpuscles certain other minute formed elements are also found in shed blood, namely the Blood- platelets. They are only from one-fifth to one-third of the diameter of the red corpuscles and they do not contain nuclei. There has been very much discussion as to whether they exist in the circulating blood as such, or are not artefacts arising out of the shedding of the blood. They have been regarded by various observers as preformed constit- uents of the circulating blood, as detritus from the destruction of leukocytes and as protein coagula or sphere crystals, which appear in the blood whenever the endothelium of the bloodvessels is injured. They have, however, been observed by Osier in the blood contained in the freshly excised capillaries of the mesentery, so that injury to the bloodvessels, or shedding of the blood from the vessels is not an essential prerequisite to their formation. On standing in shed blood the platelets swell and finally break up and disappear and there is some .indication that those agencies which prevent the disintegration of the platelets also hinder the Coagulation of the blood. They appear to consist of protein with a very high admixture of a phospholipin which resembles Lecithin. The Specific Gravity of the blood necessarily varies with its total dilution, that is, with the amount of fluid which has recently been absorbed from the intestine. As a rule it remains between the upper and lower limits of 1.060 and 1.054, averaging 1.058 in males and a little less in females. In newborn infants the blood has a higher specific gravity, about 1.066. The Chemical Composition of the Blood is very constant in certain respects and highly variable in others. Thus we have seen that the reaction, osmotic pressure and relative proportions of the various inorganic constituents are exceedingly invariable. The concentrations of proteins, glucose, cholesterol and so forth are, on the contrary, very variable. The following analytical data, cited after Abderhalden, are therefore not to be regarded as affording fixed criteria of the com- position of the blood in the different species enumerated, but simply as indications of an approximate average composition. Furthermore the estimations of the inorganic constituents are, as Abderhalden points out, merely of comparative value, since the analytical errors involved in the estimations were high, although presumably of similar magnitude in each of the types of blood investigated. At the time that the above analyses were made the whole of the Glucose in the blood was supposed to be confined to the plasma (or serum). It has since been ascertained by Rona and Masing, however, that the glucose in the blood is contained partly in the erythrocytes and partly in the plasma. It is not, however, distributed between these two elements in proportion to their relative volumes. In addition to the various substances enumerated in these analyses, it must also be remembered that blood contains small amounts of Amino-acids, derived by absorption from the intestine, and of waste products such as Ammo- nia and Urea derived from the metabolic activities of the tissues. 22 OOCD I-H TJ< >o OMO c< O OC O5 O "5 >O T}< Tf I-( (N CO CO Tjl o -O5co o * oo co o -rid -oodd * 1 ^ CDC^COCO^ ^OT^ O O CO i O Jr-J -ec' ^ ONO - CO ^ O 1-1 O O CDt~i-HO CO iO O5 1-1 OI-HI-ICD -O"*coio Or-tOO -OOOO . O b- CO OS O5 t OCOi-( .OOCO--HO ;^ d dodo OMO 'ddcidd I> rH CO CO . iO O (~iOiO . O CD CO ifi ' O -< O O 'OOOO Tj-COCOt^ COCO CDOOIOO'OCS 1-1 rH t". OOCOrHCClO rH CO -10^00000-* O O5 r-l O O >O rH O Tf CD -cDOOrHOO O CO O -OOCOOO in TH - SCOOO -^CO oooooo -rHCO OGOCOCO -rHr-n COOCDiO CD^COCOO COCOCOTfi Ot^COOO ot^com -o^ccoo oodd -dodo COOOt^ Tf 1C CD CO O5 CO >C CO O5 00 CD rH rH * O rH CO CO O (X) rH OlTtHiC O 1C -HH -^rHO rH>CCOOO -OrH rH CO rH rH COrHrH OOiCCDt^ rf* rfi CO t>. COCCOTti rHrHTtC dcod 'ddcodd COCOCO 00 .^CO 2220 'rHrH dddd 'dddd s . JHT}< QCOOOrHO OCOO 'OOCOOO COMPOSITION OF THE BLOOD 339 The Proteins of blood-serum consist of an admixture of albumins and globulins. It is quite uncertain how many different proteins the blood-serum (or plasma) may contain, but certain fractions can be readily distinguished from one another. Among the globulins the "Insoluble Globulin" or "Euglobulin" may be readily separated by simple dilution of the blood-serum with from ten to twenty volumes of distilled water, followed by acidification with dilute acetic acid or with a stream of carbon dioxide. The same fraction separates out on sub- mitting blood-serum to dialysis. An additional globulin fraction, the so-called Pseudoglobulin remains in solution, but may be coagulated by half-saturation of the serum with ammonium sulphate, and there are indications that this substance, in turn, is not a single chemical individual The Albumin fraction, which is not coagulable by half saturation with ammonium sulphate, may also not improbably consist of a mixture of proteins. Thus from the serum of the horse, but only with great difficulty from other sera, a Crystalline Serum Albumin may be obtained by first removing the globulins by half-saturation with ammonium sulphate and then adding more ammonium sulphate until coagulation of the albumins just begins, and allowing the mixture to stand for some time. Only a portion of the albumin is deposited in crystalline form, however, and we are uncertain whether the portion which does not crystallize merely represents the quantity requisite to saturate the liquid with crystallizable albumin, or whether it represents a different protein. In addition to the albumins and globulins the blood often contains very small amounts of Proteose, and also a glucoprotein, termed sero- mucoid which yields glucosamin on hydrolysis. It is present in blood- serum only to the extent of from 0.2 to 0.9 parts per thousand. It has been noted by a large number of investigators that the relative proportion of globulins to albumins in the blood-serum may present remarkable abnormalities in persons or animals afflicted with certain Infections. Normally the globulins are always less abundant than the albumin fractions, so that the ratio globulin albumin is always less than unity. In animals or human beings infected with Streptococcus or Staphylococcus , however, the ratio may be much more than unity, the globulins in some instances amounting to as much as eighty or ninety per cent, of the total proteins. The question of the origin of this remarkable change is of course one which is of great importance to our understanding of the mechanisms by which the organism protects itself against infections, more particularly since, in the case of Diph- theria at least, the Antitoxins resulting from infection or immunization have been found to be associated with the globulin-fraction of the serum. The older analyses aiming at the solution of this problem were subject to very great errors and uncertainties, because of the compara- tively large volumes of blood which were required for a single analysis. The proteins were coagulated by alcohol, dried and weighed, while 340 VEHICLES OF CHEMICAL CORRELATION in another sample the globulins were removed by half-saturation with ammonium sulphate and the resulting solution of albumins was purified by dialysis and its protein content determined by a nitrogen estimation or by coagulating, and weighing the dried coagulum. These processes were tedious, inaccurate and time-consuming, and the large quantity of blood required necessitated restriction of analyses to single samples or to samples taken at rare intervals from the same animals. The recent Refractometric Method of Robertson removes these sources of inaccuracy and permits the determination of the " non-proteins" (including proteoses), globulins, albumins and total proteins in a quantity not exceeding one and one-half cubic centimeters of serum. Samples of this volume may be taken several times in a day from the ear of a rabbit without any evident disturbance due to hemorrhage, and hence the effects of various procedures and administrations may be studied by comparing the composition of the blood-serum of the animals before and at successive intervals after the experimental condition is inaugurated. The following are average results obtained by the refractometric method with various species of mammals and birds. Per cent, of total Species. Non-protein, per cent. Globulin, per cent. Albumin, per cent. Total protein, per cent. protein. Globulin. Albumin. Horse .... 1.65 3.5 4.0 7.5 47 53 Albino rat ... 1.61 1.7 4.2 5.9 29 71 Ox 1.34 2.2 5.0 7.2 31 69 Hog 1.49 2.8 4.3 7.1 39 61 Sheep .... 1.30 1.1 5.2 6.3 18 82 Goat 1.43 1.6 4.9 6.5 24 76 Cat 1.87 2.6 5.1 7.6 34 66 Dog . 2.01 1.3 4.8 6.1 21 79 Guinea-pig . 1.28 0.9 4.7 5.7 16 84 Hen 1.42 1.1 3.5 4.6 25 75 Duck 2.77 1.9 3.2 5.1 38 62 It must, however, be recollected that the normal proportion of globulin to albumin is subject to considerable fluctuation, not only in different individuals of the same species, but from time to time in the same individual. The following are estimations made by Rowe upon sera derived from eighteen normal persons: The last four determinations were made upon samples obtained from the same individual on different dates. It will be observed that the globulins in these normal individuals never exceeded thirty-two per cent, of the total proteins, although ranging from this proportion down to sixteen per cent, in different individuals. On the other hand seventeen persons with Syphilis, and yielding a strongly positive Wassermann Reaction gave values for the proportional globulin content ranging from 26 to 49 per cent., and averaging 35.7 per cent. Eight COMPOSITION OF THE BLOOD 341 persons with Pneumonia had a globulin-ratio of from 27 to 50 per cent, and averaging 40 per cent. Other infections showed corresponding increases in the proportion of globulins to total proteins in the serum. Cases of Nephritis gave a high proportion of globulin (24 to 50 per cent.) while those in which nephritis was associated with the accumulation of salts and urea in the blood had also, of course, a high non-protein content. On the other hand a series of patients with diabetes gave normal values for the globulin-ratio excepting in one instance in which a local infection was also present. Individuals afflicted with various types of anemia, hyperthyroidism, goiter, hemophilia, chronic bron- chitis, pellagra, obesity, lead-poisoning, chronic gastro-intestinal dis- orders and neurasthenia presented normal values for the protein-ratio. Exceptionally high values of the proportion of globulin to albumin in the blood-serum, therefore, are associated with Infections or else with Toxemias. Globulin expressed Non-proteinSj Total proteins, in per cent, of total Sample No. Age. per cent. per cent. protein. 1 .... 27 1.2 7.8 25 2 . ... 30 1.3 7.4 30 3 .... 36 1.3 7.3 32 4 . .".. . . 21 1.3 7.7 26 5 24 1.1 7.6 16 6 .... 30 7 ..... 32 8 ..-.. 48 9 . , . . 19 10 . .". . . 25 11 . , . . . 48 12 28 .2 7.4 32 .1 8.0 28 .2 7.9 27 .2 8.2 27 .3 7.7 26 .2 7,3 30 .3 6.8 29 13 .... 23 1.2 7.4 24 14 .... 19 1.25 6.5 29 15 .... 48 1.25 6.7 31 16 .... 25 1.3 7.5 21 17 ..;'-. 26 1.3 6.7 25 18 .... 29 1.3 6.8 21 19 .... 26 1.3 7.5 20 20 ..... 26 1.3 8.2 21 21 . . . . 26 1.3 8.2 18 22 26 1.1 7.9 24 Averages . . . ,'... 1.24 7.5 25.5 The origin of the rise of globulins in infections is still to be sought. It is not due to, or directly correlated with the development of anti- bodies in the circulation, because as C. L. A. Schmidt has shown, a high degree of immunity to pure proteins may be induced without any rise of globulins. It is not due to the Leukocytosis or increase in white blood corpuscles which often accompanies infection, because Hurwitz and Meyer have shown that the leukocyte-count and the globulin increases do not in any degree run parallel to one another, while C. L. A. Schmidt has shown that the leukocyte-count may be reduced to one-half the normal in rabbits by the administration of Benzole without causing any significant alteration of the globulin-ratio. It is not due to alterations of bodily temperature, because, as Hanson and McQuarrie 342 VEHICLES OF CHEMICAL CORRELATION have shown, the previously reported rise of globulins after the adminis- tration of Antipyrin was due to analytical errors, and does not occur; The same observers have also shown that therapeutic agents which markedly accelerate or retard metabolism, namely Thyroid Extract and Sodium Cacodylate respectively, are devoid of influence upon the protein quotient, and Hanson has also shown that the previously reported effects of Starvation were due to individual fluctuations and that if a sufficient number of analyses be made neither starvation nor heavy feeding is found to affect the quotient in any constant manner. On the other hand Buck has shown that if Ether or Chloroform be admin- istered for very prolonged periods to animals, so that Albuminuria begins to appear, the globulin quotient rises, far more markedly than could be accounted for by an escape of serum-albumin into the urine. This observation may possibly indicate that the true source of the marked alterations in the globulin-quotient which occur in infections and toxemias resides in alterations of the Permeability of the tissue- cells. No further evidence bearing upon this possibility is as yet, however, in our possession. THE COAGULATION OF THE BLOOD. One of the most remarkable properties of the blood is that which it possesses of clotting or coagulating in a brief period after its issuance from the bloodvessels. The clot which is formed is a markedly con- tractile one and if it is loosened from the sides of the vessel to which it otherwise adheres, the clot, with its entangled blood-corpuscles, shrinks away toward the center of the vessel, expressing a clear white or pale yellow serum as it recedes. This phenomenon is known as Syneresis. If a clot be cut into pieces with a knife or rod, the pieces retract from one another and round up into separate masses. A number of different agencies are capable of preventing the clotting of blood w r hen it is shed, thus the various Calcium Precipitants, such as oxalates, citrates, sulphates and so forth will, if added in sufficient amounts, prevent or delay the coagulation of the blood and in fact a common way of preparing incoagulable blood is to receive the blood directly from the vessels into a solution of sodium or ammonium oxalate. Such Oxalated Blood as it is called, remains fluid and inco- agulable until, and unless a soluble calcium salt be added to it in suffi- cient amount to remove all of the calcium-precipitating agent. Accord- ing to Sabbatini there are minimal and maximal concentrations of Calcium Chloride below and above which coagulation is inhibited. The upper limit is a 0.162 molecular solution, the lower about one thou- sandth part of this concentration. Salts which do not actually pre- cipitate calcium, such as sodium citrate, prevent coagulation by reduc- ing the concentration of free Calcium Ions below the necessary minimal limit. Other agencies which will prevent coagulation are certain solutions CO AGVL AVION OF THE BLOOD 343 of Peptones or Proteoses. When these are injected into the circulation, in a very brief period the blood which is drawn from the vessels is found to be incoagulable. The Peptone-plasma obtained from this blood by centrifugalization may be induced to coagulate by the mere addition of a suspension of leukocytes obtained from lymph, or by the addition of calcium chloride in excess of the amount already present in the blood, or by acidification with carbon dioxide or acetic acid. Wooldridge has also drawn attention to the very interesting property possessed by some proteins which are probably Phosphoglobulins, namely that of inducing Intravascular Clotting if injected into the circulation gradually or in small doses; while they render the blood Incoagulable if they are injected more quickly or in larger doses. The former effect Wooldridge designated the Positive Phase of the action of the protein, the latter he termed the Negative Phase. It is an especially remarkable fact that, according to Pickering, albino rabbits, and the Norway hare when in its albino condition, are immune from these effects. These various phenomena have not yet received any adequate interpretation. Another agent which renders blood incoagulable is the extract of leeches' heads, known as Hirudin. Certain Snake Venoms induce a like effect. 'The clotting of the blood is in the first instance due to the trans- formation of a soluble protein, Fibrinogen, into an insoluble modifi- cation, Fibrin. This was conclusively shown by the investigations of A. Schmidt and of O. Hammarsten. If the plasma obtained from blood be mixed with an equal volume of a saturated solution of Sodium Chlo- ride a precipitate or coagulum of fibrinogen is produced which may be washed repeatedly in half-saturated sodium chloride solution, redis- solved in dilute sodium chloride, reprecipitated by half-saturation with sodium chloride and again redissolved. This solution of fibrinogen in from 1.0 to 1.5 per cent, sodium chloride will not clot, however long it may be allowed to stand. In order to induce it to clot, another substance must be added to it, to which the name Thrombin has been applied. Thrombin may be obtained from freshly-formed fibrin. It is best prepared from the strings of fibrin which are obtained by whipping freshly-shed blood; these are washed in cold water with constant knead- ing until all of the Hemoglobin has been removed. The fibrin is then squeezed dry, minced with scissors, and then covered with an eight per cent, sodium chloride solution, which does not dissolve the fibrin, but extracts the thrombin which is associated with it. The mixture is placed in a refrigerator for forty-eight hours, and then filtered through cheesecloth. A few drops of the viscous -filtrate, added to ten c.c. of the fibrinogen solution, cause immediate clotting, without the addition of any calcium salt. On the other hand, thrombin solution unmixed with fibrinogen will not clot, whether calcium salts be added to it or not. 344 VEHICLES OF CHEMICAL CORRELATION Since calcium is necessary for the coagulation of freshly-shed blood, it might seem reasonable to suppose that the thrombin solution contains combined or associated calcium, which suffices to permit the process to go forward. This is, however, not at all the case, for throm- bin may be purified by dialysis and precipitation with Acetone, and when this has been done twice the thrombin is found to be perfectly free from calcium. The true secret of the essentiality of calcium in the clotting of recently shed blood lies in the fact that thrombin, as such, is absent from the circulating blood, and from oxalated plasma. Instead, we have a mother-substance, Prothrombin which is converted by calcium salts into thrombin. This fact may be shown in a variety of ways, among which the following may be cited: Wooldridge showed that if peptone plasma be cooled for some time to zero degrees centigrade a precipitate of minute discoidal particles collects at the bottom of the container. They resemble very greatly the Blood-platelets and may, indeed, actually be identical with them. When these are removed from the plasma, clotting of the fluid is now very difficult to induce by the customary agents, by carbon dioxide, calcium chloride and so forth. Wright subsequently showed that the same precipitate occurs in oxa- lated plasma and Hammarsten showed that its removal prevented the subsequent clotting of the plasma by the addition of sufficient calcium chloride to precipitate the oxalate and furnish a favorable excess of calcium ions. If, however, this precipitate be treated with lime salts and the calcium subsequently removed by oxalates, it now is found to contain very active Thrombin which quickly induces coagulation in Oxalated Plasma. A portion at least of the material in the discoidal particles was, therefore, converted by the calcium salts into thrombin. This constituent is prothrombin. Another method of preparing prothrombin is that which has been devised by Howell. Oxalated blood is centrifugalized and the plasma is heated to 54 Centigrade. This coagulates the fibrinogen. The filtered plasma is treated with Acetone, and the precipitate is collected upon a filter and dried. When the prothrombin is required for use the filter paper is cut into small pieces and extracted for about one hour with dilute sodium bicarbonate solution. This solution does not cause clotting of pure fibrinogen or of oxalated plasma unless it is first treated with calcium chloride (0.2 per cent.). The circulating blood contains prothrombin, therefore, and it also contains calcium salts, and the question necessarily arises why the prothrombin is not converted into thrombin in the vessels, thus leading to intravascular coagulation? The reason that this does not occur is that the conversion of prothrombin into thrombin requires not only the presence of- calcium salts but also another factor, derivable from tissue extracts, which Morawitz termed Thrombokinase, but which has recently been identified by Howell as a phospholipin, namely, Kephalin. COAGULATION OF THE BLOOD 345 The prompt clotting which occurs when normal blood is shed is due to something which is added to the blood when it comes into contact with the lacerated tissues over which it flows, or which is derived from the disintegration of the leukocytes or platelets in the shed blood. This can be shown by employing the blood of Birds or Amphibians in which the white corpuscles do not disintegrate so readily after shedding as they do in the blood of mammalia. If a paraffined cannula be intro- duced into an artery of a bird and the blood be collected in a paraffined centrifugal tube and directly centrifugalized, the plasma which is obtained either does not clot at all, or only very slowly. The plasma of birds' blood which is thus obtained may be induced to clot if any of a large variety of tissue extracts, such as leukocyte extract, or extracts of the brain, testes or thymus be added to it. The active substance is soluble in ether and with difficulty soluble in alcohol, and it contains phosphorus and nitrogen. Howell, and Mc- Lean have shown that pure Kephalin, prepared from brain or other tissues has the same power of inducing coagulation as the whole tissue extract, while other phospholipins, lecithin, cuorin and sphingomyelin are devoid of activity. The activity of kephalin is dependent upon the presence of unsaturated linkages, for hydrogenated kephalin, or kephalin that has become oxidized by exposure to the air, is inactive. That some other factor besides the mere presence of unsaturated linkages determines the action of kephalin is, however, evident from the fact that the great majority of the phospholipins which are devoid of Thromboplastic Action also contain unsaturated linkages. The prothrombin in oxalated birds' plasma is not converted into thrombin by kephalin unless calcium salts are also present. Evidently therefore, both of these factors cooperate in the transformation of prothrombin into thrombin. Two views of the mode of action of thrombin upon Fibrinogen have been advanced: The older view, originally proposed by A. Schmidt, regarded thrombin as an enzyme which converted fibrinogen into fibrin by hydrolysis, just as Casein is converted by rennet into Para- casein. The foundation of this view was twofold: In the first place the thrombin in the plasma or serum of shed blood is inactivated by heating to 60 centigrade for a few minutes and the majority of the enzymes are similarly inactivated at a like temperature. In the second place very small quantities of thrombin are requisite to produce rela- tively large quantities of fibrin. Howell, however, has adduced a number of facts which militate against this view. In the first place the apparent thermolability of thrombin is due to the presence of salts or other substances in the plasma or serum, and pure thrombin, freed from inorganic salts by dialysis, will withstand boiling for five minutes or more without total loss of activity. If to the same solution 0.5 to 1 per cent, of sodium chloride be added, boiling for one minute inactivates the thrombin completely. Of course this fact, in itself, does not prove that thrombin is not an enzyme for, as we have seen, 346 VEHICLES OF CHEMICAL CORRELATION many enzymes, particularly those of bacterial origin, are known which are not inactivated by heating or in which the inactivation is tem- porary or reversible. Moreover it is not really certain that any pure enzymes are thermolabile, since, with one exception, no pure enzymes have ever been prepared. The one exception is that afforded by the Laccase or oxidizing enzyme of Medicago sativa, which has been shown by Euler to be a mixture of calcium salts of aliphatic hydroxy-acids. A synthetic mixture of calcium glycollate, citrate, malate and mes- oxalate has the same oxidizing action as the vegetable enzyme and is unaffected by boiling. The evidence afforded by the effects of heating is therefore incon- clusive either for or against the view that thrombin is an enzyme. Much more decisive is the quantitative relationship of the fibrin-yield to the thrombin which has been added to the fibrinogen solution. The following are estimates obtained by Howell: 0. 05 mgm. of thrombin yielded 10. 75 mgm. of fibrin. 0.16 " " " 34.00 " " 0.25 " " " 36.80 " 0.64 " " " 42.50 " Moreover, a submaximal quantity of thrombin acting upon a solution of fibrinogen will never furnish a full yield of fibrin, no matter how much time is permitted for the reaction to take place. Evidently, therefore, thrombin enters into and determines the final equilibrium which is attained and its action cannot be purely catalytic. The action of thrombin upon fibrinogen is specific in the sense that no other protein is similarly modified by thrombin, but it is indifferent whether the thrombin and the fibrinogen are derived from the same or related or even unrelated species of animals. Thus, Howell has found that the fibrinogen of all vertebrates is converted into fibrin by thrombin derived from pigs' blood. There remains to consider the part which is played by the various factors which contribute to the formation of thrombin from pro- thrombin. Reasoning from the analogy afforded by the conversion of Trypsinogen into Trypsin by the Enterokinase of the succus entericus, Morawitz supposed that the conversion of prothrombin into thrombin by tissue-extracts was attributable to an enzyme which he designated Thrombokinase. A fact which encouraged this view is that if tissue- extracts be heated to from 56 to 60 Centigrade, they lose their throm- boplastic activity, and it was inferred that, like the majority of the enzymes, the thromboplastic substance was thermolabile. Kephalin, however, which is very active in promoting the conversion of pro- thrombin into thrombin does not lose its thromboplastic powers when it is heated. The solution of this apparent contradiction has been supplied by the investigations of Howell, who has shown that if the coagulum which forms when tissue extracts are heated to 60 be ex- tracted with ether, the dried ether extract has all the thromboplastic COAGULATION OF THE BLOOD 347 activity of the original unheated fluid. Evidently the kephalin in tissue-extracts is carried down with the protein coagulum, either physically adherent to it or else chemically combined with it. According to Howell, the activation of prothrombin by kephalin is due to the removal from the plasma of an inhibiting substance, Antithrombin, which is present in varying amounts in the blood of dif- ferent species of animals. The proof for the existence of this substance is as follows: If thrombin in a quantity known to be sufficient to rapidly coagulate a given amount of a solution of fibrinogen be pre- viously incubated for about fifteen minutes at blood-temperature with a small amount of fresh plasma or of plasma freed from fibrinogen by heating to 54 C., the ability of the thrombin to coagulate the fibrino- gen is found to have become very much impaired. If, however, the plasma has been previously treated with kephalin, its power of inacti- vating thrombin is lost or very much weakened. This may be illus- trated by the following data, furnished by Howell. The following mixtures were prepared: Mixture A. Fresh pigs' plasma + equal volume of water. Mixture B. Fresh pigs' plasma + equal volume of kephalin solution. The mixtures were allowed to stand for thirty minutes and then heated to 54 C. to coagulate the fibrinogen. The filtrates were then tested for their antithrombin-content as follows: Mixture A, 1 drop + Thrombin 5 drops Incubation of 15 mins. + Fibrinogen 10 drops = Partial clot in 65 mins. Mixture A, 1 drop + Thrombin 4 drops Incubation of 15 mins. + Fibrinogen 10 drops = No clot in 2 hours. Mixture A, 1 drop + Thrombin 3 drops Incubation of 15 mins. -j- Fibrinogen 10 drops = No clot in 2 hours. Mixture A, 1 drop + Thrombin 2 drops Incubation of 15 mins. + Fibrinogen 10 drops = No clot in 2 hours. II Mixture B, 1 drop + Thrombin 5 drops Incubation of 15 mins. + Fibrinogen 10 drops = Clot in 5 to 10 mins. Mixture B, 1 drop + Thrombin 4 drops Incubation of 15 mins. + Fibrinogen 10 drops = Clot in 5 to 10 mins. Mixture B, 1 drop -f Thrombin 2 drops Incubation of 15 mins. + Fibrinogen 10 drops = Clot in 5 to 10 mins. Mixture B, 1 drop + Thrombin 2 drops Incubation of 15 mins. + Fibrinogen 10 drops = Clot in 10 to 15 mins. It will be seen that the inhibitive action of mixture A is totally absent in mixture B, which has been incubated with kephalin. These results have found important surgical applications, both for controlling hemorrhages, for which purpose gauze soaked in an aqueous solution (or emulsion) of kephalin is employed, and for the treatment of ab- normal tendency to prolonged hemorrhage in cases of Hemophilia, which Howell interprets as being due to an abnormal content of anti- thrombin in the blood of the patients who exhibit it. Hemophilia is a hereditary condition, and is further peculiar in 348 VEHICLES OF CHEMICAL CORRELATION that it is almost invariably displayed only by the males of the hemo- philic family, while the hereditary tendency to hemophilia is trans- mitted by the females. This peculiar mode of inheritance is also encountered in hereditary Color-blindness and in certain other instances of inherited abnormality; it is designated Sex-linked Inheritance. We may therefore sum up the processes and substances concerned in the coagulation of the blood as follows: The circulating plasma contains: Fibrinogen + Prothrombin + Calcium salts + Antithrombin Thrombin + Calcium salts Neutralized by kephalin. Hereafter unessential I Fibrin. Howell believes that in addition to antithrombin properly so called, which inhibits the action of thrombin upon fibrinogen, the circulating plasma also contains an Antiprothrombin which inhibits the conversion of prothrombin into thrombin by calcium salts and is, like anti- thrombin, neutralized or inactivated by kephalin. In regard to the chemical nature of the substances which take part in the coagulation of the blood, Fibrinogen is a globulin, being like other globulins coagulable by half-saturation of its solution with ammonium sulphate, but differing from the serum-globulins in being also coagulable by half-saturation of its solutions with sodium chloride. It is not known in what chemical respects Fibrin differs from fibrinogen, but the results of Howell and others would seem to render very prob- able the view that fibrin is a compound of fibrinogen and thrombin. The jelly which is formed by the conversion of fibrinogen into fibrin in the blood or in neutral or faintly acid salt solutions is of exceptional interest because, as Schimmelbusch and Howell have shown, it consists of an interlacing network of acicular crystals enclosing an interstitial fluid (Fig. 19). If, however, fibrinogen be clotted in alkaline solution the jelly, viewed under the microscope or ultra-microscope appears to be structureless. The crystalline jellies display the characteristic tendency of clotted blood to shrink in and express fluid, whereas the structureless jellies do not. The source of the fibrinogen of the blood appears to be in the Liver, since, as Whipple has shown, conditions associated with injury to or insufficiency of the liver, such as Phosphorus or Chloroform poisoning or hepatic cirrhosis lead to a marked diminution of the fibrinogen content of the blood. COAGULATION OF THE BLOOD 349 Thrombin may be a protein, but if so then it is protein of unusual properties, for it is not coagulable by heat, and repeated extraction with chloroform appears to remove it from its solution in water. On the other hand it yields the biuret- and Millon reactions and all of the reactions for Tryptophane, and it is coagulable by half-saturation with ammonium sulphate. Putrefaction does not destroy it and in fact often seems to increase its activity. These properties appear to indicate that thrombin may be a protein split-product, possibly a proteose. FIG. 19. Fibrin crystals viewed under the ultramicroscope. (After Howell.) The nature of Antithrombin is unknown, in plasma it is thermolabile while the antithrombin in leech extracts (Hirudin) is not. It is uncer- tain, however, whether this thermolability may not be due to asso- ciated impurities, as it is in the case of thrombin. On the other hand Antiprothrombin appears to be a phospholipin, McLean having shown that Cuorin from heart-muscle and a phospholipin resembling Jecorin 350 VEHICLES OF CHEMICAL CORRELATION from the liver possess marked ability to inhibit coagulation, the origin of the inhibition being the delaying or prevention of the formation of thrombin from prothrombin by calcium salts. THE CHEMISTRY OF HEMOGLOBIN. The red coloring-matter in the erythrocytes of the vertebrates is hemoglobin, a compound protein which is split by hydroylsis into a histone-like protein, Globin, and an iron-containing organic acid, Hematin. By reason of the power which it possesses of forming a readily dissociable compound with Oxygen, hemoglobin accomplishes the trans- portation of oxygen from the lungs to all the tissues of the body. Other pigments fulfilling a like function are found widely dispersed among invertebrate animals. Thus in the Arachnids Crustacea and Mollusca there is found a protein containing copper, which has been termed hemocyanin and which becomes blue when saturated with oxygen, and colorless when the oxygen is liberated again. The content of Iron in hemoglobin is identical in all species of animals. The following figures, for example, are given by Jaquet: Hemoglobin from the blood of: Per cent, of iron. Dog 0.0336 Horse . . . . i . . . - . . . ". ...... 0.0335 Ox ;-..... ... 0.0336 Hen . . . : . .;:.. . . . 0.0335 Assuming that each molecule of hemoglobin contains one atom of iron, this implies a molecular weight for hemoglobin of 16,669 while complete analyses indicate an empirical formula approximating to the following: C759Hl20 8 N 2 loS2FeO204 If we examine the Absorption-spectrum of well aerated or arterial blood or of a pure solution of hemoglobin which has been shaken with air or oxygen, we find that the transmitted light contains two well-marked absorption-bands, lying between the Fraunhofer lines D and E. The band nearest to D, termed the a band is narrower, but darker and sharper than the ft band lying nearer to E. On dilution, the & band is the first to disappear. On concentration the bands become broader and finally appear to coalesce. The center of the a band corresponds to the wave-length X = 579, that of the ft band to the wave-length X = 542. In the Photographic Spectrum a band may also be seen in the ultraviolet region, near to G, having its center at the wave-length X = 415. This band, which was first detected by Soret, has been proposed as a means of detecting hemoglobin in high dilutions, since it is still perceptible in solutions containing only one part of hemoglobin in 40,000, while the bands in the visible spectrum are no longer per- ceptible at a dilution of one in fifteen thousand. The absorption-band CHEMISTRY OF HEMOGLOBIN 351 in the ultraviolet spectrum is, however, not characteristic of hemo- globin. It is also shown by solutions of its protein component, Globin, and more or less distinctly by solutions of many other proteins. It is distinctly visible in the light transmitted through solutions of Tyrosine, Phenylalanine and other aromatic ammo-acids, to which radicals its presence in the protein absorption-spectrum is due. It was first shown by the English physicist, Stokes, that if blood be placed under a vacuum, or acted upon with a reducing-agent such as an alkaline solution of ferrous sulphate or ferrous tartrate (known as Stokes' Reagent), or warm solutions of the alkaline sulphides, the absorption-spectrum of the solution changes. Only one band is now to be seen in the visible spectrum, where formerly there were two. This lies between D and E, nearer to D than to E. The same spectrum is sup- plied by the blood of asphyxiated animals. This absorption-spectrum is due to hemoglobin as distinguished from the Oxyhemoglobin which is formed when hemoglobin solutions are aerated. The center of the band lies at wave-length X = 559. The band in the photographic spectrum is at the same time shifted, as Gamgee has shown, the center of this band in solutions of Reduced Hemoglobin lying nearer the visible spectrum than it does in solutions of oxyhemoglobin. The color of solutions of oxyhemoglobin is the typical scarlet of arterial blood; solutions of reduced hemoglobin are darker, with a slightly purple hue and they also exhibit the phenomenon of Dichroism, the color of light reflected from the surface of the solution being green, while transmitted light, as we have stated is red, with a slightly purple tinge. By the action of oxidiz ing-agents reduced hemoglobin is rapidly converted into oxyhemoglobin, but the further action of many oxidiz- ing-agents such as ozone, potassium permanganate, potassium ferri- cyanide and chlorates results in the formation of a modification of oxyhemoglobin which is designated Methemoglobin. The absorption spectrum of methemoglobin resembles that of oxyhemoglobin, except- ing that the /3 band is more intense than the a band and a third band is present between C and D. The color of methemoglobin solutions is chocolate-brown, changing to red when the solution is rendered acid, the absorption-spectrum changing at the same time and showing only one absorption-band between C and D. The oxygen-content of met- hemoglobin appears to be identical with that of oxyhemoglobin, but it is much more firmly combined and is not given up under a vacuum, nor is it displaced by a stream of Carbon Monoxide. When, however, methemoglobin is treated with Stokes' reagent reduced hemoglobin is reformed and this in turn forms oxyhemoglobin on shaking the solution up with air. Methemoglobin is often spontaneously formed when arterial blood is allowed to stand in sealed tubes and it may be found in transudates and cystic fluids stained with blood, or in old extravasations of blood following upon injuries. The blood of animals which have been asphyxiated by illuminating gas is of a peculiar florid cherry-red color, which does not change when 352 VEHICLES OF CHEMICAL CORRELATION Stokes' reagent is added to it. This is due to the presence of Carbon Monoxide Hemoglobin, which may also be obtained by blowing a stream of carbon monoxide or of illuminating gas through a solution of oxyhemoglobin or reduced hemoglobin. In the former case the oxygen combined with the hemoglobin is quantitatively displaced by the carbon monoxide, a given volume of carbon monoxide displacing an equal volume of oxygen. We can readily distinguish between normal arterial blood and the blood obtained after carbon monoxide poisoning, in the first place by the lack of effect of Stokes' reagent upon the color of the carbon monoxide hemoglobin, and in the second place by the effect of adding concentrated sodium hydroxide (specific gravity 1.3) in the proportion of two volumes of sodium hydroxide solution to one volume of blood. Blood containing carbon-monoxide hemoglobin yields a cinnabar-red precipitate, whereas normal blood yields a dingy brown precipitate. Furthermore, Tannic Acid yields with normal blood a brownish-green precipitate, and with carbon- monoxide blood a pale crimson-red precipitate. The spectrum shows two absorption-bands similar to those of oxyhemoglobin but nearer to the violet end of the spectrum. The carbon monoxide may be dissociated from the hemoglobin by the prolonged action of a vacuum or of a stream of oxygen or an indifferent gas. The quantity of oxygen or carbon monoxide which combines with one gram of hemoglobin is 1.34 c.c. at C. and 760 m.m. Hg. This corresponds to one molecule of oxygen or carbon monoxide for every atom of iron in the hemoglobin molecule. If, therefore, we regard the molecule of hemoglobin as containing one atom of iron, the reaction between hemoglobin and oxygen appears as a simple bimolecular reaction as follows: Hb + O 2 ^ HbO 2 The reaction proceeding from left to right when the partial pressure of oxygen is increased, as it is in the lungs, and from right to left when the partial pressure of oxygen is reduced, as it is in the tissues. Sim- ilarly the interaction with carbon monoxide may be represented as follows: Hb + CO ^1 HbCO Designating the concentration of reduced hemoglobin in any solu- tion by the symbol Cr, that of oxyhemoglobin by the symbol Co, and that of oxygen by the symbol "b," then applying the mass-law to the balanced reaction: Hb + O 2 ^ HbO 2 Cr b Co We would have, at equilibrium: Cr X b = KCo CHEMISTRY OF HEMOGLOBIN 353 where "K" is a constant which is characteristic of the equilibrium, and represents the ratio of the velocities of the opposed reactions. The concentration of oxygen in the solution will, of course, be directly proportionate to the partial pressure Po of oxygen in the atmosphere above the solution and to the absorption-coefficient at of oxygen in water at the particular temperature "t" which is employed. We therefore have : b = Po.at and:. ^ = Kat Po Hence in a solution of hemoglobin brought into equilibrium at any given temperature with a mixture of nitrogen and oxygen, such as air, by shaking or by exposure over a very extensive surface, as in the capillaries of the lungs, the ratio of oxyhemoglobin to reduced hemo- globin should be directly proportional to the partial pressure of oxygen in the atmosphere to which it is exposed. This relationship was investigated by C. Bohr who found so many irregularities which were apparently inconsistent with the equation that he inferred the existence of several different compounds of hemo- globin with oxygen. The whole question was, however, reinvestigated by J. Barcroft and his collaborators with greatly improved technique and it was ascertained that the irregularities observed by Bohr were due to inconstant contamination of the hemoglobin by crystalloids and that in properly dialyzed solutions the relationship deduced from the mass-law holds good with exactitude. The origin of the irregulari- ties in solutions containing inorganic electrolytes resides in the ten- dency of hemoglobin to polymerize in such solutions, Roaf having found that while hemoglobin in distilled water exerts an Osmotic Pressure corresponding to a molecular weight of 16,000, in sodium chloride solution the osmotic pressure corresponds to a molecular weight of 32,000. On rendering this latter solution alkaline the molec- ular weight of the hemoglobin again falls to 16,000, the weight which is also indicated by the iron- and sulphur-contents, assuming each molecule of hemoglobin to contain one atom of iron. The influence of Temperature upon the Equilibrium-constant of a balanced chemical reaction is expressed by the well-known thermo- dynamical equation: KT = K To e~T ^f) where KT is the value of the equilibrium-constant at the temperature T, KT O is the value of the constant at temperature To, " q" is the heat given out by the conversion of one gram-molecule of the substance and "e" is the base of the natural or "Napierian" logarithms. The validity of this equation for the reaction between oxygen and hemo- 23 354 VEHICLES OF CHEMICAL CORRELATION globin has also been established by Barcroft, as the following data reveal. The oxygen pressure was constantly maintained at 10 mm. Hg Percentage of hemoglobin converted into oxyhemoglobin. At 16 24 32 38 49 Observed 92- 71 37 18 6 Calculated 90 71 41 22 6 from which figures and the above equation it is easy to deduce that "q" or the heat given out when one gram-molecule of hemoglobin unites with oxygen, is 28,000 calories. Now the heat given out when one gram of hemoglobin unites with oxygen is 1.85 calories. Hence we have the simple ratios: weight in grams of Heat given out by one gram- one gram-molecule molecule 28, 000 Weight of one gram Heat given out by one gram 1.85 whence the weight in grams of one gram-molecule of hemoglobin is calculated to be 15,200, which estimate, when one recollects the number and variety of measurements which enter into it, is in extraordinarily good accord with the known molecular weight of hemoglobin, namely, about 16,000. FIG. 20. Hemin crystals, magnified. (After Preyer.) By the action of acids, alkalies or heat in the presence of oxygen, hemoglobin can readily be split up with the liberation of Hematin. If this hydrolysis is accomplished in the presence of hydrochloric acid the substance obtained is the hydrochloride of hematin or Hemin, which may be readily recognized by its characteristic crystalline form (see Fig. 20). Alkaline solutions of hematin show pronounced Dichroism, being red in thick, and green in thin layers, while acid solutions of hematin are brown. The solid substance forms glistening bluish-black amor- phous masses. The hydrochloride, however, is brown. If hematin be dissolved in strong Sulphuric Acid, on diluting the CHEMISTRY OF HEMOGLOBIN 355 solution a dark red substance, Hematoporphyrin, or iron-free hematin is deposited, the iron originally contained in the hematin molecule being left in the solution in the form of Ferric Sulphate. Hemato- porphyrin is identical with a substance known as Hematoidin which is frequently found in the form of microscopical rhombic crystals in old extravasations of blood or apoplectic clots. It is also identical with Bilirubin, the red coloring-matter of the bile. When hemoglobin is decomposed by alkalies in the absence of oxygen, we obtain Hemochromogen, or "reduced hematin." This substance yields bright red solutions in alkaline media, acids very quickly change it into hernatoporphyrin and a ferrous salt: Hematin. 2H 2 O + 2HC1 H 2 Hematoporphyrin. By reduction of hematoporphyrin we obtain, among other products, a substance known as Hemopyrrole, C 8 Hi 3 N, which is a methyl propyl pyrrole: HC - C - CH 2 .C 2 H 5 HC C CH 3 NH From its quantitative composition and the abundance of Methyl Pyrrole derivatives among its decomposition-products, it appears probable that hematin may be built up out of four methyl pyrrole radicals united by iron and oxygen. The hydrochloride, or Hemin may possibly be represented by the following structural formula: -CH=C(OH) C=C CH=CH C C CH 3 HC CH \ O FeCl \/ NH CH 3 C HC :: CH=C(OH) < ^ CH 3 \/ NH CH / HC CH NH The extensive investigations of Marchlewski, to whom we owe much of our knowledge of these pigments, have resulted in establishing the very close relationship which exists between hematin and Chloro- phyll, the green pigment of plants. Thus among the products resulting 356 VEHICLES OF CHEMICAL CORRELATION from the decomposition of chlorophyll, a substance, Phyloporphyrin, is obtained which differs from hematoporphyrin only in containing two hydrogen atoms in the place of two hydroxyl-groups. The attempt has been made to transform hematoporphyrin into phyloporphyrin by reduction, but this attempt has as yet only been partially successful, only one of the hydroxyl groups in hematoporphyrin having been replaced by hydrogen. The close relationship of hematin to chlorophyll at once suggests the possibility that the necessary radicals for the binding of hemo- globin may be obtained by animals from the decomposition-products of chlorophyll. The pyrrole grouping may of course be obtained from the Proline and Oxyproline constituents of the protein molecule, but it is a question whether the synthetic activity of the hemopoietic tissue in the red marrow of the bones goes so far as to build up hsmatin from pyrrole or whether, rather, somewhat more complex fragments of hematin may not be requisite. It is true that chlorophyll is not digestible by the hydrolytic enzymes of our alimentary system, but that does not exclude the possibility of bacterial digestion in the lower intestine, and as a matter of fact, Marchlewski has shown that chloro- phyll does actually in part disappear when introduced into the aliment- ary canal of animals. Abderhalden has suggested that the failure of inorganic-iron therapy in certain cases of Anemia may be attributable to lack of certain decomposition-products of chlorophyll in the diet, or to lack of the proper assimilation or utilization of these products which he conceives, may be necessary for the synthesis of hemoglobin. THE CRYSTALLINE FORMS OF HEMOGLOBIN IN RELATION TO THE BIOLOGICAL INDIVIDUALITY OF THE BLOOD. The constant percentage of iron in the hemoglobins derived from different Vertebrata invites, but does not establish the accuracy of the supposition that the hemoglobins from different sources are identical. While the quantitative composition of hemoglobin must be the same in all species, yet there exist a very large number of conceivable ar- rangements of the various radicals and groupings in the molecule, and of stereochemical differences not detectable by mere analysis. In fact Reichert and Brown have in recent years very strongly advocated the view that the hemoglobin of every species differs chemically or stereochemically from that of every other, basing their view upon the results of their monumental investigation of the crystalline forms of hemoglobin derived from different sources. Crystals of hemoglobin are readily obtained from the blood of cer- tain animals by the mere evaporation of blood "laked" by ether. This procedure suffices in the case of the blood of the rat, for example. In many cases it is necessary to cool the blood to zero and in some to add alcohol to reduce the solubility of the hemoglobin. Generally speaking the best method to induce crystallization is to add from one to five CRYSTALLINE FORMS OF HEMOGLOBIN 357 per cent, of Ammonium Oxalate to freshly shed blood, which not only prevents clotting but accelerates the process of crystallization, then lake the corpuscles by shaking up the blood with ether, remove the debris of corpuscles by centrifugalization and allow the fluid to evapo- rate on a microscopic slide. In some cases the nature of the agent em- ployed to lake the blood or induce Hemolysis is of importance in deter- mining the ease of crystallization. Thus if dogs' blood be laked with Toluol, an abundance of crystals of hemoglobin is easily obtained by merely cooling the laked blood in a refrigerator. The results of Reichert and Brown have shown that the crystals obtained from the blood of different species are never identical in form. From an enormous number of measurements of crystal-angles, etc., conducted upon hemoglobins derived from a very wide variety of species these observers conclude that the crystals of the different species of any one genus belong to the same crystallographic system and generally to the same crystallographic group, and they have approximately the same axial ratios, or their ratios bear a simple relation to each other. In other words the hemoglobin crystals of any genus are isomorphous, but not identical. In some cases this Isomorphism may be extended to include several genera, but this is usually not the case unless, as in the case of the dogs and foxes, for example", the genera are very closely related. On the other hand the oxyhemoglobin obtained from the same species always crystallizes in the same form, although often with a different "habit" when obtained by different methods of preparation. But upon comparing the hemo- globins from different species of a genus it is always found that they differ from one another to a greater or less degree in angles or axial ratio, in optical characters, and particularly in those characters com- prised under the general term "Crystal Habit," so that one species can usually be distinguished from another by the form of its hemoglobin crystals (Fig. 21). A clear relationship is thus seen to subsist between the physico- chemical behavior of a constituent of organisms, and their place in the phylogenetic scale of relationships as established by their gross mor- phology, and a long stride has been taken toward the establishment of a physicochemical basis for morphological distinction. The further, and entirely independent question now arises, however, as to the chemical origin of the observed physicochemical phenomena. Our experience with the crystallography of inorganic and the simpler organic substances has led us to infer with a considerable degree of confidence that substances which show differences in crystal- lographic structure are different chemical substances. Crystal form is affected even by isomeric modifications which analysis, unaided by other methods of investigations, fails to reveal. Now the enormous number of atoms in a protein molecule encourages, at first sight, the supposition that an enormous and indeed, for all practical purposes, an infinite number of isomerides are possible between which the most refined methods of analysis would not enable us to distinguish, but 358 VEHICLES OF CHEMICAL CORRELATION FIG. 21. Oxyhemoglobin crystals of various animals. 1, the goose; 2, the Tasmanian devil (Sarcophilus ursinus) ; 3, the kangaroo (Macropus giganleus) ; 4. the horse ; 5 , the guinea-pig; 6, the long-armed baboon (Papio langlceldi). (After Reichert and Brown.) CRYSTALLINE FORMS OF HEMOGLOBIN 359 which would very probably differ from one another in the morphology of their crystals. In point of fact, however, the available number of isomers would be very greatly restricted by the necessity of maintain- ing unaffected the amino-acid groupings of the protein moiety, which could not differ materially in different species without leading to de- cided differences in the chemical behavior of the hemoglobins, which have not been observed by any investigator. Further doubt is thrown upon this interpretation of the facts by the observation of Hiifner, recently confirmed with the utmost precision by Butterfield, Heubner and Rosenberg, and Schumm, that the characteristic Absorption- bands, and the ratio of the absorption of light in different parts of the spectrum of hemoglobin are absolutely identical in species so far removed from one another as the horse and man (Schumm) or the rabbit, sheep, and hog (Heubner and Rosenberg). Now these are properties which we would anticipate might be materially affected by internal differences of atomic arrangement. Further reason for doubting the correctness of referring the differ- ences of crystal structure displayed by the hemoglobins of different animals to internal differences in the molecule of the hemoglobins is supplied by the observation of Loeb and Brown that the crystal-form of the hemoglobin of the mule is intermediate in character between that of the horse and that of the donkey. For if we assume that each different crystal-form represents a different internal atomic arrangement of the hemoglobin molecule, then the number of such arrangements, even if very great, must nevertheless be limited. The number of possible forms of crystals must, therefore, also be limited, and, moreover, the possible modifications of forms must be discontinuous, i. e., there must exist forms between which no intermediate forms are possible. This being the case it would be very remarkable indeed were the hybridi- zation of two closely related species to lead to the synthesis of a new isomeric variety of hemoglobin not yet appropriated by any existing species of animal and, in addition, lying between the hemoglobins of the parent-species. If analogous phenomena should be displayed by all hybrids and by all varieties and mutations that might have arisen or might conceivably arise in the future, we would have to admit that the hemoglobins already recognizable as differing from one another in crystalline form are only a small proportion of those which are realisable. A much more reasonable supposition is that embodied in the view that the differences in crystal-form observed by Reichert and Brown were attributable, not to the internal variation of atomic grouping in the hemoglobin molecules, but to external variations in the milieu from which they are crystallized. The technique adopted by Reichert and Brown was to induce crystallization directly in the laked blood. Now we know from the observations of the immunologists that the blood-plasma from any species of animal differs antigenically from that derived from any other species, and since all known antigens are proteins, we infer that the proteins or, more probably, the compound Protein Complexes in blood-plasmas derived from different species are in 360 VEHICLES OF CHEMICAL CORRELATION certain definite respects different from each other. The crystals of each species studied by Reichert and Brown were therefore deposited from a different medium, and it is not improbable that the observed differ- ences between the crystals are attributable to these known differences in the media in which they were formed. It is well known that crystal- habit is modified by alterations of the medium from which the crystals are deposited. That modifications of this origin, so great as to prevent inclusion of the crystals formed in different media in the same iso- morphic series, have not hitherto been observed in the domain of inor- ganic chemistry is not improbably attributable to the simpler character of the conditions accompanying crystallization in inorganic or non- colloidal media. We have seen that there are many reasons for sup- posing that proteins, even in solution, are disposed in a certain reticular structure (cf. Chapter XIII), and if, as the facts which we dwelt upon in connection with the properties of the compounds of proteins with each other would seem to indicate, characteristic protein complexes, formed by the union in differing proportions of a relatively small number of simpler protein components, exist in each type of blood- plasma, we may well suppose that the reticular structure of the solu- tions comprising these plasma would likewise differ from one another. Having regard to the markedly cohesive properties of proteins, crystal- lization within the meshes of such a reticulum might very conceivably, through external strains imposed by points of attachment to the reticu- lum, modify the effects of the internal strains which find their expres- sion in crystal form. This hypothesis finds decided support in the fact, first observed by Halliburton, and confirmed by Reichert, that the crystal form of oxyhemoglobin derived from a given species may be profoundly modified by admixture with the blood of another species. The follow- ing are illustrative results obtained by Halliburton, the "normal" form of rat-hemoglobin crystals being rhombic, those obtained from guinea-pigs being normally tetragonal, and those from squirrels' blood hexagonal. Form of hemoglobin crystals deposited Blood of Mixed with that of from the mixture. Rat Squirrel Both rhombic prisms and hexagons present. Rat Guinea-pig No rhombic prisms of the shape usually seen in rats' blood present; no tetrahedra; crystals are all rhombic prisms with hexagonal habit. Squirrel Guinea-pig Hexagonal plates and retrahedra both present; many tetrahedta imperfect; the tetrahedra all reduced to about half the si2e of those prepared from the unmixed blood of the same guinea-pigs. Squirrel Fine rhombic needles and hexagonal plates both present in abundance. Guinea-pig The greater number of the crystals formed are very small tetrahedra about a quarter the size of those prepared from the blood of the same guinea-pig. The optical properties are, however, the same; rhom- bic prisms, very slender, like those of dogs' blood are also seen. THE CHEMICAL DETECTION OF BLOOD 361 According to Reichert, the degree of modification of crystal form induced by admixture of two bloods depends very greatly upon the proportion in which they are mixed. In view of these facts there can be little doubt that the nature of the milieu in which crystallization occurs does play an important part in determining the form of the crystals which are deposited, and having regard to the known individuality of the plasma from different bio- logical species, it would appear unnecessary to seek further for the origin of the differences in crystal form of the oxyhemoglobins derived from blood of different species of animals. In this way we can also interpret the changes in crystal-form which Halliburton observed to result from repeated Recrystallization of hemoglobin, for as Wichmann and more recently Katz have shown, the crystalline proteins swell in, or absorb the surrounding fluid menstruum in a manner analogous to the swelling of jellies. A number of recrystallizations are therefore required to remove completely traces of the original menstruum in which crystallization occurred. Bradley and Sansum believe that the hemoglobins from different animals are antigenically different, because guinea-pigs sensitized to ox- or dog-hemoglobin failed to display Anaphylactic Shock, or reacted but slightly to hemoglobins of other origins, while they reacted strongly to the hemoglobin with which they were sensitized. As the hemo- globin preparations employed by Bradley and Sansum were admittedly (with the exception, they believe, of dog-hemoglobin) not free from contamination by serum, the interpretation of these results is open to serious question. Doubt is especially thrown upon this evidence for the specificity of hemoglobins from different species by the fact that the animals sensitized to the purest preparation of hemoglobin em- ployed, that of the dog, reacted strongly, not only to dog-hemoglobin, but also to dog-serum. Observers are not all agreed that pure hemo- globin is antigenic; its protein component, globin, certainly is not, and having regard to the investigations of Wichmann and Kat, cited above, revealing the marked ability of crystalline proteins to absorb the menstruum from which they are deposited, and to the observation of Schulz and Zsigmondy that Egg-albumin must be recrystallized from 3 to 6 times in order to remove appreciable con- tamination by other proteins, we may infer that in all probability the specificities demonstrated by Bradley and Sansum are serum- specificities and not hemoglobin-specificities. THE CHEMICAL DETECTION OF BLOOD. The chemical detection of blood and identification of blood-stains is often of the very gravest medicolegal import. The older methods of detection depended upon microscopical identification of blood- corpuscles, and, of course, a very slight degree of putrefactive change, or the drying of a blood-stain upon a garment rendered the detection 362 VEHICLES OF CHEMICAL CORRELATION of these formed elements impossible. This was succeeded by the far more delicate and reliable Hemin Test, which consists in placing a drop of suspected fluid or saline extract of shreds of stained fabrics, upon a microscope-slide, adding a crystal of salt and a drop of glacial acid, heating the fluid to boiling by passing the slide to and fro over a small flame, and then examining the fluid, as it cools, for hemin crystals. This test may be successfully employed with samples of blood far advanced in decomposition. A still more delicate test, however, is the Benzidine reaction. This depends upon the power of an enzyme or Peroxidase, 1 which is present in blood, to decompose Hydrogen Peroxide, liberating nascent oxygen which oxidizes the benzidine with the produc- tion of a green or blue color. Properly conducted, this test will detect one part of blood in three hundred thousand, which means, in effect, that a murderer may wash his blood-stained hands in a bath full of water, and yet if any drainage remains unemptied at the bottom of the bath, the fact that he has done so may be detected with certainty. Never- theless even more delicate tests are available. TJius Buckmaster has found that if an alcoholic solution of Guaiaconic Acid be added to blood together with hydrogen peroxide, a blue color may be produced at a dilution of one in five million. This test is also given by perfectly fresh Milk collected and bottled with aseptic precautions, but it is not given by the milk which is ordinarily obtainable in the market. For the identification of the Species from which blood is derived we rely upon the antigenic Specificity of blood. The suspected fluid is mixed with anti-human serum prepared by immunizing a rabbit against human blood. The mixture is incubated, and the occurrence of a flocculent precipitate indicates that the suspected fluid contained either human blood or the blood of an anthropoid ape. Since "The Murders in the Rue Morgue" must be admitted to have constituted an entirely exceptional problem, the alternative thus presented does not furnish any serious basis for uncertainty. THE ORIGIN AND COMPOSITION OF LYMPH. The tissues are not, excepting in a very few situations, bathed by blood itself, but by the Lymph, which is derived from blood, and through the intermediation of which the substances dissolved or combined in blood are brought into physical contact with the proto- plasm of the living cells. There was formerly much discussion of the question whether lymph is elaborated from the blood by a process of active secretion, consti- tuting an Exudate, or whether, on the contrary, it is a Transudate, derived from the blood by passive filtration. Heidenhain believed it to be an exudate for the following reasons: 1 It is considered probable that hemoglobin itself is the agent which brings about this decomposition. Catalase, which is also present in blood, decomposes hydrogen peroxide with the production of inactive, or molecular oxygen. ORIGIN AND COMPOSITION OF LYMPH 363 If the lymph were derived from the blood by mere leakage or filtration through the walls of the bloodvessels, the rate of leakage should be greater, the greater the pressure of the blood. The rate of flow of lymph in the Thoracic Duct, however, does not always decrease when the arterial blood-pressure decreases, nor does it always increase when the arterial pressure increases. Then, again, the injection of strong salt solutions into the circulation might be expected to with- draw fluid from the lymph-spaces by osmotic attraction, yet the lymph- flow from the thoracic duct is actually increased by this procedure. Finally certain specific substances, particularly crayfish extract, and extracts of leeches or shell-fish, certain Proteoses and also the South American arrowhead poison Curare cause a very great increase in the flow of lymph, as Heidenhain supposed, by stimulating the secretory activity of the vessel-walls through which the lymph issues into the interstices of the tissues. Nevertheless Starling has conclusively demonstrated that the pro- duction of lymph is, after all, a process of passive filtration. The phenomena adduced by Heidenhain, convincing as at first sight they appear to be, are nevertheless simply attributable to .the fact that the Permeability of the bloodvessels for lymph varies very greatly in differ- ent parts of the body. These differences in permeability lead to differ- ences in the rate of filtration of lymph no less pronounced than the difference in the rate of filtration of water through paper and through unglazed porcelain. The most permeable vessels are the capillaries in the Liver, while the capillaries in the skeletal muscles are almost impermeable. We can render the capillaries in the leg-muscles per- meable by heating them to 56 C., and in this way cause such extensive transudation of lymph that a frog's leg, so treated, becomes rapidly edematous. If the blood-pressure in the liver be raised or lowered the lymph-flow is raised or lowered in like proportion, but the pressure in the liver and that in the general arterial system do not always run parallel, so that the departures from parallelism between arterial pres- sure and lymph-flow observed by Heidenhain were not inconsistent with the view that lymph is a transudate, mainly furnished by the vessels of the liver. Strong salt or sugar solutions simply alter the distribution of the interstitial fluids, causing a general imbibition of fluid into the vascular system, and a Hydremic Plethora which results in readjustment by more rapid filtration into the lymph-spaces in the liver. If we previously withdraw from the vascular system enough blood to equal the volume of fluid which is attracted into it by the subsequent injection of salt or sugar, no plethora results, and no increased flow of lymph ensues. The various Lymphagogues or lymph-producing substances alluded to above cause an increased transudation by the injury they cause to the walls of the blood-vessels, greatly increasing their Permeability, and producing an effect analogous to that of heating to 56 C. The composition of lymph is very variable. In general it may be 364 VEHICLES OF CHEMICAL CORRELATION regarded as resembling blood-plasma, but containing a larger propor- tion of tissue waste-products and of fatty substances derived from the chylous lymph-vessels of the intestine. REFERENCES. THE COMPOSITION OF THE BLOOD: Abderhalden: Zeit. f. physiol. Chem., 1897, 23, p. 521; 1898, 25, p. 65. SERUM-PROTEINS : Reiss: Beitr. z, chcm. Physiol. u. Pathol., 1904, 4, p. 150. Arch. f. exp. Path. u. Pharm., 1904, 51, p. 18. Munch, med. Woch., 1908, 55, p. 1853. Jahrb. f. Kinderheilkunde, 1909, 70, pp. 3, 174. Ergeb. d. inn. Med. u. Kinderheilkunde, 1913, 10, p. 531. Deutsch. Arch. f. klin. Med., 1915, 117, p. 175. Robertson: Jour. Biol. Chem., 1912, 11, p. 179; 1915, 22, p. 233. Buck: Jour. Pharm. Exp. Therap., 1913-14, 5, p. 553. Wells: Jour. Biol. Chem., 1913, 15, p. 37. Thompson: Ibid., 1915, 20, p. 1. Briggs: Ibid., 1915, 20, p. 7. Tranter and Rowe: Jour. Am. Med. Assn., 1915, 65, p. 1433. Rowe: Jour. Lab. Clin. Med., 1915-16, 1, pp. 439 and 485. Arch. Int. Med., 1916, 17, p. 455; 1917, 19, p. 354. Righetti: Univ. California Pubs., Pathology, 1916, 2, p. 205. Hurwitz and Meyer: Jour. Exp. Med., 1916, 24, p. 515. Schmidt and Schmidt: Jour. Immunology, 1917, 2, p. 343. Hanson and McOuarrie: Jour. Pharm. Exp. Therap., 1917-18, 10, p. 261. Hanson: Jour. Immunology, 1918, 3, p. 67. Clark: Ibid., 1918, 3, p. 147. Toyama: Jour. Biol. Chem , 1918-19, 38, p. 161. COAGULATION OF THE BLOOD: Wooldridge: Proc. Roy. Soc , London, 1883-4, 36, p. 417; 1884-50, 38, pp. 69 and 260; 1886, 40, pp. 134 and 320; 1887, 42, p. 230; 1887-8, 43, p. 367; 1888, 44, p. 282. Uebersicht einer Theorie der Blutgerinnung, Ludwig's Festschrift, Leipzig, 1887, p. 221. Arch. Anat. u. Physiol., 1888, p. 527. On the Chemistry of the Blood, Collected Papers, London, 1893. Martin: Jour, and Proc. Roy. Soc., New South Wales, 1895. Sabbatini: Arch. ital. de Biol., 1903, 39, p. 333. Morawitz: Ergeb. d. Physiol., 1905, 4, p. 307. Loeb, L.: Biochem. Centr., 1907, 6, pp. 829 and 889. Virchow's Arch., 1904, 176, p. 10. Biol. Bull., 1902-3, 4, p. 301. Hofmeister's Beitr., 1904, 5, pp. 191 and 534; 1905, 6, p. 260; 1906, 8, p. 67; 1907, 9, p. 185. Hekma: Int. Zeit. phys. chem. Biol., 1915, 2, pp. 279, 299, 352. Biochem. Zeit., 1916, 73, pp. 370 and 428; 1916, 74, pp. 63 and 219. CHEMISTRY OF HEMOGLOBIN: Marchlewski: Die Chemie des Chlorophylls, 1895. Gamgee: Schafer's Text-book of Physiology, Edinburgh, 1898, vol. 1. Kiister: Zeit. f. Physiol. Chem., 1898-9, 26, p. 314; 1906, 47, p. 294. Nencki and Zaleski: Ber. d. d. chem. Ges., 1901, 34, p. 997. Marchlewski: Zeit. f. physiol. Chem., 1904, 41, p. 33. Butterfield: Ibid., 1912, 79, p. 439. Heubner and Rosenberg: Biochem. Zeit., 1912, 38, p. 345. Schumm: Zeit. f. physiol. Chem., 1913, 83, p. 1. Newcomer: Jour. Biol. Chem., 1919, 37, p. 465. CRYSTALLINE FORM: Halliburton: Quar. Jour. Micr. Sci., 1887, 28, pp. 181 and 201. Reichert: Am. Jour. Physiol., 1903, 9, p. 97. Reichert and Brown: Carnegie Inst. pubs. No. 116, Washington, 1909. Loeb and Brown: Science, N. S., 1917, 5, p. 191. Robertson: The Physical Chemistry of the Proteins, New York, 1918. ORIGIN AND COMPOSITION OF LYMPH : Starling: Principles of Human Physiology, Philadelphia, 1915. CHAPTER XVI. EXAMPLES OF CHEMICAL CORRELATION. THE CHEMICAL CORRELATION OF RESPIRATORY ACTIVITIES. The normal Respiratory Movements of the diaphragm and inter- costal muscles are adjusted to the average need for oxygen which is imposed by the normal functional activities of our tissues. The per- formance of function and the maintenance of the temperature of the body necessitate an expenditure of energy which, since the Hydrolyses which occur in living tissues are usually but slightly exothermic, must be derived for the greater part from energy liberated by Oxidations. As the tissues which are primarily concerned in the performance of mechanical work are the muscular tissues, variations of their activity may most clearly be seen to necessitate corresponding variations in the rapidity and extent of the oxidations upon which their power of per- forming work depends. Of all the various tissues of the body, in fact, the muscles are subject to the most sudden and extreme variations of functional activity, being at the one moment in the state of moderate tension which is the normal condition of rest, and at the next expending all the energy required, for example, to lift the whole weight of the body up a steep incline. To provide a sufficient oxygen supply to render possible at all times, without alterations of the respiratory rhythm, the maximal expenditure of energy by the skeletal muscles, would require a very great wastage of energy by the respiratory muscles themselves, or else the relegation of an excessive proportion of the bodily volume to performance of respiratory functions. The mechan- ism actually and normally employed provides an amplitude of oxygen for customary and moderate needs and when the oxygen requirements of the skeletal muscles renders the customary means of ventilating the body insufficient, then the efficiency of ventilation is temporarily enhanced by a very decided increase in the frequency and amplitude of the respiratory movements. Now there is no immediate or obvious connection between the move- ments of the respiratory muscles and those of the skeletal muscles. There is no anatomical or mechanical connection or association be- tween them that would render it a priori probable that the motions of the one group of muscles would tend to synchronize in frequency and extent with those of the other. Moreover, the respiratory movements in the adult higher vertebrates are known to be primarily under the control of a particular region of the Medulla Oblongata, situated in floor of the fourth ventricle, and designated the Respiratory Center. 366 EXAMPLES OF CHEMICAL CORRELATION Stimulation of this area enhances the rate and amplitude of the res- piratory movements. Its narcotization or injury depresses or annuls the respiratory movements. The actual synchrony is therefore not directly between the skeletal muscles and the respiratory muscles, but between the skeletal muscles and the nervous tissues of the respiratory center. Here we have an even less obvious relationship between tissues which nevertheless act in perfection of harmony, and the source of this harmony lies in a chemical and not in a spatial or mechanical interdependence of the tissues which participate in it. The initial effect of deprivation of oxygen or of interference by mechanical or other means with the entrance of air into the lungs is an increased amplitude and frequency of the respiratory movements, a condition which is designated Hyperpnea. This is succeeded by the stage of Dyspnea, in which the still more rapid movements become almost convulsive in character, until finally every muscle which can directly or indirectly assist in the effort to fill or empty the lungs is brought into i itense activity. This activity is quite uncontrollable, as the reader may convince himself by the simple endeavor to "hold the breath" for a prolonged period. If, finally, the lack of oxygen, or obstruction to the passage of air, still defeats the object of these exertions, a relatively sudden cessation of respiratory convulsions sets in, due to paralysis of the respiratory center, and the animal or man is now said to have suffered Asphyxia. If, on the contrary, instead of deprivation of oxygen or obstruction to the intake or exit of air, we have an exceptionally efficient ventilation of the lung, by forcible and repeated inflation or by a series of rapid and very deep voluntary breathing movements, then a condition of temporary suspension of the activity of the respiratory center sets in, a condition known as Apnea, which is purposely cultivated by divers and swimmers before undertaking a period of prolonged immersion below the surface of water. Either no desire to breathe is experienced for a perceptible interval, or the desire is very easily controlled by a voluntary effort. After a somewhat longer lapse of time than usual, however, the desire to breathe is again acutely felt, and the respiratory movements there- after become again uncontrollable by any effort of the will. Now the effects of suspended breathing are twofold. In the first place the supply of oxygen to the blood, and therefore to the tissues, is cut off and the available oxygen in the body, free or combined in easily dissociable compounds like Oxyhemoglobin is soon exhausted by the irreducible minimum of oxidative change which accompanies the life of all the tissues. In the second place the carbon dioxide which ultimately results from these oxidations cannot escape from the body, and therefore accumulates in the blood and in the tissues. The stimulated activity of the respiratory center which accompanies inadequacy of respiration is due to some change in the blood which irrigates it. This is conclusively shown by the fact that if the cerebral circulations of two animals be "crossed," so that the blood from the CHEMICAL CORRELATION OF RESPIRATORY ACTIVITIES 367 carotid artery of the one animal supplies the brain of the other, then the prevention of effective respiration in the animal of which the brain is receiving normal blood produces no hyperpnea or dyspnea, while the other animal, which can breathe freely, but whose brain is supplied with blood from an asphyxiated animal, shows every sign of respiratory distress. Two possibilities evidently exist, therefore. Either the stimulation of the respiratory center which results from prevention of the normal ventilation of the lung is due to a lack of sufficient oxygen in the blood which supplies the respiratory center, or else it is attributable to the accumulation of carbon dioxide in the blood-supply. It may be that both of these factors play some part in determining the total result, 1 but by far the predominant part is that which is played by the accu- mulation of Carbon Dioxide, as will be clear from the following con- siderations : In the first place it has long been known that a very slight increase, relatively speaking, in the carbon-dioxide content of the inspired air leads to a considerable acceleration and increase in amplitude of the respiratory movements. To bring about a like increase, by a mere decrease of oxygen, provided thorough ventilation of the lungs be secured by unobstructed breathing movements, requires a very much greater diminution of oxygen pressure than the requisite increase of carbon-dioxide pressure. Then, again, in the performance of muscular work there is little or no deficiency of oxygen in the blood, but the content of carbon dioxide must be increased, for the output of carbon dioxide in the lungs is increased and, furthermore, the carbon-dioxjde content of the air contained in the alveoli of the lungs, the Alveolar Air, is increased by work. The performance of work being in fact accomplished by means of the liberation of energy derived from oxi- dations, the end-products of these oxidations, among which carbon dioxide and water are predominant, must accumulate in the tissues during the performance of work, and therefore be more abundantly contained in the blood than during rest. The supply of oxygen to the tissues, on the other hand, is normally superabundant, and the muscular tissues, moreover, contain a certain reserve-store of oxygen, and when they are excised from the body, they can contract and perform work for a considerable period in an atmosphere which is devoid of oxygen. In fact, the saturation of the arterial Hemoglobin with oxygen is so nearly complete in normal respiration that the hyperpnea which results from energetic exercise would be devoid of utility if its object were the introduction of more oxygen to the tissues. Finally, we have seen that Apnea may result from enhanced ventilation of the lungs, but this is not due to an increased intake of oxygen or of satur- ation of the tissues therewith, for it may be brought about as well by 1 It is improbable that lack of oxygen is in itself a stimulus to the respiratory center or any other tissue. The apparent stimulation, if it occurs at all, is only indirectly attributable to deficiency of oxygen. 368 EXAMPLES OF CHEMICAL CORRELATION repeated and forced filling and emptying of the lungs by an indifferent gas, such as hydrogen or nitrogen, and when this has been done, it is found that the carbon-dioxide tension, in the alveolar air and therefore in the arterial blood, is decidedly lower than normal. The increased carbon-dioxide tension of the blood in obstructed breathing is therefore the stimulus which excites the activity of the respiratory center. But the carbon dioxide may conceivably act in either of two ways upon the center, namely as a specific chemical stimu- lant, or else indirectly by the increase in the hydrogen ion concentra- tion of the blood which it brings about. The experiments of Winterstein were designed to elucidate this question. This investigator introduced acids into the blood which was passing by the carotid artery to the brain, and he obtained a decided acceleration of the respiratory rhythm as a result. Prior to these experiments of Winterstein, it had also been shown that in frogs in which the floor of the fourth ventricle has been exposed, the direct application of acids to this area of the medulla causes acceleration of the respiratory rhythm, while that of alkalies slows it. Both experiments were inconclusive, however, because they did not enable us to ascer- tain whether the acids administered excited the center by virtue of the hydrogen ions which they contributed to the blood, or by setting free carbon dioxide from bicarbonates and thus increasing the carbon- dioxide tension of the blood. Subsequent experiments by Laqueur and Verzar threw more light upon the question, tending to show that carbon dioxide is a specific stimulant for the respiratory center, for they found, using Winterstein's technique, that the nature of the acid added to the cerebral circulation profoundly affected the result, and the efficiency of the various acids did not run parallel to their "strength" or dissociation into ions. Carbon dioxide, lactic acid and various fatty acids are much more efficient stimulators of respiration than the strong mineral acids. Evidently, therefore, we have here to deal with an effect which is not wholly a hydrogen-ion effect, but also in part an effect involving the undissociated molecule or the anions of the acid employed* THE CHEMICAL REGULATION OF THE CIRCULATORY SYSTEM. Removal of the Suprarenal Glands in animals, or their destruction by disease (usually tubercular) in man, is followed by the rapid appearance of intense prostration and muscular weakness. The blood-pressure falls to an extremely low level, and death finally supervenes. In man, the destruction of the glands by disease is usually somewhat gradual and the symptoms are correspondingly slow to develop. They are of the same description as those which develop in animals when these glands are excised, but, in addition, a peculiar patchy bronze-like pig- mentation of the skin occurs. The nature of the pigment which is deposited in these patches is unknown, but it is highly probable that CHEMICAL CORRELATION OF RESPIRATORY ACTIVITIES 369 it is chemically related to Adrenaline, for adrenaline, like the Tyrosine from which it is probably derived, is readily converted into highly colored substances by oxidizing-agents and by oxidizing-ferments, especially by the Tyrosinase which occurs in many vegetable tissues, particularly those of fungi, and also in certain animal tissues, as, for example, in tumors arising in the suprarenal bodies (melanomas) and in the ink-sac of the cephalopod sepia. The blood-pressure raising substance, adrenaline, which occurs in the medulla, or inner portion of the suprarenal gland, is capable, when administered intravenously, of correcting the excessively low blood- pressure in animals with the suprarenals excised, or in Addison's disease, but it does not avail to prevent the ultimate death of the animals and it is probable that other substances essential to life are produced by these glands besides adrenaline. It is possible that the cortex of the gland, which has an epithelial origin and differs both in structure and embryological development from the medulla, may play an equally essential part in the bodily economy. This is indicated in the first place by the fact that serious symptoms of adrenal insufficiency may accompany degenerative changes affecting the cortex alone, and further- more by the remarkable effect of extensive superficial Burns upon the cortex. Burns or scalds, if at all extensive, are followed by lesions of the suprarenal cortex and especially by minute hemorrhages therein. These changes are progressive for several days following the injury, and are prominent in instances of deferred death resulting from extensive burns or scalds. The unusual abundance of Lipoids and especially of Cholesterol Esters in the suprarenal cortex is suggestive of a function related to the lipoid metabolism, but the nature of this function remains unknown. It is of the active physiological principle of the medulla, namely Adrenaline, that our knowledge is most extensive. This substance, when injected intravenously in minute amounts (0.001 mg. and upward in a dog), causes a marked rise in blood-pressure (Fig. 22). This phenomenon is one of many consequences of the general action of adrenaline in stimulating the Myoneural Junctions of the muscles inner- vated by the sympathetic system. The action is not upon the nerves themselves, or upon their anatomically visible endings, for it is more and not less pronounced when the nerve is cut and allowed to degener- ate up to and including its anatomical connection with the muscle. The action is not upon muscle-fibers themselves, for in the first place muscles not innervated from the sympathetic system are not affected by adrenaline, and in the second place the muscles innervated by the sympathetic system are not all affected alike, for if inhibitory fibers predominate the muscle is relaxed, while if stimulatory fibers predomi- nate the muscle is contracted. The glandular tissues are variously affected, for if the adrenaline stimulates their secretory activity, the contraction of the bloodvessels and the consequently diminished blood- supply operate in a contrary direction. In the kidneys the diminished 24 370 EXAMPLES OF CHEMICAL CORRELATION blood-supply at first reduces the output of urine, but when the blood- pressure effect passes off, which it does rather rapidly, a decided Diuresis follows. Local subcutaneous administration of adrenaline so constricts the adjacent vessels that its absorption is thereby much delayed and its action is prolonged. It is upon this fact that the extensive employ- ment of adrenaline in minor surgical operations depends. Bleeding is prevented, and an unobstructed view of the tissues is secured for the period of the operation. Furthermore, local Anesthetics simultaneously applied share in the difficulty of absorption, and therefore continue their local analgesic action for a longer period than would otherwise be attainable. The tendency to post-operative hemorrhage is, however, said to be enhanced by adrenaline and it is also to be remembered that FIG. 22. Blood-pressure (B.P.} and bowel volume (7.7.) of cat. At A injection of adrenaline. The blood-pressure rises and bowel volume diminishes, indicating con- striction of the mesenteric vessels. As these relax again the blood-pressure falls. The vagi had been divided previously, so that there is no secondary slowing of the heart. (After Cushny.) the normal defense of the tissues against infections is supplied by the blood and by the leukocytes which the blood and lymph contain, so that a measure of natural protection against bacterial invasions is denied the tissues by this procedure. An important affect of intravenous injections of adrenaline is the appearance of Glucohemia and its resultant, Glycosuria. The power of the liver to polymerize glucose is apparently rendered deficient and the normal equilibrium between glycogen and glucose in the liver-tissues is shifted in favor of the glucose. It has been established in many ways that minute quantities of adrenaline are constantly present in the blood. That this must be continually supplied to the blood by the suprarenal glands follows from the fact that injected adrenaline very rapidly disappears from the circulation and the tissues, being apparently destroyed or, at all events, CHEMICAL CORRELATION OF PROCESSES OF DIGESTION 371 converted into substances devoid of the typical activities of adrenaline. It is, however, a question that is still being debated whether or not the small amounts normally present in the blood-stream actually influence the tone of the vascular system, and help to maintain the normal blood-pressure. The extremely low blood-pressure in Addison's disease, however, and the marked effect of adrenaline in raising it, stated even to be more marked than in normal individuals, would seem to point rather decisively to a constant relationship between the functional activity of the suprarenals and the maintenance of normal blood-pressure. According to Cannon, however, one of the most important functions of the suprarenals is to assemble a group of conditions appropriate for the defense of the organism in an emergency. Violent Emotional States, such as fear, rage or pain (and also anesthesia) lead to a marked discharge of adrenaline from the suprarenal glands, and to all the effects which arise from intravenous injection of adrenaline. The intravenous injection of adrenaline in the cat will, as a matter of fact, elicit very many of the most easily recognizable external signs of fear without the application of any other stimulus. Thus the hair of the back and tail is raised, and the pupils of the eyes are widely dilated. It is to the presence of an excess of adrenaline in the blood that the glycosuria of violent emotions, the so-called Emotional Glycosuria, is partly due. It has been shown by Macleod that stimulation of the splanchnic nerves, which innervate the suprarenal glands, results in the production of glucohemia which is partially attributable, however, to the direct stimu- lation of the liver through the hepatic branches of the splanchnics. The effect of emotional stimulation, operating through the splanch- nics, is to increase the adrenaline in the blood and thereby to increase the blood-pressure, quicken the heart-beat and thus enhance the mobil- ity of the blood and the rate of access and exit of the raw materials and products of metabolic activities. The liability to external hemor- rhages is reduced owing to the constriction of peripheral vessels, and also to a definite reduction of the coagulation-time of the blood, which is another result of adrenaline administration. The instantly avail- able nutritive materials for the muscle-cells are increased by the mobilization of sugar-reserves. In short the animal is placed in the best attainable condition for a sudden extreme effort and the sustain- ment of possible injury. In conflicts, or in efforts to escape from more powerful predatory forms, the suprarenal glands probably constitute an essential factor in success or failure. THE CHEMICAL CORRELATION OF THE PROCESSES OF DIGESTION. The arrival of foodstuffs in the stomach is preceded by a considerable secretion of Gastric Juice, and, in consequence, the processes of gastric digestion are enabled to go forward without delay. The correlation 372 EXAMPLES OF CHEMICAL CORRELATION between the acts involved in the intake of foodstuffs and the secretory activity of the glands of the gastric mucosa is, however, as the classical researches of Pawlow have shown, nervous in origin, and not chemical, arising in part from reflexes arising from optical and olfactory stimuli and in part from gustatory and tactile impressions. The detailed consideration of their mechanism belongs therefore to the domain of physiology rather than to that of biochemistry. The subsequent steps in the process of digestion involve, however, a very remarkable series of chemical correlations. During gastric digestion the pyloric sphincter remains closed, and it opens to permit the discharge of the stomach contents only when the digestion of the proteins has attained the stage of nearly complete conversion into Proteoses or Peptones. The mechanism which regulates the tone of the sphincter is nervous, but the stimulus which releases the reflex dilatation is chemical, and consists of the presence in the lower end of the stomach of foodstuffs containing a definite excess of hydrogen ions. This is very clearly shown by the investigations of Cannon, who found that the period which elapses before the first open- ing of the sphincter and discharge of Chyme into the intestine, is pro- portional to the quantity of substances in the food which are capable of neutralizing acids. Thus, solutions of sugar or starches are retained for but a brief time in the stomach, but the period of their retention may be enhanced very greatly by admixture with substances which neutralize free acids. Meat and other dietary constituents which contain proteins on the contrary are retained for a relatively prolonged period. When the acid chyme has been discharged into the duodenum in sufficient quantity to induce a certain acidity of the contents of the upper part of the small intestine, the pyloric sphincter again closes in accordance with the general law governing the musculature of the intestine, namely, that any localized stimulus causes relaxation below and contraction above the stimulated point. When the Chyme is being discharged from the stomach through the dilated pyloric sphincter, an augmented outflow of Pancreatic Juice is already travelling down the pancreatic duct to meet it. The time- relations of the production of the two digestive fluids, gastric juice and pancreatic juice, is illustrated by the following data obtained by Pawlow. Time after Gastric secretion Pancreatic secretion partaking of food. after 100 grams of meat. after 600 c.c. of milk. 1 hour 11.2 8.8 2 ' 8.2 7.5 3 ...... 4.0 22.5 4 \ - ... 1 . 9 9.0 5 ........... 0.1 2.0 the maximal secretion of pancreatic juice coinciding, in time with the moment when the maximal quantity of chyme is leaving the stomach. CHEMICAL CORRELATION OF PROCESSES OF DIGESTION 373 The immediate origin of this phenomenon resides in the acidity of the gastric contents which, upon the opening of the pylorus, come in contact with the mucosa of the upper part of the duodenum, and, in fact, a copious secretion of pancreatic juice may be elicited by simply bathing the duodenum with dilute acids, for example 0.4 per cent, hydrochloric acid. The same result is obtained if the acid be intro- duced into the jejunum, but not when it is introduced into the ileum. The exciting agent, however, is not the acid itself, for the injection of 0.4 per cent, hydrochloric acid (one-tenth normal) into the circulation is without effect upon the secretion of pancreatic juice. The excitation of the pancreas is, on the other hand, not accomplished through a nervous reflex because it occurs, and is undiminished when the portion of the intestine which is treated with acid is isolated from all nervous connections, and furthermore, it continues after the administration of Atropine, which paralyzes the endings of the secretomotor nerves. The actual intermediary which brings about this correlation is a substance Prosecretin which is present in the mucous membrane of the duodenum and the jejunum, and which is changed by acids into Secretin, a diffusible, water-soluble, heat-resistant substance, which has the property of specifically stimulating the secretory cells of the pancreas. If the mucous membrane be scraped from the surface of the duodenum and rubbed up in physiological saline solution (0.9 per cent. NaCl) the filtered extract which is thus obtained may be injected into the circulation without eliciting any secretion of pancreatic juice. If, however, the extract be previously boiled, or acidified and then neutralized, the injection will now be followed by a copious secretion of pancreatic juice. In normal digestion the transformation of the prosecretin in the duodenal mucosa into secretin is accomplished by the acid chyme, and the secretin which is formed is carried by the blood- stream to the cells of the pancreas. Secretin occurs in the mucosa of the intestine in all vertebrates and even in the intestines of fishes. It is diffusible, is not destroyed by boiling, and is soluble in acidified solutions of mercuric chloride, being precipitated on neutralization. It appears to be a nitrogenous base, and is probably an amine derived by Decarboxylization from an amino- acid or from an amino-acid derivative. Acidified extracts of the intestinal mucosa and of many other tissues, contain /3-Iminazolyl Ethylamine but this substance is devoid of action upon the secreting cells of the pancreas. The chemical identity of secretin has therefore not been established. A nitrogenous base having a similar action upon the pancreas is known, however, namely Pilocarpine, a trimethyl ammonium derivative obtained from the leaves of Pilocarpus jaborandi. It must be stated, however, that acids are not the only substances which will bring about a secretion of pancreatic juice when they come into contact with the duodenal mucosa. Fats are particularly active in causing secretion of the pancreatic juice after their entry into the duodenum, probably, however, only after they have been partially 374 EXAMPLES OF CHEMICAL CORRELATION converted into soaps. The origin of this effect is unknown. The Soaps, like other Calcium Precipitants are strong stimulators of nerve fibers and nerve endings, and the contention of Pawlow, that their action upon pancreatic secretion arises reflexly through stimulation of nerve endings in the intestine, is therefore not unfounded. On the other hand it has been suggested that the soaps formed from fats in the intestine, convert prosecretin into secretin or into some substance of like action, which is carried to the pancreas by the blood-stream. Other substances causing an especially abundant flow of pancreatic juice are Chloral Hydrate and Ethyl Alcohol. The chemical coordination of the processes of digestion does not end, however, with the coordination of the secretory activities of the digestive glands. If care be taken to excise the pancreas without allowing the tissues to come into contact with the mucous membranes of the intestine, or if the secretin is collected by means of a cannula placed in the duct, so that it is obtained before it touches the intestinal surface, it is found that the fluid is devoid of proteolytic activity. Yet the moment after it arrives within the intestine a very intense proteolytic activity is developed. The reason for this is that Trypsin is not present within the tissues or secretions of the pancreas as such, but in the form of a proteolytically inactive precursor which is desig- nated Trypsinogen. The conversion of trypsinogen into trypsin will not occur spontaneously, under aseptic conditions, even after a period of weeks or months. If, however, the fluid is momentarily acidified and then neutralized, the conversion of trypsinogen into trypsin is found to have been completed within the brief period of exposure to the action of hydrogen ions. A more prolonged exposure results in partial or complete destruction of the trypsin, and since the rate of secondary destruction of the enzyme is proportional to the dissociation or " strength' ' of the free acid, it is safer to employ, for the conversion of the trypsinogen, a weakly dissociated acid, such as Salicylic Acid, which furnishes a sufficiency of hydrogen ions to activate the trypsino- gen but decomposes the active trypsin relatively slowly. In actual digestion the activation of the trypsinogen may be brought about in part, it is true, by the admixture of the pancreatic juice with the acid chyme, for the contents of the duodenum are acid to Litmus, although alkaline to Methyl Orange, throughout the greater part of its length. But that another chemical mechanism exists in the intes- tine which is capable of bringing about very rapid and complete activa- tion of trypsinogen is shown by the fact that when the pancreatic secretion is poured into the empty intestine, the trypsinogen which it contains is found to have been activated within a very brief period after its arrival within the intestine; in fact mere contact with the surface of the intestinal mucosa for a few moments suffices to bring about a considerable degree of activation, and under such circumstances, of course, the reaction of the fluid remains consistently alkaline. This activation is brought about by a substance which is contained CHEMICAL CORRELATION OF PROCESSES OF DIGESTION 375 in the secretions of the intestinal glands comprising tlje so-called Succus Entericus. The activating constituent is designated Enterokinase, and because of the fact that it is destroyed or inactivated by heat- ing, and is furthermore active in very small quantities, it has been generally assumed to be an enzyme, and in fact Pawlow has termed it a "ferment of ferments." Nevertheless the proof that enterokinase is an enzyme is very imperfect. Many substances are modified by heat which are not enzymes, of course, and the small amount of the material required to activate a large volume of pancreatic juice may merely be expressive of the minute quantity of trypsin which is actually present in the secretion of the pancreas. We have no method of quantitatively estimating trypsinogen and enterokinase except in terms of each other and we have no data which could enable us to arrive at an estimate of the actual weight of trypsinogen which is activated by a given weight of enterokinase. More conclusive evidence of the enzymatic character of enterokinase would be afforded if we were to find that a limited quantity of succ is e itericus will activate very large quantities of pancreatic juice provided, only, that sufficient lapse of time be allowed for the completion of the process. But from the results of Hamburger, Hekma and others, it appears that the contrary is actually the case, and that there is a quantitative relationship between the amount of succus entericus which is added to pancreatic juice and the amount of trypsin which is produced. We have stated that the pancreatic juice, as produced by the secre- tory cells of the pancreas, is proteolytically inactive. While this is generally the case, it is*not necessarily or invariably so. The juice obtained by the action of Secretin is, it is true, invariably inactive, but juice obtained by stimulation of the secretomotor fibers in the vagus usually contains active trypsin, and juice containing preactivated trypsin may also be obtained after the administration of certain food- stuffs, particularly diets containing a high proportion of meat. The seat and mechanism of this activation is unknown. In general, however, it is evident that the proteolytic powers of pancreatic juice must be much enhanced by admixture with. succus entericus, in part through the activation of trypsinogen by entero- kinase and in part owing to the fact that succus entericus contains Erepsin, an enzyme capable of splitting peptones or casein, but not other proteins, to amino-acids. The digestion of protein by mixed proteolytic enzymes is always more rapid and complete than when a single enzyme is present, because different enzymes attack different linkages preferentially so that in the presence of two or more enzymes a larger number of amino-acid linkages are rendered susceptible to rapid disruption. This being the case it is a fact of interest and importance that pancreatic juice itself, according to Pawlow, stimulates, by its presence, the secretion of succus entericus. At all events during gastric digestion the secretion of fluid from the glands of the intestine is very small, but after passage of the chyme into the intestine and the coinci- 376 EXAMPLES OF CHEMICAL CORRELATION dent inflow of pancreatic juice and bile, the secretion of succus entericus is greatly increased. According to some observers, however, this increase is attributable to secretin, which is believed by them to stimulate the intestinal glands as it does the glandular cells of the pancreas. It is difficult at present to disentangle these alternative possibilities, and further investigation is evidently required before the relative parts played by secretin and the pancreatic juice itself in promoting the secretion of succus entericus can be correctly evaluated. THE CHEMICAL CORRELATION OF THE ORGANS OF GENERATION. The secondary sexual characters of the male, such as the growth of the beard and the deepening of the voice in man, the development of horns ia the ram and of the comb and tail-feathers of the cock, have long been known to be attributable to the development of the Testes. Castration has long been practised both in man and in animals for the purpose of preventing the development of secondary sexual characters, and of bringing about the psychic and metabolic modifications which also accompany the excision of these organs. The removal of the testes in man before the onset of puberty prevents the appearance of the beard and the deepening of the voice which characterises that period of development, and hardening of the epiphyses of the bones is delayed, so that the legs and arms grow to an unusual length in proportion to the size of the whole body. In certain varieties of sheep only the males are possessed of horns, and in these varieties castration of the young male altogether suppresses the development of the horns. Similarly the castration of cocks suppresses the development of the comb. If, however, the excised testicle be implanted in another part of the body, as, for example, in the peritoneal cavity, then the secondary sexual characters develop normally, the penis grows to its normal dimensions, the seminal vesicles and the prostate develop as if the testes were actually functioning as a generative organ, and yet, not only are the testes prevented by lack of communication with the Vas Deferens from discharging spermatozoa but, as a matter of fact, the spermatogenic tissues of the testes dwindle away, and the production of spermatozoa actually ceases. The effect of this organ upon the development of the secondary sexual characters is therefore, evidently, not attributable to its spermatogenic tissues, and appears to be due to the Interstitial Cells which are normally present between the seminal tubules and become increased in number in the transplanted organ. Since these tissues are provided with no duct for the conduction of their products to the exterior, the channel of transmission of the substances from the interstitial cells, or, as Steinach calls them collectively " The puberty- gland," to the tissues which they affect, can only be the general circu- lating media, the blood and lymph. The puberty-gland is, in fact, an example of the Ductless Glands, or Endocrine Organs. CHEMICAL CORRELATION OF ORGANS OF GENERATION 377 In the female, the excision of the ovaries leads to a more or less pronounced tendency toward the acquirement of masculine character- istics. Very marked effects upon the male, however, are elicited if the ovary be transplanted into the tissues of a castrated animal of the same species. In this case not only do the secondary sexual characters of the male fail to develop, but those of the female take their place, even to the development of the Mammary Glands. Here, again, the effect appears to be attributable rather to the interstitial elements of the ovary, than to the reproductive elements. A remarkable instance of the converse effect, namely, suppression of female characteristics by secretions from the male organs of generation, is supplied by the sterility which is almost the invariable rule in the females of heterosexual twins in cattle. A female of this type is known to cattle-breeders as a Free-Martin. It has been ascertained by F. R. Lillie that in cattle a twin pregnancy is almost always a result of the fertilization of an ovum from each ovary and development begins separately in each horn of the uterus. The ova, in the course of devel- opment, however, meet and fuse, and the bloodvessels from each side anastomose in the connecting part of the chorion, so that each embryo receives part of its blood-supply from the other. Both the arterial and venous circulations overlap, so that a constant interchange of blood takes place. If both are males or both are females no harm results; but if one is a male and the other female, the reproductive system of the female is largely suppressed in its development, and certain male organs even develop in the female. The effect of this is to render the female incapable of reproduction. A recurrent cycle of changes occurs in the Ovary of the adult female which results in the intermittent discharge of mature egg-cells from the ovarian tissues into the Fallopian tubes leading into the cavity of the uterus. The ovarian tissues contain a number of vesicles, lined with epithelium and each containing an ovum, which migrate toward the surface of the ovary, at the same time increasing in size. These are the Graafian Follicles, which periodically rupture, discharging the ova which they contain. The discharge of the egg into the Fallopian tubes may or may not coincide with the period of menstruation, in fact such evidence as we possess tends to show that the two processes, while coinciding approximately in frequency, do not occur with strict syn- chrony. The ruptured Graafian follicle, after the discharge of the ovum, undergoes a series of degenerative changes which culminate in the formation of the Corpora Lutea, which when mature appear as spherical masses of yellowish cells, disposed in a more or less columnar manner, the columns of cells radiating from the center. The Menstrual Fluid in ma consists of blood and shreds of cast-off uterine epithelium, diluted by the secretions of the mucous glands of the uterus. It contains a very high percentage of Calcium and for this reason Blair Bell has suggested that it may be related phylogenetically to the egg-shell of birds or of a remote common ancestor of the birds 378 EXAMPLES OF CHEMICAL CORRELATION and mammals. However this may be, a considerable storage of cal- cium occurs in the tissues of the female prior to menstruation, and this excess of calcium is suddenly discharged during the period of menstrua- tion. Having regard to the immense importance of the precise value of the ^ ratio in determining the susceptibility of nervous tissues to stimuli, it appears not unlikely that some of the nervous accompani- ments of menstruation, and particulirly the hyperirritability of the uterus which leads to the phenomenon of painful menstruation or Dysmenorrhea may be attributable in part to the sudden reduction of the calcium-content of the tissues which occurs at this period. The menstrual blood usually does not clot at all, or if it clots it does so very slowly. This remains the case even if fibrinogen be added to it, and, as .we have seen, calcium is not lacking. It can hardly be deficient in kephalin, or thrombokinase, since the fluid contains so much material arising from the breaking down of the tissues lining the cavity of the uterus. It appears likely, therefore, that the mucous secretions of the uterus contain a substance similar to Antithrombin or hirudin in its action upon the coagulation of blood. When the fertilized egg becomes imbedded in the wall of the uterus a proliferation of the uterine wall results in the outgrowth of a Placenta which subsequently provides the developing embryo with circulating blood derived from the mother. We have here a remarkably exact coincidence of events and we are led to inquire why the tissues of the uterus are aroused to the production of this outgrowth at the very moment when it is about to be required? An answer to this question has been afforded by the very important discoveries of L. Loeb. This observer has found that in the female guinea-pig, for a period of some ten days following the phenomenon of Ovulation, any injury to the uterine wall results in the outgrowth of a placenta. The injury may be of the nature of a slight incision, in which case a localized growth occurs which may be duplicated at other points in the uterus, so that as many as twenty different placentae may be formed in this way in a single uterus. Or the injury may consist of the irritation afforded by the presence of a foreign object, such as a thin glass rod or a number of particles of paraffin. In this case the growth of placental tissue may become so great as to interfere with the nutrition of the newly formed tissue and induce its degeneration and autolysis. The formation of placentae is prevented if the ovaries are extirpated or even if the Corpora Lutea which they contain are excised. The stimulus which arouses this reaction of the uterus to mechanical irritation comes, therefore, from the corpora lutea. If the corpora lutea are not excised at once, and placentse are permitted to form, they attain a smaller size and degenerate more rapidly if the ovaries or the corpora lutea are excised before their full development is attained. Among the many correlations which underlie and render possible the development of the embryo, the next into which we have attained some measure of insight is that which obtains between the development CHEMICAL CORRELATION OF ORGANS OF GENERATION 379 of the embryo and the development of the Mammary Glands of the mother. .As the fetus grows the mammary glands of the pregnant female hypertrophy until a portion of the hypertrophic tissue begins to break down^and give rise to a secretion of milk, and this stage of development is attained at the moment when the fetus is approaching the full term of gestation, and is about to be delivered. It has been ascertained that this remarkably exact synchrony of the development of such widely separated organized bodies as the fetus and the mammary glands of the mother is brought about by the circu- lation in the blood of some as yet unindentified substance which is elaborated by the tissues of the Placenta. If a saline extract of the placentae of rabbits be injected repeatedly into the circulation of virgin rabbits, the mammary glands hypertrophy just as they would if the animal were pregnant, and finally secrete milk which may be expressed from the nipples. Another factor, however, which may possibly contribute to the development of the mammary glands and their secre- tion of milk is the slight measure of hypertrophy of the Pituitary Gland which invariably accompanies pregnancy. The boiled aqueous extract of the posterior lobe of the pituitary gland contains a nitrogenous base of unknown constitution, designated Pituitrin, which increases the irritability of the muscular walls of the uterus, causes an increase in the volume of the urine and stimulates the secretion of milk, the latter effect being a very unusual one for any pharmacological agent to bring about. The large and repeated dosages of placental extract which Starling and Lane-Claypon found to be necessary to bring about the degree of hypertrophy of the mammary glands which is requisite for the production of milk may possibly have been attributable to the absence of the assistance, in these experiments, which is afforded in actual pregnancy by the enhanced activities of the pituitary body. That the hyperdevelopment of the mammary glands of the mother is due to the presence of stimulators circulating in the blood, and not to any reflex nervous stimulation of the glandular tissues, is shown, not only by the above-cited experiments, but also by the fact that the effect of these substances is not confined to the mother, but extends to the embryo, which is not connected by any nervous channels with the tissues of the mother. It is a familiar fact that the breasts of newborn infants frequently secrete a few drops of milk or may be made to do so by brief manipulations of the nipples. The milk thus obtained was known in former days as "witches' milk" and was accredited by the lady practioners of a hundred years ago with many important proper- ties of a supernatural description. When the development of the embryo has reached a certain stage, Uterine Contractions bring about the expulsion of the fetus. We have here another example of curiously exact coincidence in time. It is not a question of the size of the developing fetus ultimately bringing about such a degree of distention of the uterus as to induce a special tendency to contraction, for even the same individual may deliver infants in 380 EXAMPLES OF CHEMICAL CORRELATION successive births of very varying size in proportion to the bodily dimen- sions of the mother. The moment of delivery is, in fact,. primarily determined by physiological factors in the mother, rather than by the stage of development of the fetus at term. This may be very clearly seen by comparing the Variability of the duration of gestation with the variability of the weights of the infants which are delivered. The ordinary method of measuring the variability of any quantity which is adopted by statisticians consists in expressing it in terms of the percentage ratio of the Standard Deviation of the quantity measured to its average value. The standard deviation is the square root of the mean square of the observed deviations from the average. Thus, consider the following illustrative sets of measurements. i 11 101 2 12 102 3 13 103 4 14 104 5 15 105 It is obvious at a glance that the figures in the first column are very variable, those in the second column moderately so, and those in the third volumn relatively invariable or approximately constant. When we wish to express this impression in arithmetical terms we proceed as follows : Average of the first Average of the second Average of the third column. column. column. 3 . 13 103 the deviations from the average are in each case 2, 1, 0, 1 and 2. The sum of the squares of these deviations is 4+1+0+1+4=10. The mean square is therefore 2 and its square-root, which is the standard deviation, is 1.414. The variability of each of the columns of figures is the ratio of this quantity to the average, expressed as a percentage, which works out as follows: Variability of first Variability of second Variability of third column. column. column. 47. 1 per cent. 10.9 per cent. 1 . 37 per cent. Our impression of the relative variability of the three columns of figures is thus expressed in quantitative terms, the actual meaning of the results being, that in the first set of figures two-thirds 1 of the recorded values will be found to differ by less than 47.1 per cent, from the mean, in the second set two-thirds of the recorded values will differ from the mean by less than 10.9 per cent, of its value, and in the third set of recorded values two-thirds will fall within 1.37 per cent, of the mean. Applying this method to the study of the comparative variabilities of the period of gestation and of the weights of the infants delivered thereafter, we find that the two variabilities bear no proportion to 1 Or, more precisely, 68.27 per cent. CHEMICAL REGULATION OF METABOLISM 381 one another, for while the variability of the weight of newborn infants is 14 per cent., that of the length of the period of gestation is only 4 per cent. There can be little influence exerted by the size of the fetus upon the length of gestation, therefore, for otherwise the variability of the period of gestation would be nearly as great as the variability of the size of the infants delivered. It is evident that heavy infants are carried in utero for a longer period and light infants for a snorter period than w r ould correspond to their relative development. We must therefore look to maternal rather than to fetal events for the source of the determination of the period of gestation. Now the investigation of the physiological condition of the mother yields indica- tions of two factors which, as the term of pregnancy approaches, must enhance the muscular irritability of the uterus. The first is the hyper- trophy of the Pituitary Gland, to which reference has been made above. The aqueous extract of the posterior lobe exerts a very marked effect upon the excised uteri of animals, inducing powerful contractions, especially in the pregnant uterus. The active constituent is related to but not identical with /3-Iminazolyl Ethylamine. We may assume with probability that the hypertrophy of the gland which accompanies pregnancy may result in the presence of this substance in increasing amounts in the blood-stream until, finally, the hyperirritability of the uterus, with the assistance of the second active substance about to be noted, reaches a stage culminating in contractions which expel the fetus. The second factor which operates in the direction of promoting contraction of the uterus, is the presence of a substance io the Colostrum or first secretion of milk, which causes contractions of the pregnant uterus. In fact abortion has been brought about in pregnant cattle before the normal period of delivery, by injections of colostrum from a normal cow. Colostrum differs in many respects from the milk which is subsequently secreted. This will be clear from the following analyses of cows' milk, by Konig. Per 1000. Water. Solids. Casein. Other Fats. Sugar. Salts. protein. Colostrum . . 746.7 253.3 40.4 136.0 35.9 26.7 15.6 Milk .... 871.7 128.3 30.2 5.3 36.9 48.8 7.1 It has been recognized from a remote period that colostrum has a cathartic action upon the infant, so that the substance inducing uterine contractions may possibly be a general muscular stimulant. Its chemical nature is, however, unknown. THE CHEMICAL REGULATION OF METABOLISM. The activities of our various tissues are so closely interwoven with one another, and the various organs of the body are so intimately dependent upon one another for the raw materials which they elaborate into finished products, or the disposal of waste products which might otherwise be deleterious to the well-being of the whole bodily economy, 382 EXAMPLES OF CHEMICAL CORRELATION that the complete analysis of the coordinate factors of our total metab- olism would involve a survey, necessarily incomplete at the present stage of our knowledge, of the whole gamut of physiological activities. Without attempting to embark upon such an ambitious review, there are certain outstanding factors in the regulation of metabolic activity which compel our attention here, because the regulatory action which they exert would appear to constitute the prime function of the tissues concerned. FIG. 23. Cachexia strumipriva following total extirpation of thyroid; eleven years after operation. (After Kocher.) The most striking effects upon the general metabolism of the body are those which are exerted by the tissues of the Thyroid. Our attention was first drawn to the importance of this gland in the bodily economy by pathological conditions which are endemic in certain localities and sporadic in all human communities. The disorders resulting from improper functioning of the thyroid fall into two main classes, those namely, which result from subnormal development or activity of the gland, and those which result from its overactivity. The condition of Myxedema arises when the thyroid fails to develop CHEMICAL REGULATION OF METABOLISM 383 V properly, or, in later life, is extirpated, or injured by degenerative changes. If the failure of the gland occurs in childhood, intellectual development is arrested, and the condition known as Cretinism super- venes. The expression is idiotic, the skin is greatly thickened through the overdevelopment of connective tissue, and the features are conse- quently coarsened and brutalized. In adults, extirpation or destruc- tion of the gland by disease results in similar symptoms (Figs. 23 and 24) but the intelligence, although it becomes very sluggish, remains FIG. 24. The same patient as in Fig. 23, five months after thyroid administration. (After Kocher.) far above the level of an idiot. The temperature of the body is sub- normal, the total metabolism is much reduced, and the daily nitrogen output is subnormal. These conditions, if taken in hand early, are completely curable by the administration of extracts or dried prepara- tions of the thyroid gland. This is, in fact, the most completely success- ful instance of organotherapy to which we are as yet able to point, and provided the administration of the glandular preparations in appro- priate dosage be continued, individuals who would otherwise exhibit most extreme symptoms of the disorder remain in satisfactory health, with unimpaired intelligence and vigor. 384 EXAMPLES OF CHEMICAL CORRELATION The active and remedial constituent of the gland is associated with the Iodine which the thyroid contains and which distinguishes it chemically from all other tissues of the body. 1 While the iodine-con- tent of the thyroid varies very much, not only in different species of animals, but in different individuals of the same species, yet the minimal content of iodine which is consistent with normal functioning of the gland is very nearly constant and, on the other hand, the remedial value of a thyroid preparation tends to be proportionate to its iodine- content. The nature of the active iodine compound has been the subject of very many and extensive investigations. The experiments of Oswald showed that the active substance, as it exists in the glandular tissue, is either an iodized protein or closely associated with a protein which he termed Thyreoglobulin. Bauman found, however, that the partial hydrolysis of Oswald's thyreoglobulin by means of sulphuric acid did not destroy its therapeutic activity, but that a fraction of the hydrolytic cleavage -products which he termed lodothyrin retains the original activity of the thyreoglobulin. This substance, according to von Fiirth, is related to the "humin" substances which form in acid hydrolyses of protein in the presence of carbohydrate radicals, and are considered by Gortner and Blish to arise from the Tryptophane groups of the protein molecule. This fact has received peculiar significance as a result of the recent researches of E. C. Kendall who has succeeded in still further fractionating the hydrolytic cleavage-products of thyreoglobulin without destroying its therapeutic activity. By hydrolyzing thyreoglobulin in alkaline alcohol two groups of products are obtained. The one group is insoluble in dilute acids, the other is soluble. The acid-soluble substances are physiologically and thera- peutically inert and they contain very little iodine. The acid-insoluble substances contain a high proportion of iodine, and are physiologically and therapeutically potent. By further fractionation Kendall obtained a white crystalline product containing 60 per cent, of iodine, which was very active therapeutically and proved to be a derivative of Indol, being therefore related to tryptophane. Kendall believes that this compound which he designates Thyroxin is a tri-iodo-oxy-indol-pro- pionic acid, and has tentatively suggested the following constitutional formula: HI IHC C = =C.CH 2 .CH 2 .COOH IHC C C=O H 1 The alleged presence of iodine in the pituitary gland has not proved possible to confirm. CHEMICAL REGULATION OF METABOLISM 385 The administration of an excess of thyroid tissue to animals or man is accompanied by a very marked acceleration of metabolism. On a normal mixed diet the total heat-output may be raised 100 per cent. The effect of this enhanced metabolism is to cause a reduction of weight due to loss of tissue, and especially of fat, and it is for this reason that thyroid extract is the chief and only effective constituent of a variety of Obesity-cures. Unfortunately, however, the nitrogenous output is proportionately increased, so that the obese person loses not only fat, but also tissue-protein, which he frequently can ill afford to spare. Furthermore, distressing or even dangerous cardiac symptoms are liable to supervene with overdoses of thyroid extract, or even with moderate doses if the thyroid of the patient is normally active, so that the unrestricted use of thyroid preparations by the public is attended by serious danger. The stimulation of the destruction of nitrogenous tissue-constituents which follows the administration of thyroid is extremely striking. Thus, Rhode and Stockholm have found that in dogs receiving only sugar as a diet, so that the nitrogenous output was minimal, the output was increased fifty per cent, by so small an amount as 0.10 to 0.15 grams of dried thyroid tissue per kilogram body-weight of the animals. Arguing chiefly from the fact that his crystalline active fraction reacts with amino-acids, combining with the amino-group and liberating carbonic acid, Kendall has advanced the view that the thyroid secretion catalyzes the process of Deaminization of amino-acids. The power of deaminizing amino-acids is known to be shared by all the tissues and the stimulating effect of thyroid extract is likewise common to all tissues. The question is an extremely difficult one to decide, for when we recollect that the proteins of the tissues stand in a relation of equilibrium to the reserve amino-acids which they contain, and that these in turn are in equilibrium with the amino-acids circu- lating in the blood it is evident that anything tending to break down the amino-acids which have not yet become integral living tissue must also indirectly lead to the breaking down of tissue-protein, and the stimulation of endogenous catabolism. In support of Kendall's theory, however, may be cited the facts that hyperthyroidism, as in exophthalmic goiter, is aggravated by a high protein diet, and that the effects of thyroidectomy are more serious in carnivorous than in herbivorous animals. A remarkable effect of administration of thyroid tissue to mice is the extraordinarily increased tolerance for Acetonitrile to which it leads. Reid Hunt has found that if 0.1 milligrams of dried thyroid tissue be administered to mice on ten successive days, they will with- stand ten times the normal lethal dose of acetonitrile, administered subcutaneously, and indeed he proposes this enhanced tolerance to a specific substance as a test for the activity of various thyroid prepara- tions. The significance of this effect is, however, uncertain because it is not universal; in fact in such a closely allied animal as the rat, 25 386 EXAMPLES OF CHEMICAL CORRELATION administration of thyroid tissue, so far from enhancing the tolerance for acetonitrile, actually renders the animals more sensitive than usual to intoxication by this poison. Hyperthyroidism occurs spontaneously in the condition known as Basedow's Disease, or Exophthalmic Goiter. This condition is accom- panied by enlargement of the gland and a marked increase of secreting cellular elements, the interspaces filled with colloidal material which are characteristic of the structure of this gland being much reduced in size. There is a greatly enhanced metabolism, the calorific output being frequently twice the normal; there is a slow progressive loss of weight, incoordination o the heart-beat (Tachycardia), the tempera- ture is supernormal, the nervous system hyperirritable, and the blood- pressure is usually abnormally high. The rate and intensity of living is in fact increased in all its aspects, and frequently to a dangerous extent. The administration of thyroid preparations, or in fact of any iodine-containing substance, leads to a reduction of the Hyperplasia of the epithelium of the gland, and an increase in the quantity of col- loidal material, that is, to a return toward the normal structure. It is a question whether the symptoms of Basedow's disease are altogether attributable to hyperfunctioning of the gland. The remedial effects of iodine would point rather toward a deficiency of the iodine-contain- ing principle as the origin of the hyperplasia of the secreting epithelium which characterizes the disease. In fact the iodine content of the hyperplastic gland may actually be below normal, and a similar condi- tion may be aroused in the residue by excision of a considerable portion of the gland, as if the effort of a small part of the thyroid tissue to assume the functions of the whole stimulated a proliferation of the epithelial elements. On the other hand it must be recollected that a deficient content of any substance in a secreting gland does not neces- sarily mean that the production of the substance is diminished; it may merely mean that its rate of discharge from the gland is abnor- mally high, so that it has no opportunity to accumulate within the tissues of the gland itself. The prevalence of Myxedema and goiter in certain geographical areas and particularly in mountainous or hilly regions, and the com- parative rarity of such conditions elsewhere, has led us to ascribe the endemic forms of thyroid disease, directly or indirectly, to localized physiographical or geological conditions. Even in the days of Marco Polo, the prevalence of Goiter was attributed to a peculiar quality of the water in the localities affected, 1 and this impression still prevails, both in medical and in lay circles. Notwithstanding the clue this offered, however, it has not yet proved possible to establish the nature " Departing from thence " (Samarcand) " you enter the province of Karkan . . . The people ... are in general afflicted with swellings in the legs and tumors in the throat, occasioned by the quality of the water they drink." The "swellings in the legs" are attributable to a nematode worm, Filaria medinensis of which the "carrier," or intermediate host, is a minute fresh-water crustacean, Cyclops. CHEMICAL REGULATION OF METABOLISM 387 of the abnormality in drinking-water which causes disorders of the thyroid. It cannot even be definitely stated whether the abnormality consists in the presence of an infecting agent, or in a chemical compon- ent or its absence. The numerous circumscribed and yet widely sepa- rated areas of endemic occurrence, however, speak against the view that the disease is communicated by an infecting organism. The goiter which occurs among fishes in hatcheries, has been traced to overfeeding with a high protein diet. Lying just above the thyroid, or, in some animals, imbedded in the thyroid tissue, are a variable number (two pairs in man) of small glands, known collectively as the Parathyroids. Structurally they differ essentially from the thyroid and evidently they also differ from the thyroid very decisively in function, for their excision leads to quite a different sequence of events from those which follow thyroidectomy. The removal of the parathyroids, if complete, results in acute neuro- muscular symptoms which are collectively designated Tetany, and which resemble very closely a condition which not infrequently arises spontaneously in young children. For a little time succeeding para- thyroidectomy, no abnormalities appear, but within forty-eight hours tremors are observed in the extremities, followed by involuntary con- tractions of more and more muscles of the body until, finally, convul- sions supervene, terminating after several days in death. The condi- tion is completely relieved, according to W. G. Macallum, by the administration of Calcium Salts, and for this reason it was thought probable, for some time, that the special function of the parathyroids consists in the regulation of the Calcium Metabolism. Many facts, however, speak against this view. In the first place observers are not agreed that the excision of the parathyroids leads to increased excre- tion of calcium or a reduction of calcium in the blood and tissues, and in the second place other disturbances of metabolism to which attention has been directed in recent years offer a more probable origin of the neuromuscular symptoms. The remedial effect of calcium salts is regarded merely as an example of the general action of calcium in reduc- ing the irritability of nerve fibers. On the other hand some disturbance of the calcium metabolism unquestionably accompanies parathyroidec- tomy, for it has been found by Erdheim that parathyroidectomy in rats (probably not complete) leads to deficient dentine-formation in the teeth of the operated animals, and Erdheim and Carrel have found that callus-formation in injured bones is delayed by parathyroidectomy. The effect of parathyroidectomy upon the nitrogenous metabolism is very marked. The output of Ammonia is much increased, and for this reason Kendall and others have suggested that the parathyroids control the transformation of ammonium carbonate into Urea, which is the normal end-result of the deaminization of amino-acids, and occurs primarily in the liver. There is a decided Alkalosis or increased alka- linity of the blood in parathyroidectomy, and the symptoms may be alleviated by the injection of acids. On the other hand it has not 388 EXAMPLES OF CHEMICAL CORRELATION proved possible to induce tetany by injections of ammonia or ammo- nium carbonate. The urine of children between the ages of two and fifteen normally contains Creatine, which is absent from the urine of adults, 1 and it is between these ages that children are most liable to develop symptoms of tetany. On the other hand the content of creatine in muscular tissues is definitely connected with their Tonus or degree of tonic contraction and is increased by all measures which increase tonus. It has therefore been suggested by many observers that the tetany arising from parathyroidectomy may originate in a disturbance of the normal metabolism of creatine. In this connection it is of especial interest to note that Landois and Maxwell have found that while the gray matter of the motor-areas of the cerebral cortex is remarkably insensi- tive to the ordinary chemical stimuli which increase the irritability of nerve-fibers (calcium precipitants), it is powerfully stimulated by applications of creatine, with the effect of inducing convulsions. It has not, however, proved possible to induce tetany in animals by injections of creatine. Creatine is methyl guanidine acetic acid, and is therefore related to the amino-acid, Arginine, the relationship of arginine, methyl guanidine and creatine to one another is shown by the following formulae: /CH 3 ,NH.CH 2 .CH 2 .CH 2 .CH.NH 2 COOH /NH.CHs N CH 2 COOH C=NH C=NH C=NH \ S NH 2 \NH 2 ^NHz Arginine. Methyl guanidine. Creatine. Methylguanidine and Dimethyl Guanidine occur in small amounts in blood, muscular tissues 'and urine. It has recently been shown by N. Paton that the quantity of methylguanidine in the blood and urine is decidedly increased after parathyroidectomy in animals, and in the spontaneous tetany which occurs in children. The following figures are illustrative: Guanidine + Methylguanidine in milligrams per liter. A. BLOOD. DOGS. Normal Parathyroidectorny. . 1 . 00 (average of 5) 8.7 (average of 8) B. UBINE. DOGS. Nor mal. Parathyroidectomy. LO. 25 (average of 6) 1.1 (average of 6) CHILDREN. Idiopathic tetany. 0.12 (average of 8) (Average of 3 cases) Active tetany . . . . 0.58 Latent tetany . . . . 0.38 Recovery . . . . . 0.12 1 Occasionally present in the urine of women. CHEMICAL REGULATION OF METABOLISM 389 The subcutaneous or intravenous injection of Guanidine or Methyl- guanidine was found by Paton to lead to marked symptoms of tetany. Previous observers had established the fact that guanidine causes fibrillary twitchings of muscular tissue through stimulation, followed by paralysis of the myoneural junctions, and Fuhner, in 1906, demon- strated that this action is antagonized by calcium salts. The origin of parathyroid tetany would therefore appear to reside in a disturbance of nitrogenous metabolism, and especially in the metabolism of the guanidine derivatives. The aggravation of symptoms which accom- panies the administration of a high meat-diet is thus accounted for. Whether the parathyroids control the metabolism of other nitrogenous constituents of the diet besides those which contain a guanidine nucleus, is unknown, but the alkalosis which accompanies parathyroidectomy suggests that the products of metabolism which the parathyroids remove or elaborate are strongly basic substances such as might be derivable from the decomposition of Diamino Acids, of which, of course, arginine is an example. It has recently been shown by Uhlenhuth that tetany may be induced in amphibian larvae which do not possess parathyroids (Amblystoma) ; by the administration of thymus tissue, and he suggests that the func- tion of the parathyroids is to remove or render non-toxic substances produced by the Thymus. This would also explain the prevalence of tetany in children, since the thymus degenerates as maturity is attained. While this is very possible, it must also be remembered that the thymus is unusually rich, among animal tissues, in Thymus Nucleic Acid, which yields Guanine among its decomposition-products. Now guanine, when oxidized, yields, among other products, guanidine, so that the tetany observed by Uhlenhuth may have had a dietary rather than a specific glandular origin. REFERENCES. GENERAL: Biedl: The Internal Secretory Organs, their Physiology and Pathology, trans., Forster, London, 1913. Swale, Vincent: Internal Section and Ductless Glands, London, 1912. Schajer: The Endocrine Organs, London, 1916. Paton: The Nervous and Chemical Regulators of Metabolism, London, 1913. Starling: The Principles of Human Physiology, Philadelphia, 1915. RESPIRATION: Pembrey: Respiratory Exchange, Recent Advances in Physiology and Biochem- istry, by Leonard Hill, London, 1906. Robertson: Biochem. Zeit. Festband fur H. J. Hamburger, 1908, p. 287. Arch. Int. de Physiol., 1908, 6, p. 388. Pfliiger's Arch., 1912, 145, p. 329. Winterstein: Pfliiger's Arch.,- 1911, 138, pp. 159 and 167. Laqueur and Verzar: Ibid., 1912, 143, p. 395. Douglas, Haldane, Henderson and Schneider: Phil. Trans. Roy. Soc., 1913, 203B, p. 185. Douglas: Ergeb. d Physio!., 1914, 14, p. 338. Scott: Am. Jour. Physiol., 1917, 44, p. 196. CIRCULATION: Stewart; Jour. Exp Med , 1912, 15, p. 547. MacLeod and Pearce: Am. Jour. Physiol., 1911-12, 29, p. 419. 390 EXAMPLES OF CHEMICAL CORRELATION CIRCULATION : Folin, Cannon arid Denis: Jour. BioJ. Chem., 1912-13, 13, p. 477. Cannon and Gray: Am. Jour. Physiol., 1914, 34. p. 232. Cannon, Gray and Mendenhall: Ibid., 1914, 34, pp. 243 and 251. Cannon: Bodily Changes in Hunger, Fear and Rage, New York, 1915. Cannon and Caltell: Am. Jour. Physiol., 1916, 41, p. 74. Stewart and Rogoff: Jour. Lab. and Clin. Med., 1918, 3, p. 209. DIGESTION: Starling: Recent Advances in the Physiology of Digestion, Chicago, 1907. Cannon: Am. Jour. Physiol., 1907-8, 20, p. 283. The Mechanical Factors of Digestion, London, 1911. Pawlow: The Work of the Digestive Glands, London, 2d ed., 1910. ORGANS OF GENERATION: Lane-Claypon: Jour. Physiol., 1905, 32, p. xli. Blair-Bell: British Med. Jour., 1909, 1, pp. 517 and 592; 1913, 1, p. 652. Marshall: Physiology of Reproduction, London, 1910. Steinach: Centr. f. Physiol., 1910, 24, p. 551; 1913, 27, p. 717. Pfliiger's Arch., 1912, 144, p. 71. Loeb, L.: Proc. Soc. Exp. Biol. and Med., 1910, 7, p. 90. Jour. Morphology, 1911, 22, p. 37. Biol. Bull., 1914, 27, p. 1. Godlewski: Physiology der Zeugung in Winterstein's Handbuch de r vergleichenden Physiologie, Jena, 1914, vol. 3, Pt. 2. Robertson: Am. Jour, of Obstetrics, 1915, 71, p. 916. Lillie, F.: Science N. S., 1916, 43, p. 611; 1917, 23, p. 371. METABOLISM: Maxwell: Jour Biol. Chem., 1906-7, 2, p. 183. Macallum and Voegtlin: Jour. Exp. Med., 1909, 11, p. 118. Kocher: Les Prix Nobel en 1909, Stockholm, 1910. Cooke: Jour. Exp. Med., 1910, 12, p. 45. Kendall: Jour. Biol. Chem., 1915, 20, p. 501. Jour. Am. Med. Assn., 1915, 64, p. 2042; 1916, 66, p. 811. Endocrinology, 1918, 2, p. 81. Wilson and Kendall: Am. Jour. Med. Sc., 1916, 151, p. 79. Paton and Findlay: Quar. Jour. Exp. Physiol., 1916-17, 10, p. 324. Rhode and Stockholm: Jour. Biol. Chem., 1918, 37, p. 305. PART IV. THE CHEMICAL PROCESSES WHICH UNDER LIE AND ACCOMPANY LIFE-PHENOMENA. CHAPTER XVII. PROCESSES INFERRED FROM DIRECT OBSERVATION. THE INTERMEDIATE METABOLISM OF THE CARBOHYDRATES: MUSCULAR CONTRACTION. We have seen, in considering the chemical regulation of the respira- tory movements, that the energy-expenditure in muscular exertion is derived from oxidations. This follows immediately from the low heat- value of the hydrolyses which occur in the body, and which render them insufficient sources of energy, and from the greatly increased consumption of oxygen and output of carbon dioxide which accompanies the performance of muscular work. It remains to consider, however, what class of foodstuffs undergoes the oxidations which release muscular energy. That Carbohydrates afford a proportion of the necessary heat-units is evident from the fact that during the performance of muscular work the Glycogen which is normally stored up in muscular tissues, is greatly diminished in quan- tity, and even the further reserve which is stored up in the liver becomes much reduced by the performance of severe and long-sustained muscu- lar work. The potential energy contained in these reserves of glycogen is very considerable. Thus the liver of a man, when fully stocked with glycogen, contains about 150 grams of this polysaccharide, while the muscles, at rest and after feeding, contain a like amount. The total available reserve of carbohydrate material in the body is therefore about 300 grams, having a heat- value of 4.1 calories per gram or 1230 in all. If only one-fifth of this potential energy were converted into -mechanical work, its remainder being dissipated as heat, it would lift a weight of one hundred tons to a height of over three feet. 1 The exhaustion of glycogen by the performance of Muscular Work 1 The equivalent of one calorie in mechanical work 'is 426.5 kilogram-meters. 392 PROCESSES INFERRED FROM DIRECT OBSERVATION may be observed in a variety of ways. In the first place we may excise the two corresponding leg-muscles of a frog, analyze one to serve as a standard for resting muscle, and stimulate the other with a tetaniz- ing current until exhaustion supervenes, and the muscle will contract no longer, and then repeat the analysis upon this exhausted muscle. The content of glycogen in the stimulated muscle is invariably found to be lower than in the resting muscle, as much as fifty per cent, of the glycogen being generally found to have disappeared. Another way of approaching the problem is to cut the motor-nerves supplying one set of leg-muscles and, after the lapse of a definite period, to compare the glycogen-content of these muscles deprived of nervous connections with the glycogen-content of the corresponding normal muscles on the other side of the body. The muscles of our skeleton, while their nervous connections remain intact, are in receipt of con- stant slight nervous stimuli, insufficient to elicit actual contractions, but maintaining a condition of Tonus or constant tension which is a favorable precedent to rapid and forcible movements. This tonic contraction of the muscles of the skeleton consumes energy, not in the performance of external work, it is true, but in the performance of Internal Work; the overcoming of resistances analogous to friction or to the resistance to extension which is displayed by a liquid surface. This tonus and its resultant expenditure of energy are prevented by cutting off the stimuli which maintain it, so that a muscle with its motor-nerves severed, relaxes, and consumes less energy than a normal muscle with its nervous connections intact. Corresponding with this we find that the glycogen reserves in the paralyzed muscles tend to accumulate and to exceed the glycogen-content of the innervated muscles on the opposite, unoperated side of the body. This is clearly shown by the following determinations by Marcuse upon rabbits, the sciatic and crural nerves having been severed upon one side: . Percentage of glycogen. Experiment Paralysed Innervated Number muscles. muscle. 1 0.748 0.539 2 0.749 0.461 3 0.589 0.395 4 0.542 0.341 Average 0.657 0.434 The glycogen-reserve, through lack of expenditure, was therefore increased fifty per cent, in the paralyzed and demobilized muscles. Again, we may compare the glycogen-content of all the tissues in two similar animals, in the one after a period of rest, and in the other after a period of intense muscular exertion, and we obtain again the same result, namely a disappearance of glycogen with the performance of muscular work. Thus Kiilz forced a large and well-fed dog, weighing 45.5 kilos, to draw a heavy cart for nine hours and forty minutes. The animal was then killed, and the total glycogen-content of all its tissues INTERMEDIATE METABOLISM OF CARBOHYDRATES 393 was determined. Fifty-two grams of glycogen were obtained, corre- sponding to 1.16 grams of glycogen per kilogram of body-weight. A normal well-fed dog of similar dimensions contained 3.8 grams of glycogen per kilogram of body-weight. Even after four weeks of starvation a similar dog was found to contain 1.5 grams of glycogen per kilogram of its body-weight, so that somewhat less than ten hours of severe muscular exertion reduced the glycogen reserves of the body to a greater extent than four weeks of sheer starvation. So far, then, we have proved that muscular energy may be and is derived, in part at least, from the consumption of carbohydrate mate- rials. The question now remains, what proportion of the energy of muscu- lar work is provided by the carbohydrates of the food? For while the experiments which we have cited show that a part, and probably a large part of the energy expended in muscular work is certainly derived from carbohydrates, they do not preclude the possibility that an important proportion of the necessary heat-units may be supplied by other foodstuffs, for example by Proteins. This question was answered as early as 1865 by a classical experiment which was performed by Fick and Wislicenus. These observers ascended Mount Faulhorn, climbing to a height of 1956 meters above the starting-point. For seventeen hours before they started, during the six hours occupied in the ascent, and for six hours following the completion of the ascent they consumed no food which contained nitrogen. The urine passed during the ascent and in the six hours succeeding the ascent was collected and from its nitrogen-content the total quantity of body-protein which had been decomposed was estimated. It was found that Fick had decomposed 38.3 grams of protein while Wislicenus had decomposed 37.0 grams. Now if we assume, which, of course is not the fact, that all of the protein was decomposed so completely as to produce the end-products of perfect combustion, namely CO 2 , H 2 O and nitrogen, this quantity of protein would have liberated 250 calories, equivalent, if it were wholly con- verted into mechanical work, to 106,000 kilogram-meters. But Wisli- cenus, for example, weighed 76 kilograms, and the work which he actually performed in the mere effort of raising his body through 1956 metres was 76X1956=148,656 kilogram-meters, so that upon the most excessively liberal computation the protein which was decom- posed during and after the ascent could not possibly have furnished the energy consumed in the ascent. As a matter of fact, the actual yield of calories when protein is burnt in the body is much less than that which would be derived if combustion were complete, for instead of nitrogen being formed the oxidation stops with the production of Urea which has a very considerable heat-value of its own and which is voided from the body and not utilized. Furthermore no machine is known, not even a living machine, which can quantitatively convert heat into mechanical work. In fact actual measurements have shown that only twenty per cent, of the heat-value of foods is, as a rule, 394 PROCESSES INFERRED FROM DIRECT OBSERVATION available for the production of mechanical work. If we apply these various corrections to the above 'estimate of the work available from the proteins destroyed by Fick and Wislicenus, we find that it actually amounts to only 13,000 kilogram-meters, or less than nine per cent, of the work required merely to lift the weight of their bodies to the top of the mountain. Now it must be remembered that the ascent of their bodies was by no means the whole of the mechanical work which was performed by these experimenters, for apart from the tonus of their skeletal muscles, the work of the secretory and excretory organs, and the movements of the digestive canal, expenditures of energy that cannot very easily be computed, their circulations had to be maintained by the beating of their hearts and their respiratory movements by contractions of the diaphragm and intercostal muscles. These sources of expenditure of energy alone can be estimated to have accounted for no less than 30,000 kilogram-meters of work during the ascent of the mountain. All the energy actually procurable from the protein they decomposed, therefore, would not have half sufficed to maintain the respiratory movements and the heart-beat, leaving nothing over whatever for the ascent of the moun- tain. The proportion of muscular energy furnished by the proteins must therefore have been very small. That under normal conditions the whole of the energy consumed in muscular exertion is derived from non-protein sources, is rendered very probable by the discovery of Voit, that work upon the treadmill by a dog fed upon mixed rations does not increase the nitrogen output. Not only is the total nitrogen output unaffected by muscular work upon a mixed diet, but the entire Protein Metabolism pursues its normal course, undisturbed by the large expenditure of energy which is occur- ring. This is shown by an experiment by Shaffer, who investigated the urine of a man fed upon a purine-free diet, containing a minimal allow- ance of nitrogen, in three different periods, namely, a rest-period of six days which he spent in bed; a normal period of five days which he spent in performing light work about the laboratory, and a work period of four days in which he added to the laboratory work long daily walks. The following were the results obtained : Period. Food. Urine. N. grams. Calories. Total N. Nitrogen present as: Sulphur. Ammonia. Creatinin. Uric acid. Undeter- mined. I. Rest II. Normal III. Work . 5.9 6.0 5.9 2300 3000 3200 4.77 4.40 3.94 0.35 0.38 0.42 0.605 0.600 0.560 0.11 0.106 0.12 0.35 0.42 0.42 0.438 0.424 0.414 The question arises, however, whether, if placed under practical compulsion to do so, by the scarcity or absence of other source of INTERMEDIATE METABOLISM OF CARBOHYDRATES 395 energy, the muscular tissues may not be able to utilize proteins for the performance of mechanical work. Experiments of Kellner, conducted upon horses, render this very probable, for this observer found that while muscular work upon a mixed diet, as Voit had previously shown, does not increase the nitrogenous output of the horse, yet muscular work upon a diet which contained an insufficient allowance of carbo- hydrates did result in a notable increase of nitrogen elimination. This fact may be paralleled by the oft-repeated observation that while Bacteria will preferably obtain their energy from carbohydrates in the culture-medium, yet if these be insufficient in amount, proteins are attacked and energy is derived from the hydrocarbon radicals which they contain, nitrogenous fragments being split off as by-products of the process. Now proteins, being an abnormal source of muscular energy, may very possibly give rise to some unusual products when necessity com- pels their utilization for this exceptional purpose. We recognize that the protein metabolism of muscular tissues is peculiar. The abundance of Creatine in the muscles and the presence of Methylguanidine, Dimethyl- guanidine, Carnitine and other physiologically active nitrogenous bases in muscular tissues show that the degradation of protein in these tissues does not follow the channels normal to other tissues, and arouses the suspicion that rapid and extensive breaking-down of muscle-pro- teins might lead to the production of toxic bases in dangerous amounts and to notable physiological disturbances. We are reminded, in this connection, of the fact that the dangerous toxemia of pregnancy, Eclampsia, is often accompanied by sudden involution (degeneration) of the muscular tissues of the uterus. Nor are there wanting facts which tend directly to show that extreme muscular exhaustion upon a high protein diet may be dangerous. The experiences of Mawson and Mertz in the Australian Antarctic expedition of 1912-1913, which culminated in the tragic death of Dr. Xavier Mertz, may be instanced. In severe antarctic weather and heavily crevassed country, involving extraordinary expenditures of energy to maintain bodily heat and make progress over the ground, at a distance of three hundred miles from headquarters, these explorers, through loss of a companion and a sledge in a crevasse, found themselves with a bare one and a half weeks' food for themselves, and none at all for the dogs. They started to walk back to their headquarters, killing the dogs from time to time and consuming their necessarily excessively lean flesh. After eighteen days Mertz began to fail, and during several days expressed especial aversion to the dogs' meat; he displayed great muscular weakness, and complained of Violent abdominal pains from which Mawson also suffered. Seven days later symptoms of central nervous intoxication appeared. The following are notes from Mawson's diary: "January 7. It was a sad blow to me to find that Mertz was in a weak state and requited helping in and out of his bag. He needed rest for a few hours at least before he could think of travelling. I have 396 PROCESSES INFERRED FROM DIRECT OBSERVATION to turn in again to kill time and also to keep warm for I feel the cold very much now." "At 10 A.M. I get up to dress Xavier and prepare food, but find him in a kind of fit. Coming round a few minutes later, he ex- changed a few words and did not seem to realize that anything had happened . . ." "During the afternoon he had several more fits, then became delirious and talked incoherently until midnight, when he appeared to fall off into a peaceful slumber. . . After a couple of hours, having felt no movement from my companion, I stretched out an arm and found that he was stiff." 1 These are not symptoms of mere inanition. Definite intoxication was also present, and it appears not improbable that the extraordinary exertions necessitated by their situation, carried out as they were upon an almost exclusively protein diet, may have led to the abnormal disintegration of food- and tissue-proteins by the muscular tissues, with the production of poisonous nitrogenous, fragments. The employment of a high protein diet as a preparation for muscular exertion and endurance is therefore in the highest degree irrational, more especially since the rate of loss of heat from the body on a protein diet is diminished, so that the cooling necessary for the maintenance of prolonged bodily effort is rendered more difficult than usual. The only possible ground for the formerly popular dietary of beefsteak for athletes is the fact that on a diet purely of flesh the muscular machine is more efficient, i. e., produces less heat per unit of external work per- formed. In fact in a dog fed upon pure flesh Pflueger obtained the highest work-yield that has ever been observed, nearly fifty per cent, of the heat-value of the food appearing as mechanical work. For a short, sharp "dash" or brief effort, therefore, a high protein diet may possess advantages, but for prolonged extreme exertion a mixed diet containing an exceptionally abundant allowance of carbohydrates is the only rational prescription. This is, in fact, the actual dietary which, in the absence of suggestion or direction, is voluntarily chosen by those classes and groups of individuals whose mode of earning a living compels great and sustained muscular effort. The normal source of muscular energy is therefore the carbohydrates of the dietary. That the Fats may also be utilized for this purpose is evidenced by the fact, first established by Rubner, that fat and carbo- hydrate are Isodynamic Foodstuffs, i. e., that equicalorific amounts of these substances can replace one another in the diet. There has been some discussion of the question whether or not the fats are directly utilized for the performance of work, or whether they may not have to undergo a preliminary transformation into carbohydrates. This question has been experimentally investigated by Zuntz, who found that when carbohydrates predominate in the diet the total amount of 1 The Home of the Blizzard, Sir Douglas Mawson, London, 1915, vol. i, pp. 258-259. INTERMEDIATE METABOLISM OF CARBOHYDRATES 397 energy liberated by the body (work plus heat) corresponds to 9.33 small calories 1 for every kilogram-meter of work performed, while if the carbohydrate be replaced by fat, the total liberation of energy is 10.37 calories for the same amount and kind of work. Now 2.35 small calories are equivalent to one kilogram-meter of mechanical work, so that on a carbohydrate diet 25 per cent, of the excess of energy-dissipa- tion due to work was actually converted into mechanical work, and on a fat diet 22.7 per cent. There is thus little difference of efficiency whether fats or carbohydrates furnish the source of energy. Now if fats had first of all to be converted into carbohydrates, before they could be utilized for work, a great deal of oxygen would have to be introduced into the molecule, since the fats contain a much higher pro- portion of hydrogen to oxygen than the carbohydrates. If all this preliminary oxidation were unavailable for the production of muscular energy, not less than 29 per cent, of the energy of the fat would be wasted, and we would expect the performance of mechanical work on a fat diet to be only two-thirds as efficient as upon a carbohydrate diet. It is highly probable; therefore, that fats undergo but little preliminary modification before they are available for muscular work. They are not the first choice of the muscles, however, if all dietary materials are available, carbohydrates are used first. Fats are pressed into the service when carbohydrates begin to fail, and proteins form a last resource. The performance of muscular work involves a considerable increase of oxygen-intake, and carbon-dioxide output. The final products of muscular exertion are therefore carbon dioxide and water. The oxidation of glycogen or its hydrolytic cleavage-product, glucose, is not accomplished in a single step, however. Intermediate products are transiently formed, and of the nature of many of these we can only form conjectures which, however, are gradually becoming more and more clearly defined as persistent research reveals, one after another, the various substances which may arise from the oxidations of glucose in the animal body. One of the first of these intermediate products to be clearly recognized was, however, Lactic Acid. The lactic acid which is found in muscular tissue is not the ordinary racemic acid which may be obtained by synthesis in laboratory-glass- ware. It is the dextrorotatory acid, or Sarcolactic Acid: CH 3 I CHOH I COOH which, when pure, forms a viscous, acid syrup, forming crystalline salts with a variety of bases. The zinc salt is the one usually employed for the isolation and estimation of lactic acid in muscular tissues. 1 The small calorie is the heat required to raise the temperature of 1 gram of water one degree. The large calorie is the heat required to raise the temperature of a kilogram of water one degree. 398 PROCESSES INFERRED FROM DIRECT OBSERVATION There has been some discussion of the question whether the lactic acid of muscular tissues actually arises from the partial oxidation of carbohydrates or whether it may not, on the contrary arise from Proteins, as, for example, by the deaminization of the Alanine radical of proteins: CH 3 CH 3 I I CHNH 2 + H 2 O = CHOH + NH 3 I COOH COOH Alanine. Lactic acid. While such an origin of sarcolactic acid must be admitted to be possible, yet it is more probable that the major part of the lactic acid produced by the muscular and other tissues of the body, arises from a carbohydrate source. Thus Mandel and Lusk have shown that in Phosphorus-poisoning there is a great increase in the lactic-acid output in the urine. If, however, the body has previously been drained of its carbohydrate reserve by inducing Glycosuria through the administra- tion of Phloridzin, then phosphorus-poisoning results in no hyper- production of lactic acid. The lactic acid in excised muscles of the frog rapidly diminishes on standing. This is due to its oxidation by the muscle-tissues. Now the oxidation of lactic acid is evidently a more difficult step to accomplish than its production from glucose or glycogen, for, if the oxygen supplied to the muscles be interfered with by asphyxia, by inhalation of air poor in pxygen, or by poisoning with Carbon Monoxide, the lactic-acid con- tent of the tissues and of the blood and urine is enormously increased. One of the characteristics of extreme muscular Fatigue is the stiffen- ing and inextensibility of the muscles which ensues. After death the Rigor Mortis or postmortem stiffening of the muscles occurs with extreme rapidity if the animal has immediately prior to death been engaged in extreme and prolonged muscular exertion. The stiffening and increased opacity of the muscles which occurs after extreme fatigue or death is due to the coagulation of certain proteins which the muscle- fluids contain, the semifluid Muscle-plasma being converted into a jelly. It was found by Halliburton that if muscles be frozen and minced and then subjected to pressure at a temperature slightly above freezing, an opalescent fluid is obtained which clots spontaneously upon warming to a little above bodily temperatures, or upon standing for some time at room-temperatures. According to von Fiirth the gelatinization of this fluid is due to changes which occur in two proteins, the one a globu- lin, Myosin and the other an albumin, Myogen. The myogen fraction is much the more abundant of the two. Upon heating or acidification these soluble proteins are transformed, respectively, into Myosin Fibrin and Myogen Fibrin. The process is not reversible; the jelly cannot be liquified by cooling or by neutralization. It is believed that the partial gelatinization of these proteins which constitutes rigor in INTERMEDIATE METABOLISM OF THE FATS 399 muscles is brought about by the lactic acid and even, in part, by the carbon dioxide which accumulates in fatigued muscles. The Creatine content of muscular tissues is not decisively affected by muscular work. It appears that the increase, if any, is very slight, a fact which corresponds to the subordinate part which is normally played by proteins in the development of muscular energy. Neverthe- less Van Hoogenhuyze and Verploegh have found a definite increase of creatine in muscular tissues after severe work, provided the work was performed by starving animals. In other words if protein is of necessity employed by the muscles as a source of energy, then creatine is numbered among the chemical products of muscular work. The production of creatine appears, however, to bear an especial significance in relation to muscular Tonus; any agent tending to increase the tonic contraction of the muscles leading to an increased creatine-content. Thus the creatine-content of the muscles is increased by drugs such as Cinchonine which increase tonus, and in pregnancy the creatine-content of the muscular tissues of the Uterus is very greatly increased. THE INTERMEDIATE METABOLISM OF THE FATS; DIABETES. The normal products of the oxidation of the fats and sugars are finally, as we have seen, carbon dioxide and water. In animals with normal metabolism, but few of the intermediate products of oxidation can be perceived, because the various stages are passed through rapidly when the oxidation is once begun, and intermediate products of the process, therefore, have no opportunity to accumulate. One stage which is easily recognizable is that afforded by the production of Lactic Acid because the next step in the oxidative processes is evidently accomplished with relative difficulty, so that a proportion of this prod- uct accumulates in the tissues, especially if the oxidative processes are interfered with so as to increase the difficulty of further transfor- mation. Our knowledge of other stages, in the oxidation-processes of the body is, however, very largely derived from an experiment which is performed for us by nature in the disease or group of diseases known as Diabetes Mellitus. Glycosuria, the excretion of sugar in the urine, may be induced by the injection of physiologically unbalanced Salt Solutions and particu- larly by solutions containing Magnesium Salts. The origin of this glycosuria, whether it arises from an unusual discharge of sugar from the muscles or the liver, or from an increased permeability of the kidneys for sugar, has not as yet been ascertained. A glycosuria without any accompanying Glucohemia, that is, without any increase in the normal percentage of sugar in the blood, may be induced by the administration of the glucoside Phloridzin. This glycosuria is evidently due to an alteration of the normal Permeability of the kidney for sugar. The epithelium of the normal kidney interposes an impassable barrier 400 PROCESSES INFERRED FROM DIRECT OBSERVATION to the passage of sugar into the urine provided that the sugar in the blood does not much exceed the normal concentration of 0.10 to 0.15 per cent. After treatment with phloridzin, however, this barrier breaks down. The normal sugar-content is drained out of the blood, and the liver and muscles, in the endeavor to restore the normal equilibrium between Glycogen and Glucose, release glucose continuously to the blood, so that the ultimate result is the drainage of the carbohydrate reserves of the body. That the effect is a purely local one upon the epithelium of the kidneys is shown by the fact that if the phloridzin be supplied only to one kidney by perfusion into the corresponding renal artery, that kidney, but not the other, will eliminate glucose. It has been supposed that phloridzin, being a glucoside, acts as a carrier of glucose across the kidney-epithelium, liberating glucose on the one side and combining with it upon the other, but of this we have no definite proof. Yet again, glycosuria may result, temporarily, from an excessive ingestion of carbohydrates, and particularly of sugars. This form of glycosuria, known as Alimentary Glycosuria is not serious unless, indeed, it occurs too readily, when it may indicate a slight or incipient diabetes. It is stated by Gushing that alimentary glycosuria tends especially to occur in conditions of Hyperpituitarism or overactivity of the pituitary gland, of which condition, in fact, he considers a readily elicited alimentary glycosuria to afford confirmatory diagnosis. In the opposite condition of Hypopituitarism he finds, on the contrary, an extraordinary tolerance for ingested sugars and alimentary glycosuria fails to appear 'after a dosage of glucose or levulose which, in normal individuals, would inevitably be followed by an excretion of sugar in the urine. Other observers, while confirming Cushing's observation that pituitary disease is accompanied by disturbances in the carbohy- drate-tolerance, do not concur with him in his view of the relationship of the disturbance to hyper- or hypo-functioning of the pituitary gland. It must be recollected in this connection, however, that our means of distinguishing between hyper- and hypo-activity of the pituitary gland are rendered very imperfect by the fact that the physical effects of previous hyperactivity of the pituitary body persist, and may in fact constitute the most prominent symptoms, long after the condition has passed into one of deficient activity of the gland. The possible involvement of the nervous system in the etiology of ^jt diabetic conditions was very strikingly brought into prominence by the discovery of Claude Bernard in 1854 that injury of a certain area in the medulla oblongata induced a transitory but severe glycosuria. The particular area concerned lies between the level of the origins of the auditory nerves and the vagi. The Diabetic Puncture is most suc- cessful in animals that have been well fed with carbohydrates and may fail in ill-nourished animals. The immediate cause of the excretion of sugar which follows this operation is a pronounced Glucohemia, the sugar in the blood rising from the normal level of 0.10 or 0.15 per cent. INTERMEDIATE METABOLISM OF THE FATS 401 to 0.3 per cent, or more, and the kidneys simply excrete that proportion of the blood-sugar which constitutes an excess over the normal amount. The glucohemia which ensues after the diabetic puncture is evidently due to a failure of the normal power of the liver to store up glucose in the form of its anhydride, glycogen. The efficiency of the operation is proportional to the glycogen-content of the liver at the time it is per- formed, and at the end of the process the liver is found to have been drained of its glycogen-reserves. It appears that the storage-capacity of the liver is subject to control by the nervous system. The afferent path in the reflex arc is contained in the vagi. If the vagus is cut and the peripheral end is stimulated no glycosuria ensues, but if the central end is stimulated a decided discharge of sugar from the liver occurs. The efferent paths lie in the splanchnic nerves, and if these be previously severed the diabetic puncture is without effect. The greatest advance toward the interpretation of spontaneous diabetes, however, occurred when in 1889 von Mering and Minkowski discovered that extirpation of the Pancreas in animals produces a pro- found glucohemia and glycosuria terminating ultimately in the death of the animal. The effects of this operation have been very exhaus- tively studied in recent years by F. M. Allen who finds that glucohemia and glycosuria may be induced by partial removal of the pancreas. If nine-tenths of the gland be excised a severe diabetes ensues, but if only a small part of the pancreas, for example one-eighth, be removed, a mild diabetes ensues which is modifiable by diet. Thus if a sufficiency of the pancreas be left in situ no glycosuria at all may appear in the urine. If the remnant of gland be larger glycosuria may be absent on a meat-diet or even on a diet containing bread, but glycosuria will ensue if sugars be added to the diet and, once started, may continue on a bread-and-meat diet. In turn, continued glycosuria upon a bread- and-meat diet may culminate in a condition in which glycosuria con- tinues on meat alone, and the experiment terminates fatally. The interesting observation has been made by Carlson, that if glyco- suria be induced in a female animal by depancreatization, and the animal subsequently becomes pregnant, the glycosuria ceases at the time that the pancreas begins to develop in the embryo. It is not certain, however, whether this is due to the mother being enabled to utilize glucose herself through transmission of a pancreatic hormone from the fetus to the maternal circulation, or whether, which is perhaps more probable, the drainage of carbohydrates from the mother by the needs of the fetus deprives her of the excess which she is unable to utilize herself. In fatal cases of diabetes it has repeatedly been observed that degenerative changes are present in certain elements of the pancreatic tissues, namely the Islets of Langerhans, and it is particularly to the removal of these elements that the diabetes following total or partial extirpation of the pancreas is due. Thus injection of paraffin into the ducts arising from the secretory tissues of the pancreas results in 26 402 PROCESSES INFERRED FROM DIRECT OBSERVATION complete atrophy of the secreting epithelium, the Islets of Langerhans alone remaining unimpaired. Under these circumstances no glycosuria occurs, but if this atrophied remainder of the gland be removed typical pancreatic diabetes at once occurs. When the pancreas is only parti- ally removed the overstrain upon the remainder of the tissues leads to their degeneration and the symptoms, possibly slight at first, become progressively more severe. According to Allen, however, if the residue of pancreatic tissue be sufficient and overstrain be avoided by a diet low in carbohydrates and in fats, the incidence of progressive degenera- tive changes in the residual tissues may be avoided. The occurrence of spontaneous Diabetes in human beings has been recognized from very ancient times, but the actual identification of the sweet constituent of the urine as Glucose was not accomplished until 1838. It is characterized, it would appear, almost if not quite invari- ably by a distinct Glucohemia. It is improbable that any cases of spontaneous and persistent glycosuria are due solely to increased permeability of the renal epithelium such as may be brought about experimentally by the administration of phloridzin. The light forms of diabetes resemble alimentary glycosuria except in the fact that the Assimilation-limit for carbohydrates is unusually low so that glycosuria recurs whenever a normal abundance of carbohydrate is ingested. In such cases the mere performance of muscular work may arrest the gly- cosuria. Between this light form of diabetes and the more severe forms every intermediate stage may be observed, and not infrequently the same patient may pass through all degrees of severity of the disease successively. In most severe forms of diabetes sugar continues to be eliminated on a pure protein diet and the urine may contain over ten per cent, of glucose, being usually, but not invariably, dark and dis- colored from the presence of other abnormal constituents arising from the disordered metabolism. The sugar which is excreted in the severe forms of diabetes does not arise from carbohydrates in the diet or in the tissues, for not only does it continue on a carbohydrate-free diet, but the quantity excreted per diem may be far in excess of the carbohydrates in the food and in the tissues of the body added together. Thus in one experiment upon a depancreatized dog Pfliiger found that out of a total excretion of 3097 grams of sugar only 422 grams could possibly be accounted for as arising from carbohydrate reserves of the animal. The dif- ference, namely 2675 grams, must have arisen from some other source. Liithje even went so far as to feed a depancreatized dog com- pletely upon casein. In eight weeks it excreted nearly 1200 grams of sugar, only a small proportion of which, of course* could have been derived from glycogen in the tissues of the animal. Since the fats, upon a diet such as this, are very quickly used up, we have no alternative but to assume that the sugar was derived in part from the decomposition of proteins and, as a matter of fact, in the severer forms of diabetes there is a decided tendency for the ratio of the sugar INTERMEDIATE METABOLISM OF THE FATS 403 (dextrose) to the nitrogen eliminated in the diet to approach a constant level. This ratio, designated usually by the symbol ^ is regarded by Lusk as affording valuable indication of the severity of the diabetes, for he finds that upon an exclusively fat-and-protein diet the ^ ratio in the severest cases of diabetes approaches a critical value of 3.65 : 1. If the sugar excreted were wholly derived from protein this would mean that from 6.25 grams of protein decomposed in the tissues of the diabetic, 3.65 grams, or 58 per cent, of the weight of the protein, was transformed into glucose. This Lusk believes to be the maximal quantity of carbohydrate which is obtainable from protein, and he illustrates this by reference to the following figures: Maximum ratios observed in: Phloridzin diabetes. Diabetes mellitus in man. In dog. Lusk. In man. Benedict. Mandel and Lusk. Grunwald. Foster. Mosenthal. Joslin. 3.65 3.66 3.62 3.64 3.58 3.82 3.66 3.68 3.60 3.65 3.66 3.75 3.56 3.70 3.64 3.58 3.38 3.48 3.75 3.85 3. 44 3.66 3.69 3.67 3.67 3.68 3.64 According to Joslin these high ratios, which usually only precede death by a brief interval, are never observed if Fats be excluded from the diet, a fact which is a very striking illustration of the deter- minative part played by fats in the evolution of diabetic symptoms, a part, however, which has only in recent years come to be fully appre- ciated, thanks to the work of Allen, Joslin, Bloor and other investi- gators. The excretion of sugar in diabetes mellitus is not attributable to loss of glycogen storage-capacity on the part of the liver, for eve a in fatal cases, and after a prolonged excretion of sugar, appreciable quantities of glycogen may still be found in the liver. It is naturally a difficult matter to ascertain whether or not the storage capacity of the liver in diabetics is fully normal, but there can be no question but that the main abnormality of the carbohydrate metabolism in diabetes is essentially a failure to utilize the glucose in the diet. The tissues which are unable to utilize glucose are nevertheless starving for it and every possible mechanism for manufacturing glucose from other foodstuffs, even as we have seen, from proteins, is pressed into service, but the product of these efforts is still glucose and, therefore, worthless or even worse, for it is excreted from the body and involves a corresponding wastage of the fuel-value of the dietary. The failure of the diabetic to oxidize glucose does not by any means originate in a failure of oxidative powers in general. On the contrary the relationship of the condition to glucose and also as we shall see, to 404 PROCESSES INFERRED FROM DIRECT OBSERVATION the fats is highly specific and the oxidation of other and even much more difficultly oxidizable substances may be normal; thus Lactic Acid, Mannitol and even Inosite or Benzene are oxidized just as well by the diabetic as by the normal individual. Even a very slight degree of oxidation of glucose itself suffices to enable the tissues to overcome the obstacle. Thus gluconic acid, glucuronic acid, saccharic acid and mucic acid are all readily oxidized by a diabetic. The relationship of these substances to glucos*e may be seen from the following formulae CHO COOH CHO COOH COOH ill!! HCOH HCOH HCOH HCOH HCOH I ' I I ! I HOCH HOCH HOCH HOCH HOCH I I I I I HCOH HCOH HCOH HCOH HOCH I I I I I HCOH HCOH HCOH HCOH HCOH r i i i i CH 2 OH CH 2 OH COOH COOH COOH Glucose. Gluconic acid. Glucuronic Saccharic Mucic acid. acid. acid. Even more surprising is the fact that sugars other than glucose may be very much better utilized by a diabetic than glucose itself. Cane- sugar is badly tolerated, as might be expected from the fact that it yields glucose on hydrolysis. For the same reason Maltose, which yields two molecules of glucose when hydrolyzed, is even less well tolerated by diabetics than cane-sugar. Lactose is very badly tolerated probably because it gives rise, on hydrolysis, not only to glucose but also to Galactose which is very poorly assimilated by diabetics. Levu- lose, on the contrary is comparatively well assimilated. In many cases it is possible to administer levulose to diabetics without untoward symptoms when similar quantities of glucose would precipitate a pro- found glycosuria. Depancreatized dogs will store up glycogen on a levulose diet when they cannot do so on a diet containing equal quanti- ties of glucose. For this reason, since levulose is somewhat expensive, it has been proposed to administer Inulin to diabetics. Inulin is a polysaccharide of levulose which occurs in the tubers of dahlias, the tuberous artichoke and the sweet potato. It is, however, indigestible by any of the alimentary juices and simply increases the bulk of the feces and provides a culture-medium for intestinal bacteria. The bacteria in the lower intestine certainly attack inulin and the products of their activity may be absorbed or utilized, but these products are not of a carbohydrate nature, for if inulin be administered to an animal with phloridzin glycosuria, no increase of sugar-output is observed. Inulin is therefore of little if any value to a diabetic. Now it is a very significant fact that when levulose is tolerated by a depancreatized animal, it is converted into glycogen in the liver. This would point, seemingly, to a failure of the liver to convert glucose into glycogen in diabetics, although it is well able to store the glycogen INTERMEDIATE METABOLISM OF THE FATS 405 when it has once been formed. A slight change in the configuration of the molecule of sugar which is absorbed and carried to the liver enables the liver to perform its customary function. The Urine of diabetics has very frequently a pronounced fruity odor, and is usually decidedly acid in reaction. These characteristics of diabetic urine are due to the presence therein of extraordinary amounts of Aceto-acetic Acid, CH 3 COCH 2 COOH, Acetone, CH 3 COCH 3 and Hydroxybutyric Acid, CH 3 CH(OH)CH 2 COOH. These products are all closely related to one another and unquestionably arise from the same source. Thus aceto-acetic acid may be derived from hydroxy- butyric acid by oxidation, water being split off, while aceto-acetic acid, with the loss of carbon dioxide, is convertible into acetone. It is probable that hydroxy butyric acid is the parent substance of all the "acetone-bodies" which are found in the urine of diabetics. The production of these substances rapidly and in large amounts, produces the extreme Acidosis which is characteristic of the later stages of untreated or improperly treated diabetes, and which culminates in the Diabetic Coma or acid-intoxication which formerly was the invariable and still is the very frequent termination of the disease. The amount of acetone in diabetic urine is comparatively small and it is of minor significance. The aceto-acetic acid may be detected by the deep red color which is communicated to urine containing this substance if Ferric Chloride solution be added to it in excess of the amount necessary to precipitate the phosphoric acid as ferric phosphate. It was formerly believed that the acetone bodies in urine were derived from the imperfect oxidation of Carbohydrates and that they probably represented intermediate stages in the degradation of carbohydrates to carbon dioxide and water. This view has now been abandoned with the recognition of the fact that Fats play a predominant part in the genesis of diabetic Acidosis. The very slight change in the glucose molecule which suffices to render it assimilable and utilizable points, in any case, to the improbability that succeeding stages in the oxidation of glucose are exceptionally delayed in the tissues of the diabetic. If oxybutyric acid were in truth an intermediate stage in the oxidation of carbohydrates, as lactic acid, for example is known to be, then the accumulation of this substance in the blood and in the tissues must mean that the subsequent steps of oxidation have become exceptionally difficult. But the very slightest initial oxidation of glucose renders it readily utilizable, so that we must infer that all stages of oxidation succeeding the formation of gluconic or glucuronic acids, for example, are readily performed by the diabetic. Now oxybutyric acid, if it were formed at all from glucose, must succeed the formation of gluconic or glucuronic acids, so that the accumulation of this substance in the tissues of a diabetic evidently cannot be due to the arrested oxidation of carbohydrates. As a matter of fact, Macleod and Pearce have found that the oxidation of glucose in the tissues of depancreatized or even in eviscerated animals is in no way defective, and Meltzer and Kleiner 406 PROCESSES INFERRED FROM DIRECT OBSERVATION have shown that a large part of the glucose which circulates in the blood of a diabetic is actually utilized by his tissues. The examination of the blood in diabetics very frequently reveals, not only glucohemia, but also a pronounced Lipemia, which may be so severe as to give to the centrifuged blood-serum a distinctly milky appearance. The following are results obtained by Bloor in estimating the lipoids in normal and in diabetic blood : Total fatty acids, grams per 100 c.c. Lecithin, grams per 100 c.c. Cholesterol, grams per 100 c.c. Whole blood. Plasma. Cor- puscles. Whole blood. Plasma. Cor- puscles. Whole blood. Plasma. Cor- puscles. Diabetic extremes Diabetic average (34 analyses) Normal average (19 analyses) Normal extremes .41-. 76 .52 .37 .29-. 42 .46-. 93 .59 .39 .30-. 47 .33-. 62 .43 .34 .27-. 45 .26-. 50 .36 .30 .28-. 33 .17-. 48 .30 .21 .17-. 26 .32-. 60 .46 .42 .35-. 48 .19-. 44 .29 .22 .19-. 25 .16-. 65 .36 .23 .19-. 31 .17-. 24 .20 .20 .17-. 24 It will be observed that the percentage of all the lipoidal constituents of the plasma is much increased in diabetics, while the lipoidal con- stituents of the corpuscles remain comparatively unaffected. The increase is especially marked in the Neutral Fats (estimated as fatty acids) and in the Cholesterol fractions. The lecithin or Phospholipin fraction increases also but in much less proportion than the others, so . fatty acid cholesterol that the ratios : r-p or = T-T-. are abnormally high in lecithin lecithin diabetic blood-plasma. For this reason it has been suggested that part at least of the failure of diabetics to utilize fat is due to an inability to convert neutral fatty acids into phospholipins. The attention of earlier investigators of diabetes was focussed upon the intolerance of these patients for carbohydrates, and the main objective of the physician was to decrease the output of glucose in the urine. Carbohydrates were therefore necessarily excluded from the diet, and to replace the deficient calorific value thus entailed the fats in the diet were not unusually increased. This procedure frequently had the gratifying result, for the time being, of reducing or even elimi- nating the output of glucose in the urine, but sooner or later the patient, whose condition at first seemed much improved, would again begin to excrete glucose; a severe acidosis developed and the case became hope- less, terminating in diabetic coma. This result has been duplicated by F. M. Allen in partially depan- creatized dogs, and he attributes it to the progressive degeneration of the Islets of Langerhans in the residual tissue due to overstrain. As a source of protein he administered beef-lung to the animals, and suet was employed as a means of administering fats. The following is his description of a typical result : "We may take the customary treatment of moderate diabetes and INTERMEDIATE METABOLISM OF THE FATS 407 illustrate it in dogs. Suppose that suitable operation and overfeeding have produced a condition where there is marked glycosuria on a kilo- gram of lung, but sugar-freedom on 800 grams of lung, together with a fair state of nutrition and entire absence of ketonuria. Now place the dog on 600 to 800 grams of lung and 100 to 200 grams of suet, according to the classical method. There is no glycosuria, weight is gained, and the .condition is splendid for weeks and possibly months. The treatment is highly successful. Closer examination shows the presence of hyperglycemia and slight ketonuria 1 which are usual in the patients of corresponding type. Glycosuria follows, illustrating the spontaneous downward progress which the authorities describe. This is cleared up by a few fast-days on the Naunyn plan, and the diet is again adjusted; it may now be 400 grams of lung and 200 grams of suet. The gain in weight continues as before, with hyperglycemia, ketonuria and subsequent glycosuria. Again the fast days are used and the protein diminished, so that the diet is perhaps 200 grams of lung and 200 grams of suet. The same cycle is repeated. Now the dog is in splendid condition and spirits, the coat sleek, the appearance such that he might create a good impression out walking in the park, only he has a difficulty in remaining sugar-free on even the protein minimum, and the fat may be pushed higher to maintain nutrition against the repeated fast days. If the dog has actually been kept fat, a fasting period about this time may diminish the glycosuria or it may remain high. The previously lively and hungry animal begins to show a curious little mournfulness, and complete repugnance to food. A day or two later, vomiting of clear mucus begins, and the dog drinks and vomits water. The acetone-reaction is heavy; the ferric chloride may be heavy or slight. The alkali-reserve of the blood falls low, and the complete picture of patients who go into fatal acidosis on fasting is reproduced." As Joslin has pointed out, patients with severe diabetes may struggle on, contending against many complications, and surviving for years on an "atrocious diet," but let a doctor intervene, eliminate carbohydrates from the diet and replace them by an equicalorific allowance of fat, and the patient promptly dies in diabetic coma. The treatment is completely successful, no doubt, in the sense that glucose temporarily disappears from the urine, but the patient nevertheless dies. Diabetes is, in fact, a multiple metabolic disorder of which the failure to utilize glucose is merely one manifestation which only indirectly induces the fatal outcome. The exclusion of carbohydrates from the diet renders calorific equilibrium and the maintenance of tissue- and body-weight impossible, unless fat be partaken of, not in usual, but even in unusual quantities. The diabetic, however, has a genuine inability to oxidize fats, and intermediate products, of which the lead- ing examples are oxybutyric and aceto-acetic acids, are formed and accumulate in dangerous and ultimately fatal amounts. 1 "Acetone bodies" in the urine. 408 PROCESSES INFERRED FROM DIRECT OBSERVATION In this dilemma the only feasible procedure is to take advantage of the long-recognized fact that the tissues may be educated by habitude to the proper utilization of carbohydrates, but the slightest overstrain upon the carbohydrate-utilizing mechanism produces a directly con- trary result and accelerates the downward course of the diabetic. This is the foundation of "Allen's Paradoxical Law," namely, that "whereas in normal individuals the more sugar is given the more is utilized, the reverse is true in diabetes." The treatment suggested by Allen con- sists essentially in freeing the urine from glucose by starvation, bearing in mind, however, the fact that starvation increases acidosis and that if the preceding acidosis was high the additional acidosis of too severe or too prolonged starvation may precipitate Diabetic Coma. The starvation-period is succeeded by a period in which proteins are admitted to the diet. Carbohydrates are now admitted, at first in very small, and then in gradually increasing amounts, until a tolerance is built up. Fats are admitted last of all, and with great caution, the allowance never being a large one. Patients treated in this way cannot commit dietary indiscretions, but they may maintain a tolerably normal and healthy existence for a number of years. Whether the "expectation of life" of a diabetic may, by a systematic regimen of this kind, be rendered equal to that of a normal individual of like age and antecedents, cannot as yet be stated, for the treatment of diabetes based upon a full realization of the part played by fats in the genesis of fatal symptoms has only recently come into being, and statistics are therefore not available. Furthermore the number of psychological factors which enter into the successful treatment of any chronic disease must be carefully borne in mind in adjudging the statistics when they do become available. The physician may know very well what ought to be done, but in practice he may rarely achieve it. The fluctuating cooperation of attendants, and the fragmentary attention of the busy practitioner to any individual case; the thousand personal details of means, circumstances, behavior, temperament, and metabo- lism, which render every individual case a separate problem which differs from any other, these factors combine to detract from the success of any method of treatment of a chronic disease-condition, however theoretically perfect the method may chance to be. It is probable, indeed, that to correctly evaluate any method of treatment of a chronic condition we should look to the successful cases rather than to the failures. The ideal means of attaining success would be, of course, to educate the patient to become his own doctor. Unfortunately, however, many patients are unteachable, and most physicians are bad pedagogues. To revert to the questions of intermediate metabolism which render the phenomenon of diabetes of such exceptional interest to the bio- logical chemist; it appears very probable that /3-Hydroxybutyric Acid is one of the normal intermediate steps in the oxidation of fats, just as lactic acid is an intermediate step in the oxidation of carbohydrates, INTERMEDIATE METABOLISM OF THE FATS 409 and that in diabetics, through failure of a particular tissue, namely the islets of Langerhans in the pancreas, the further stages of oxidation are hindered, just as, in asphyxia, the oxidations of carbohydrates subsequent to the production of lactic acid are hindered. The appear- ance of abnormal quantities of Cholesterol in the blood of diabetics suggests the possibility that the metabolism of the Hydroxyaromatic Derivatives is also disordered in diabetics. The other u acetone- bodies" in diabetic urine are undoubtedly derived from /3-hydroxy- butyric acid. Thus, if the liver be perfused with blood containing this substance, the blood which issues from the liver contains aceto-acetic acid, and even minced liver will bring about the same transformation. Butyric acid is converted quantitatively by oxidation into /3-hydroxy- butyric acid, whereas Magnus-Levy has pointed out that 100 grams of Neutral Fat made up of tristearin, tripalmitin and triolein can yield a maximum of only 36.2 grams of fl-hydroxybutyric acid. Hence cream or Butter Fat, with its high content of butyrates, is a much more dangerous source of "acetone bodies" than mutton-fat or bacon-fat or butter-substitutes such as oleomargarine. This fact is illustrated very strikingly in the intolerance which infants frequently display to cream or butter, exhibiting decided symptoms of Acidosis when these are administered in what, for other children, would be moderate amounts. These infants not infrequently tolerate a higher fat, or even olive oil, much better than they will tolerate cream or butter. The oxidation of the fats appears to take place in a series of similar successive steps, the point of attack at each stage in the oxidation being the ^-carbon atom, that is, the second carbon atom ia the hydrocarbon chain, counting from the carboxyl-group. Thus Stearic Acid is con- verted into Palmitic Acid in the following way: 410 PROCESSES INFERRED FROM DIRECT OBSERVATION CH 3 CH 3 CH 3 CH 3 | 1 1 CH 2 CH 2 i CH 2 CH 2 | CH 2 CH 2 i CH 2 CH 2 | CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 I 1 1 CH 2 CH 2 CH 2 CH 2 1 CH 2 CH 2 CH 2 CH 2 1 1 CH 2 CH 2 CH 2 CH 2 I 1 CH 2 CH 2 CH 2 CH 2 1 CH 2 CH 2 CH 2 CH 2 I 1 CH 2 CH 2 CH 2 CH 2 1 1 CH 2 CH 2 CH 2 CH 2 1 1 CH 2 CH 2 CH 2 CH 2 1 CH 2 CH 2 CH 2 CH 2 I CH 2 CH 2 CH 2 CH 2 i 0CH 2 + = CHOH + O = CO + 4O = COOH f CH 2 CH 2 CH 2 2CO 2 1 + COOH COOH COOH H 2 H 2 Stearic acid. Intermediate Intermediate Palmitic acid, hydroxy-acid. keto-acid, carbon dioxide, water. water. In a similar manner palmitic acid yields myristic acid, the next pro- duct is lauric acid and this is followed in succession by capric, caprylic and caproic acids. This acid , on oxidation of its /5-carbon atom yields butyric acid : CH CH 3 CH 3 CH 3 1 1 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 1 1 /SCH 2 + = CHOH + O = CO + 4O = COOH aCH 2 CH 2 CH 2 2GO 2 1 | + COOH COOH COOH H 2 H 2 O Caproic acid. Intermediate hydroxy-acid. Intermediate keto-acid, water. Butyric acid, carbon dioxide, water. INTERMEDIATE METABOLISM OF THE FATS 411 It is at the next succeeding stage of this process that trouble origi- nates in the diabetic. In normal tissues the intermediate hydroxy- and keto-acids are present only evanescently, being immediately oxi- dized to the lower acid, carbon dioxide, and water. In the diabetic or in the depancreatized animal there is exceptional difficulty in accom- plishing this, it would appear, especially when the stage of butyric acid has been reached and the result is that the partial products of butyric acid oxidation, /3-hydroxybutyric acid and aceto-acetic acid are permitted to accumulate : CH 3 CH 3 CH 3 I I I /3CH 2 + O CHOH + O = CO + H 2 O I I I CH 2 CH 2 CH 2 I I I COOH COOH COOH Butyric acid. |3-hydroxybutyric acid. Aceto-acetic acid. If, however, the number of carbon atoms in the original fatty-acid molecule had chanced to be uneven instead of even, the final product of this process would have been Propionic Acid, CH 3 CH 2 COOH instead of butyric acid. Now the important discovery has been made by Ringer, that pro- pionic acid is completely converted into Glucose in animal tissues, an ^intermediate stage in the process being, not improbably, the formation of 0-Lactic Acid: 2CH 3 CH 2 COOH + O 2 = 2CH 2 OH.CH 2 COOH Propionic acid. j8-lactic acid. He furthermore finds that when fatty acids having an uneven number of carbon atoms are administered they are similarly trans- formed, in part, into glucose. It happens, however, that the acid radicals of the normal tissue-fats of our dietary always contain even numbers of carbon atoms. The fatty acids possessing uneven numbers of carbon atoms are comparatively rare, and do not occur to any important extent in the fats of the normal dietary, otherwise their administration to diabetics would enable them to transform the residual unoxidized fragment of the fatty molecule into glucose, which is harmless, instead of hydroxybutyric acid which is toxic. The normal products of the complete oxidation of butyric acid according to the above scheme, would be, successively, Acetic Acid, carbon dioxide and water: CH 3 CH 3 CH 3 CH 3 I I I I /3CH 2 + O = CHOH + O = CO + 4O = COOH I I I CH 2 CH 2 CH 2 2CO 2 I I I - 4- COOH COOH COOH H 2 O +H 2 O Butyric acid. /3-hydroxybutyric acid. Aceto-acetic acid, Acetic acid, carbon water. dioxide, water. 412 PROCESSES INFERRED FROM DIRECT OBSERVATION In accordance with this view, Knoop has found that if aromatic derivatives of fatty acids containing an even number of carbon atoms be administered to animals, Phenyl-acetic Acid appears in the urine, while if aromatic derivatives of fatty acids containing an odd number of carbon atoms in the molecule be administered, the phenyl-group is split off as Benzole Acid which, as usual, combines with glycocoll in the tissues and appears in the urine as Hippuric Acid. OXIDIZING ENZYMES. In a variety of animals and plants there are to be found substances which are capable of accelerating certain oxidations. These sub- stances, in the majority of cases, resemble the hydrolyzing enzymes in the minute quantities in which they are effective, and in their insta- bility toward heat. In other cases they are thermostabile and even resist boiling. The discovery of the oxidizing enzymes we owe to the versatile investigator Schonbein (1799-1868), who employed Guaiacum Tincture as a means of detecting them. This substance is tinged blue, a coloration due to oxidative changes, by many tissues and tissue- fluids in the presence of peroxides, such as, for example, Hydrogen Peroxide. It is found, however, that the oxidizing ferments do not by any means act upon all oxidizable substances equally, on the contrary there is a high degree of Specificity in their effects. Thus the enzyme or group of enzymes occurring in the liver and in the spleen which oxi- dizes Purines, converting, for example, Xanthin and Hypoxanthin into Uric Acid, does not attack alcohols, aldehydes or polyphenols. On the other hand the alcohol oxidizing ferment or Alcoholase which oxidizes ethyl alcohol to acetic acid, does not attack purines or polyphenols. The best-studied examples of the oxidizing enzymes are those which are afforded by the Laccases, which bring about the hardening of lacquer varnish. A very active enzyme has been prepared from the sap of Rhus succedanea by Bertrand, who coagulates the sap with alcohol, redissolves the coagulum in water, and then recoagulates with alcohol. The coagulum is dried in vacno and is then obtained as a white powder which is readily soluble iu water, and is characterized by its high content of Manganese. The activity of the laccase in oxidiz- ing polyphenols is, in fact, dependent upon the presence of manganese. Thus Bertrand, in studying the action of a similar substance from lucerne with and without the aid of manganous salts, obtained the following results: Manganous salt alone . . . . ... . . 0.3 c.c. oxygen absorbed Laccase from lucerne, alone ....... 0.2 " " " Laccase plus manganous salt 6.3" " " We know that in many cases the oxides of polyvalent metals may act as carriers of oxygen, through the intermediate formation of Peroxides which are more active oxidizing agents than free oxygen itself. An OXIDIZING ENZYMES 413 example which is very familiar to biological chemists is that afforded by the action of alkaline copper salts upon glucose. If a limited quantity of Fehling's Solution be run into a boiling solution of glucose the solution is decolorized and, the red cuprous oxide is precipitated, but upon exposing the mixture in a shallow vessel to the air, the cuprous oxide again takes up oxygen, passes into solution and tinges the fluid blue. If the mixture be now boiled, the cupric hydroxide again parts with its oxygen to the excess of glucose, so that if the process be repeated a sufficient number of times a limited quantity of Fehling's solution will oxidize a relatively unlimited quantity of glucose. This is, in fact, the chief pitfall in the practical employment of Fehling's method of sugar estimation. In a similar manner many other metal oxides are capable of acting as activators or carriers of oxygen. According to Bach and Chodat the oxidizing enzymes which occur in the majority of living tissues in reality consist of two parts: the one part, the Oxygenase, playing the role that cuprous oxide plays in the oxidation of sugar, namely that of a carrier of oxygen, while the other part, the Peroxidase, facilitates the transfer of the oxygen from the oxygenase to the material which is undergoing oxidation. Hydrogen Peroxide may in many cases take the place of the oxygenase, and hence we obtain the blueing of guaiacum tincture when blood or a tissue- extract together with hydrogen peroxide act upon it. The function of the manganous salt in Laccase appears to be associated with the oxygenase fraction, while in many oxidizing ferments found in animal and other plant tissues, Iron plays the role which manganese plays in determining the activity of laccase. In this connection it is of great interest to note that Hemoglobin is itself an oxygenase, and that, according to Bertrand, the Benzidine and Guaiacum tests for blood are, in actuality, tests for hemoglobin. The majority of the oxidizing enzymes appear to be substances of a complex character, in many cases either protein in nature or closely associated with protein. It is probable that the majority of the oxygenases or oxygen-carriers are bodies analogous in complexity to hemoglobin and, like hemoglobin, containing iron or some other polyv- alent metal as an integral portion of the molecule. On the other hand Euler and Bolin have shown that the Laccase from Medicago saliva is a relatively simple substance, being a mixture of the calcium salts of aliphatic hydroxy-acids. A synthetic mixture of the calcium salts of glycollic, citric, malic and mesoxalic acids was found by them to exert the same action in accelerating the oxidation of polyphenols as the natural laccase. This enzyme is, of course, thermostabile. An important group of oxidizing enzymes is that of the Tyrosinases which convert tyrosine into dark-colored substances of complex struc- ture known as Melanins, which are probably identical with or closely allied to many naturally-occurring pigments. These enzymes are found in many vegetable tissues and von Furth has also found them in the tissue-fluids of many insects and in the ink-sac of the cephalopod 414 PROCESSES INFERRED FROM DIRECT OBSERVATION Sepia. These oxidases are also able to accomplish the oxidation of other hydroxy-aromatic compounds, such as Catechol and Quinol. An enzyme which is often mistakenly regarded as an oxidizing enzyme is the Catalase which occurs in nearly all living tissues and which possesses the property of decomposing peroxides, without, however, liberating active oxygen. It is to this enzyme that the frothing of hydrogen peroxide when added to blood or saliva is due. It is in fact a retarder of oxidations and not an accelerator, for it antici- pates the action of peroxidase upon peroxides and decomposes them, thus depriving them of ability to transfer oxygen to oxidizable mate- rials. It is probably to be regarded as a controlling agent or check upon overactivity of oxidizing enzymes. It has been shown by Burge that the catalase-content of different tissues varies very greatly, that of the liver being greatly in excess of the catalase-content of muscular tissues. The curious observation has been made by Burnett, however, that if a small proportion of liver-tissue be mixed with muscle-tissue the power of this mixture to decompose hydrogen peroxide is equal to that of an equal weight of pure liver-tissue. This looks either as if catalase really consists, like the oxidases, of two parts, of which only one is contained in considerable amount in muscle-tissue, or else the catalase in muscle-tissue is present therein, not as such, but in the form of a proenzyme or zymogen, which is activated by liver-tissue. BIOLUMINESCENCE. The phenomenon of bioluminescence or "phosphorescence" which is displayed by so many organisms, both vegetable and animal, has recently been subjected to very careful study and analysis by N. Harvey. The peculiarity of bioluminescence is the extraordinary intensity of the light which is developed, without any perceptible waste of energy, in the form of heat, an ideal unattainable by any means of illumination at present within our control. The luminescence is dependent upon the occurrence of oxidations, for it disappears when the luminescent system is deprived of oxygen, even when the lumines- cence is made to occur independently of the life of the organism, as in extracts made from the luminous tissues. Both Dubois and Harvey, the two leading investigators of this phenomenon, are agreed that the production of luminescence in animal or plant-tissues or tissue-extracts requires the interaction of two substances. The one of these is, according to Harvey, the sub- stance from which the luminescence proceeds, and it is progressively consumed in the process; this he terms the Photogenin. The other substance facilitates the oxidation of the photogenin and is termed by Harvey Photophelein. The photogenin is colloidal, i. e., does not pass through a dialyzing membrane of parchment, and its light-producing ability is destroyed by heating. The photogenin from the luminous crustacean, Cypridina, at all events, appears to be a protein. It is BIOL VMINESCENCE 415 associated with iron, copper and manganese, but whether the presence of these metals is essential to its luminescence has not yet been ascer- tained. The proteolytic enzymes destroy its light-producing power. Photophelein, on the other hand, appears to be a substance related to the proteoses or peptones in many of its properties, but it is not digest- ible by proteolytic enzymes and it is soluble in alcohol. The separation of photophelein and photogenin from one another may be accomplished by extracting the luminescent animals or organs with hot water. This extracts the photophelein and destroys the pho- togenin. A solution of photogenin may be prepared by extracting a luminous organ with cold water and allowing the extract to stand until all luminescence has disappeared, when the photophelein has been apparently exhausted. On now mixing these two non-luminous solu- tions a bright luminescence at once appears. The actual source of light has been shown by Harvey to be the photogenin, in the following ingenious manner: The light emitted by the Eastern American firefly Photinus is orange in color, while that emitted by Photuris is greenish-yellow. If, now, photinus photogenin is mixed with photophelein from either Photinus or Photuris the color of the luminescence is that emitted by Photinus, namely orange, and conversely Photuris photogenin yields greenish-yellow light whether the source of photophelein be Photuris or Photinus. Evidently, therefore, the character of the light emitted is determined by the photogenin and not by the photophelein. The action of photophelein is, to a limited extent, specific. Thus firefly-photophelein will cause emission of light by photogenin derived from other insects, but none from photogenin derived from crusta- ceans. On the other hand photogenin may be caused to luminesce by many substances which are not of animal and vegetable origin, and particularly by fat-solvents and other Cytolytic Agents. Thus lumi- nescence of photogenin may be caused by ether, chloroform, saponins or bile-salts. Harvey believes that these substances promote oxidation of the photogenin by increasing the fineness of the subdivision of the colloidal particles of which it is composed, and thus increasing the area of exposure to oxygen. The part played by photogenin itself may also be imitated by a variety of reagents. Thus many aldehydes, polyphenols such as pyrogallol, terpenes, waxes, glucose, lecithin, cholesterol, cetyl and myricyl alcohols, tannic and gallic acids, certain peptones and the bile- acids will emit luminescence when treated in certain concentrations with specific oxidizing-agents. Pyrogallol, for example, will luminesce when treated with plant Peroxidases or with Hemoglobin or by certain salts such as potassium permanganate and potassium ferrocyanide, if hydrogen peroxide is also present. For each oxidizer and oxidizable substance there is an optimal concentration above and below which the light-emission diminishes. Thus one-molecular pyrogallol solution will give no light if mixed with ^ potassium ferrocyanide and a little 416 PROCESSES INFERRED FROM DIRECT OBSERVATION three per cent, hydrogen peroxide, but T ^ or T ^ pyrogallol will give a bright light, while the light from T ^-?W75- pyrogallol is only just visible. On the other hand f potassium ferrocyanide gives no light with a mixture of -nnr pyrogallol and hydrogen peroxide, while f^- potassium ferrocyanide causes bright light-emission. The phenomenon of bioluminescence therefore depends upon the simultaneous presence, in solution, of a special type of oxidizable substance, and an oxidizing agent which presents some analogies to an oxidase, but is thermostabile and diffusible, and to some extent used up in the reaction which it accelerates. REFERENCES. INTERMEDIATE METABOLISM: Pfluger: Pfluger's Arch., 1903, 96, p. 1. Van Hoogenhuyze and Verploegh: Zeit. f. physiol. Chemie, 1905, 46, p. 415. Knoop: Beitr. z. chem. Physiol. u. Pathol., 1905, 6, p. 150. Von Noorden: Metabolism and Practical Medicine, English edition, edited by Walker Hall, London, 1907. Shaffer: Am. Jour. Physiol., 1908, 22, p. 445. Dakin: Oxidations and Reductions in the Animal Body, London, 1912. Taylor: Digestion and Metabolism, Philadelphia, 1912. Bloor: Jour. Biol. Chem., 1912, 11, pp. 141 and 429; 1913, 15, p. 105; 1913-14, 16, p. 517; 1914, 17, p. 377; 1914, 19, p. 1; 1915, 22, p. 133; 1915, 23, p. 317. Sansum and Woodyatt: Ibid., 1915, 21, p. 1. Wilder: Ibid,, 1917, 31, p. 59. Hoagland and Mansfield: Ibid., 1917, 31, p. 501. (Glucolysis in Tissue, consult foi literature.) Lusk: The Science of Nutrition, Philadelphia, 1919. PRODUCTS OF MUSCULAR ACTIVITY IN STARVATION, ETC : Cathcart: Jour. Physiol , 1906-7, 35, p. 500. Benedict: Carnegie Institution of Washington Bull., 1907, 77. Mendel and Rose: Jour. Biol. Chem., 1911-12, 10, p. 213. Mellanby: Proc. Roy. Soc. B., 1912-13, 86, p. 88. Meyers and Fine: Jour, Biol. Chem., 1913, 15, p. 305. Morse: Jour. Am. Med. Assn., 1915, 65, p. 1613. DIABETES: Allen: Glycosuria and Diabetes, Boston, 1913. Macleod: Diabetes, its Pathological Physiology, 1913. Joslin: The Treatment of Diabetes Mellitus, Philadelphia, 1917. OXIDIZING ENZYMES: Bach and Chodat: Centr. f. Biochemie, 1903, 1, pp. 417 and 457. Batelli and Stern: C. Rend. Acad. d. Sc., 1905, 140, pp. 1197 and 1352; 1905, 141, p. 139. Biochem. Zeit., 1914, 67, p. 443. Kastle: The Oxidases, Hygienic Lab., U. S. A. Pub. Health and Marine Hospital Service Bull., No. 59, Washington, 1910. Euler: General Chemistry of the Enzymes, trans, by Pope, New York, 1912. CATALASE: Burge; Am. Jour, Physiol., 1916, 41, p. 153; 1916-17, 42, p. 600; 1917, 44, p. 290. Burnett: Proc. Soc. Exp. Biol. and Med., 1918, 15, p. 80. BIOLUMINESCENCE : Dubois: La Vie et la Lumiere, Paris, 1914. 4 Harvey: Science, N. S., 1916, 44, p. 652; 1917, 46, p. 241. Am. Jour. Physiol., 1916, 41, p. 454; 1916-17, 42, p. 318. Jour. Biol. Chem., 1917, 31, p. 311. Jour. Gen. Physiol., 1918, 1, pp. 133 and 269. CHAPTER XVIIt. PROCESSES INFERRED FROM INDIRECT OBSERVATION; THE ENERGY-TRANSFORMATIONS IN LIVING ORGANISMS. THE INFLUENCE OF TEMPERATURE UPON LIFE-PROCESSES. The influence of elevation of temperature upon a chemical reaction may be twofold. If the reaction is at all exo- or endothermic, that is, if any heat is liberated or absorbed during the progress of the reaction, an elevation of temperature will bring about a definite change in Equilibrium so that at the conclusion of the reaction the final relative proportion of the various components is altered. On the other hand a rise in temperature always accelerates the attainment of equilibrium whatever the station of equilibrium may chance to be. Thus, not- withstanding the fact that the majority of the hydrolyses which occur in living tissues are exothermic, so that a rise in temperature tends to shift the equilibrium in the direction of less complete hydrolysis, yet the rate of Hydrolysis being more than proportionately accelerated, enzymatic hydrolyses which are barely perceptible at low tempera- tures become extremely rapid at the body-temperature of warm- blooded animals. The effect of temperature upon the velocity of a chemical reaction may be expressed by the equation : , /Ti-To\ in which "ki," and "k " signify the velocity-constant at the absolute temperatures "Ti" and "T " respectively, "e" is the base of the Napierian logarithms and ^ is a constant, differing in different reactions, but almost invariably possessed of such a value that the ratio ^ ko exceeds 2 when TI T = 10. The Temperature-coefficient of a chemical reaction therefore, or the ratio : Velocity of the reaction at T + 10 Velocity of the reaction at T is in almost every case greater than 2 and may be very greatly in excess of this value. 27 418 PROCESSES INFERRED FROM INDIRECT OBSERVATION The behavior of physical, that is to say molecular phenomena rather than atomic, which are affected by temperature, is quite different. The effect of temperature is in these phenomena quantitatively much less than it is in phenomena which arise from chemical transforma- tions. Thus the Viscosity of a liquid is diminished by an elevation of temperature, it is true, but the reduction of viscosity which is brought about by a rise of ten degrees in temperature does not exceed about twenty per cent., so that the ratio: Viscosity at T +.10 Viscosity at T is 1.2 or thereabouts. Consequently all the physical phenomena in solutions which are dependent upon the viscosity of the solvent, such as electrical Conductivity, and Diffusion are affected in a similar degree by elevation of temperature. Those phenomena of which the rate is determined by changes of Surface-tension have, in fact, a temperature- coefficient of less than unity, the velocity of changes in capillary tension being actually reduced by elevation of temperature. One consequence of this decided quantitative difference between the effects of temperature upon chemical and physical phenomena is that we may, with a fair degree of confidence, employ the temperature- coefficient of a complex phenomenon which involves physical as well as chemical changes as a means of gauging the extent to which the velocity of the process is governed by the chemical transformations which it involves. If the pace is set by the rate at which some chemical change transpires, then the rapidity of the process will be at least doubled and not improbably more than doubled by a rise of ten degrees in temperature. But if the chemical transformations are subordinate to some physical process and must await its development before they can proceed, or if they are simply consequent upon physical changes such as electrolysis, or alterations in surface-tension, then the pace of the whole process will be set by this physical event and the temperature- coefficient of the process may be expected to be less than 2 or even very considerably less than 2. We have already seen that the various enzymatic hydrolyses which occur in the digestion of the foodstuffs yield temperature-coefficients which lie between 2 and 4; all of them exceeding 2 at temperatures which are not too far above the temperature of the warm-blooded animals. The temperature-coefficient of enzymatic processes neces- sarily declines very rapidly at temperatures which are much in excess of 40, because at these temperatures the acceleration of the auto- destruction of the Enzyme itself is so great that its loss of activity more than compensates for the gain in the velocity of the hydrolysis which the residual undestroyed enzyme is able to bring about. We have, in fact, to deal with the resultant of two opposed processes both of which are accelerated by elevation of temperature. At lower temperatures INFLUENCE OF TEMPERATURE UPON LIFE-PROCESSES 419 the acceleration of hydrolysis is the predominant result of raising the temperature, but at higher temperatures, destruction of the enzyme becomes the controlling factor. The temperature-coefficient for enzyme destruction is exceptionally high, so that the rate of auto- destruction may be imperceptible between 30 and 40 and extremely rapid at temperatures lying between 40 and 50. Even in a single uncomplicated chemical transformation the tem- perature-coefficient is not constant, for, reverting to the equation: ko we see that the temperature-coefficient for 10 temperature-interval is given by: i n(-M-\ ' KI = e ^VTi T ) k it is therefore not independent of the absolute magnitude of the tem- perature employed; in fact the temperature-coefficient must invariably decrease as the temperature rises. Assuming a value of /z ( = 13, 200) which would yield a coefficient of 2 between the temperatures of 30 and 40, the following table shows the coefficients which might be anticipated at other temperatures : Temperature- Temperature interval. coefficient. 0to 10 ................. 2.34 10 to 20 ................. 2.22 20 to 30 ................. 2.11 30 to 40 ................. 2.00 40 to 50 . . ... ...... ...... 1-92 The reduction of the coefficient for enzyme reactions at temperatures above 40 is, however, much more extreme than could be accounted for in this fashion, the coefficient ultimately falling to zero at the thermal limit for the activity of the enzyme. The actual phenomena of life are almost invariably of a mixed character, involving physical as well as chemical processes and changes, and we may inquire through the investigation of their temperature- coefficients whether the physical or the chemical factors predominate in determining the rate of performance; whether the chemical trans- formations, in other words, are consequent upon preceding physical changes or whether, on the contrary, the physical modifications of protoplasm await and are the resultant of the chemical transformations which accompany the performance of vital activities. The first investigator to apply this criterion to the study of life- phenomena was Cohen, who in 1892 pointed out that the previous 420 PROCESSES INFERRED FROM INDIRECT OBSERVATION measurements by Clausen of the rate of production of Carbon Dioxide by germinating seeds showed that this process is approximately doubled in velocity by an elevation of 10 in temperature until an upper limit somewhat exceeding 40 is attained, when the rate of the tissue-respira- tion falls off owing to heat-injury. This method of inquiry was extended to animal tissues by C. D. Snyder, who investigated the influence of temperature upon the Rate of the Heart-beat in the isolated heart of the Pacific terrapin, Clemmys marmorata. The following are illus- trative results : Number of heart-beats per minute. Time of exposure to the temperature Heart 1. Heart 2. Heart 3. Heart 4. Heart 5. Heart 6. minutes. T. = 10. T. = 20. T. = 30. 5 9.5 9.5 21.5 21.0 48 48 10 7.0 9.0 21.0 24.0 48 44 15 6.7 8.7 19.0 18.0 48 40 20 7.0 8.2 19.0 16.5 41 30 7.0 7.0 16.0 14.0 40 6.5 7.9 15.5 15.5 50 6.5 7.9 13.5 16.0 60 6.2 7.4 13.0 15.0 It is evident that the rate of the beat is approximately doubled for each 10 rise in temperature. From the data quoted and others obtained by Snyder the following average coefficients may be com- puted: HEART-BEAT OF CLEMMYS MARMORATA Temperature-coefficient Temperature-interval. for 10 intervals. 10 to 20 2.3 20 to 30 22 30 to 37 '. ... 1.6 at temperatures exceeding 37 the rate of the beat, instead of increas- ing, diminished until the heart came to a standstill owing to irreparable heat-injury. These experiments were subsequently repeated upon the isolated heart of another species of terrapin, Emys europea, by Galeotti and Piccinini, and by Snyder upon the isolated heart of the frog, and by Kanitz upon the isolated mammalian heart. The heart in situ is, however, considerably modified in its behavior and particularly in the rate of beat by the nervous control to which it is subjected. The study of the heart-beat in the intact animal there- fore involves more numerous and more complex factors than that of the beat of the excised heart. Nevertheless in this case also the rate of the beat is primarily determined by the velocity of underlying chemical changes. Thus in the minute transparent fresh-water crustacean, Ceriodaphnia, the heart can be viewed through the body-wall of the INFLUENCE OF TEMPERATURE UPON LIFE-PROCESSES 421 animal and the beats counted at a variety of temperatures. The following are illustrative of the results obtained by this method : Temperature interval. Temperature-coefficient. Ilto21 2.76 15 to 25 2.24 17 to 27 2.05 19 to 29 2.06 21 to 31 1.14 at a temperature slightly above 31 the heart-beat ceases and the organism dies. In the case of the crustacean Limulus the Heart-ganglion can be heated or subjected to other manipulation without directly involving th,e heart-muscle itself, and Carlson has found that by heating the ganglion alone the heart-beat is accelerated, the unusually high tem- perature-coefficient of 4 being obtained. On the other hand, in the Embryonic Heart, in which the mechanism of nervous control is probably not yet established, the rate of the heart-beat is similarly affected by temperature, being doubled or trebled by a rise of ten degrees. The following are results obtained by Loeb and Ewald, employing the embryos, still enclosed within the egg, of the marine fish Fundulus: Time required for 19 heart-beats in the Temperature. embryo; seconds. 30 . . '.' 6.25 25 8.5 20 ./.,,,; 11.5 15 . 19.0 10 32.5 5 61.0 10 33.5 15 . ;_ 18.8 20 12.0 25 . '. 10.0 30 .... . . . . , 6.0 It is evident, therefore, that both the muscular and nervous mechan- isms involved in the regulation of the rate of the heart-beat are pri- marily conditioned, as to their velocity, by underlying chemical transformations. Loeb and Ewald have drawn attention to the fact that in Fundulus embryos the rate of the heart-beat is almost the same in all the embryos exposed to the same temperature, provided they still remain enclosed within the egg. This is because of the elimination of all secondary disturbing factors. As soon as the embryos begin to move, this equality disappears, because the motility of different embryos differs and the products discharged from the contracting muscles influence the rate of the heart-beat. In man and in other higher animals, the number of the disturbing factors, while the heart remains in situ, are so great that no uniformity of rate at any given temperature can be expected. " Differences in emotions or the internal secretions following 422 PROCESSES INFERRED FROM INDIRECT OBSERVATION the emotions, differences in metabolism, differences in the use of nar- cotics or drugs, and differences in activity are only some of the number of variables which enter" (Loeb). Hence the attempt to compute the temperature-coefficient of the heart-beat in situ in man from the acceleration of the beat in Fevers is illogical, and we find, as a matter of fact, a great deal of discordancy in the values computed from data of this kind, coefficients varying from 1.8 to 5 having been reported. The Respiratory Rhythm is even more susceptible to modification by sensory stimuli, muscular exertion and so forth, than the cardiac rhythm, and consequently the coefficients which have been observed for the rate of the respiratory movement at different temperatures are not of so uniform a character as those which are cited above. Never- theless the influence of temperature upon the Respiratory Center is extremely striking. It has long been a familiar fact that warming the blood in the carotid artery, by causing it to flow through a heated tube, results in a marked acceleration of the respiratory rhythm, and in frogs it has been shown that the direct application of heat to the floor of the fourth ventricle leads to a very decided increase in the rapidity of respiratory movements. It is an extremely interesting fact that the effect of temperature upon the respiratory rhythm of cold-blooded animals is very much greater at a low oxygen-tension than at a high, possibly because when the oxygen-tension is low and the consumption of oxygen by all of the tissues is accelerated by an elevation of tempera- ture, the effect of the temperature elevation itself is aided by the stimulation of the respiratory center which lack of oxygen indirectly entails, while when oxygen is abundant there is sufficient for the needs of all the tissues even at high temperatures, and the secondary stimula- tion of the center does not occur. The influence of temperature upon the velocity of "Basal Metab- olism" or tissue-respiration can only be studied in cold-blooded animals under conditions which exclude muscular movement, which would, of course, introduce irregular fluctuations in the rate of con- sumption of oxygen. This problem has been approached by Krogh in several ingenious ways. One method was to employ the pupae of insects in which tissue-respiration is of course maintained but muscular movement is arrested. The following were results obtained with the pupae of the mealworm, Tenebrio molitor: Oxygen-consumption Temperature-coefficient lemperature. per kilogram-hour. per 10 C. 10 43.5 5.7 15 104. 3.2 20 185. 2.6 300. 2.2 445. 2.2 25. 30. 32. 5 C 529. It will be observed that the temperature-coefficient is very high at low temperatures and falls rapidly as the temperature rises. A similar INFLUENCE OF TEMPERATURE UPON LIFE-PROCESSES 423 characteristic distinguished the temperature-coefficient of the time consumed in the pupal stage of development: Temperature. 13.45 . 15.55 . 17.0 18.8 20.9 23.65 . 27.25 . 32.7 . 32.95 Hours spent in pupal condition. Temperature-coefficient per 10 C. 1116. 742. 593. 439.6 320. 234.1 172.5 137.9 134.25 6.2 4.9 2.6 1.5 We may infer that the time spent in the pupal stage depends upon the extent of tissue-oxidation which has occurred. By employing curarized frogs and decerebrated turtles Krogh was also enabled to investigate the effect of temperature upon the Tissue - respiration of these animals. The values of the coefficients obtained lay between 2 and 4, but the values were not found to be so greatly affected by the position on the temperature-scale of the temperature- range employed as in the case of the insect-larvae. The influence of temperature upon the Rate of Development of organisms is again of a similar character. Thus Hertwig has investi- gated the influence of temperature upon the time taken to reach seven different arbitrarily chosen stages of development of the larvae of a frog, Rana fusca. The following were the results obtained: Temperature-coefficient for 10. Temperature- interval. Stage I. Stage II. Stage III. Stage IV. Stage V. Stage VI. Stage VII. 2. 5 to 6 10. 13. 15. 14. 6. to 15 2.6 2.6 2.5 2.6 3.1 3.5 4.5 10. to 20 2.9 3.3 3.2 2.9 3.5 3.4 3.3 20. to 14 1.5 1.4 2.0 2.0 2.0 2.0 1.8 We have seen that the rate of development in the pupal stage of insects and the rapidity of their basal metabolism are very similarly influenced by temperature, so that we may infer with probability that oxidations determine the duration of this period of development. This is not the case in the earliest stages of development, however, for Loeb and Wasteneys have investigated the influence of temperature upon the time which elapses between insemination and the first cell- division in sea-urchin eggs and have compared with this the effect of the same temperatures upon the oxygen-consumption of the eggs. The two sets of temperature-coefficients are unmistakably of the magnitude of the coefficients of chemical reactions, but they are very diversely affected by alteration of the position of the temperature- range, as the following figures show: 424 PROCESSES INFERRED FROM INDIRECT OBSERVATION Temperature- interval. Temperature-coefficient of rate of segmentation in : 3 to 13 4 to 14 5 to 15 7 to 17 8 to 18 9 to 19 10 to 20 11 to 21 12 to 22 13 to 23 15 to 25 16 to 26 17.5 to 27. 5 20 to 30 Slronglycentrotu s . 3.91 Arbacia. ...... 3.88 . ... . 3.52 . . . 3.27 , 7.3 -'! '. 2.04 . 1.90 6.0 4.7 3.8 1.74 3.3 3.1 2.8 2.5 2.6 2.2 1.7 Temperature-coefficient of rate of oxidation in : Arbacia. 2.18 2.16 2.00 2.17 2.45 2.24 2.00 1.96 Not only oxidations, therefore, but some other chemical factors even more susceptible to temperature change are involved in deter- mining the rapidity of the cell-divisions in the early stages of develop- ment. Other forms of growth, for example Regeneration, as A. R. Moore has shown, are also affected by temperature to the extent characteristic of chemical reactions. In all of the life-processes hitherto mentioned the general order of magnitude of the temperature-coefficients has been the same. When, however, we come to study the temperature-coefficients for the Dura- tion of Life we meet with a startling disparity of quantitative effects, for whereas, for example, it takes a rise of nearly ten degrees to double the rate of the heart-beat, or the rate of respiratory movements or the rate of cell-division or regeneration or tissue-oxidations, yet a tempera- ture-elevation of merely one degree, as J. Loeb has shown, serves to halve the duration of life of fertilized or unfertilized eggs of the sea- urchin, and lowering of the temperature by ten degrees prolongs the life of the organism 2 10 , that is to say over a thousandfold. The temperature-coefficient of the processes underlying the thermal death of the cells is therefore, no less than 1000. A. R. Moore has investi- gated the influence of various temperatures upon the duration of life of a hydroid, Tubularia crocea, judging viability by the retention of the power of regeneration. The following are illustrative results: Temperature. 25 26 27 28 29 30 31 32 33 34 35 36 Dura 55 to 25 to 15 to 7 to 130 to 50 to 30 to 14 to 7 to 3 to 2 to Uto Aon of life. 60 hrs. Coef IV . . . 2.0 icient for tempe interval of: 30 " * * . . 1.7 18 " ' ' . . 2.2 8 " * ' . . 3.3 140 mins. ' . . 2A\ 60 " ' ' 40 ' '" 15 " ' ' - 8 " ' 4 " ' ' . . 1.6 . . 2.4 . . 1.9 . . 2.1 1 4 3 " ' ' 2 " ' . . 1.5 rature 10. 3900 485 INFLUENCE OF TEMPERATURE UPON LIFE-PROCESSES 425 It is obvious that here we are dealing with a phenomenon of quite a distinct nature from the other phenomena of life which we have hitherto being considering, and the question immediately suggests itself whether any clue exists as to the origin of this remarkable susceptibility to temperature. Now on comparing the temperature- coefficients of various reactions involving Enzymes, one group stands out from all the rest by reason of the extraordinary magnitude of the temperature-coefficients, and that is the group afforded by the Auto- destruction which various enzymes undergo in solution. The following data are cited after Arrhenius : Numerical value Nature of process. of /*. Hydrolysis of sugar by acids 25,600 invertase 9,080 Saponification of ethyl acetate by NaOH 11,150 triacetin by lipase 16, 700 cottonseed oil by lipase Digestion of gelatin by pepsin trypsin .... " egg-white by pepsin .... Coagulation of milk by rennet .... Spontaneous destruction of trypsin in solution " " pepsin in solution 7,540 10,750 10,570 15,570 20,650 62,034 75,600 rennet in solution . ...... 90,000 vibriolysin in solution ...... 128,000 tetanolysin in solution ...... 162,000 hemolysin in solution . . . . % . .198,500 The relationship between the value of M in the equation: and the temperature-coefficient for the ten-degree interval between 20 and 30 C. is shown in the following table: A temperature- Corresponding to the coefficient of value of 2 ................ 13,200 10 ................ 44,000 100 ................ 88,000 1,000 ......... ....... 132,000 10,000 ......... ....... 176,000 It is evident therefore that the temperature-coefficient of the duration of life corresponds not at all with that of enzymatic hydrol- yses, but it is, on the other hand, of precisely the order of magnitude encountered in the autodestruction of enzymes or of specific anti- bodies. It is to the destruction of enzymes, consequently, that we may attribute the thermal death of organisms excepting in those cases, as in spores of seeds, in which the essential tissue-enzymes are thermo- stabile and the temperatures required to kill the tissue are those which 426 PROCESSES INFERRED FROM INDIRECT OBSERVATION suffice to coagulate Proteins. For example, Goodspeed, who investi- gated the thermal death of barley-seeds, exposed them to temperatures ranging from 55 to 70 C. and obtained a temperature-coefficient of 10 for the duration of life, a coefficient which is very close to the value 8 obtained by Chick and Martin in estimating influence of tempera- tures of similar magnitude upon the rate of coagulation of Hemoglobin. The remarkable disparity between the effects of temperature upon the Life-duration and the Development of organisms has been applied by Loeb to the explanation of what would otherwise be an exceedingly puzzling fact, namely the extraordinary density of the population of the polar seas. In his account of the Valdivia expedition, Chun 1 calls especial attention to the quantitative difference in the surface fauna and flora of polar and temperate or tropical regions : " In the icy water of the Antarctic, the temperature of which is below C, we find an astonishingly rich animal and plant- life. The same condition with which we are familiar in the Arctic seas is repeated here, namely, that the quantity of plankton material exceeds that of the temperate and warm seas." And again, in describing the pelagic fauna in the region of the Kerguelen Islands he states: "The ocean is alive with trans- parent jellyfish, Ctenophores (Eolina and Callianira) and of Siphono- phore colonies of the genus Agalina." This observation, which has been repeatedly made by Arctic and Antarctic travellers, would appear paradoxical in consideration of the effect of temperature upon development, for the rate of development of organisms is, as we have seen, halved or even reduced to a greater extent by a drop of 10 C. in temperature. When, however, we reflect that the duration of life of these slowly developing organisms is pro- longed a thousandfold, the density of the polar population becomes explicable, for the net result of these opposed effects would be a great increase in the number of surviving individuals and in the number of successive generations simultaneously inhabiting the cold waters. The temperature-coefficient of the life-processes which we have hitherto considered have all been of such a magnitude as to clearly invite the supposition that the velocities of the phenomena are deter- mined by the rate at which underlying chemical transformations occur. We now come to a life-phenomenon of peculiar character in which the testimony of the temperature-coefficient is far from being so unequivo- cal, namely the Conduction of Stimuli along the fibers of a motor-nerve. The influence of temperature upon the rate of conduction of the nervous impulse was first investigated by S. S. Maxwell, who employed for this purpose the pedal nerve of a large slug, Ariolimax columbianus. This nerve was selected on account of its considerable length and the slowness of the propagation of the impulse permitting a much greater exactitude of measurement than is possible in the shorter and more 1 Cited after J. Loeb: The Mechanistic Conception of Life. INFLUENCE OF TEMPERATURE UPON LIFE-PROCESSES 427 rapidly conducting sciatic nerves of a frog. The following table summarizes the results: Temperature-interval . -0.5to 9.5 0. to 10. 1. to 11. to 13. to 15. to 16. to 19. to 21. 11.5to 21.5 12.5to 22.5 13. to 23. 14. to 24. 16. to 26. Temperature-coefficient. . . 2.14 .79 .98 .07 .29 .57 .32 .47 .54 .67 .65 .32 .81 It will be seen that almost the only coefficients approaching the numerical value of 2 are those obtained at the lowest temperature- ranges, 1 nor is this a peculiarity of the type of nerve-fiber employed by Maxwell, for the later investigations of Lucas and Gantor on the transmission of impulses in the motor-nerves of frogs bear similar testimony. The following are the results obtained by Gantor: Temperature-coefficient for 10 interval. Experimental series number. Temperature. 1 2 3 4 5 0. . . . 2.35 .86 2.5 . . 2.28 .77 5. . . . 2.03 1.79 .79 1.97 2.09 7.5 . . . 1.82 1.64 L.65 1.95 1.51 10 1 79 1.60 57 1.95 1 59 12.5 '. V . 1.66 1.53 ]62 1.81 1.75 15 1 59 1.48 .55 1.77 1.67 17 5 . .50 1.68 20. 1.47 1.61 Average. 1.87 1.77 1.71 1.64 1.61 These coefficients are intermediate in value between those usually obtained in physical phenomena and those which may characterize chemical transformations. We are therefore led to v suspect that physi- cal events play a large part in determining the rate of transmission of nervous impulses. This view is rendered the more probable by the historical difficulty which has been encountered in demonstrating the existence of any metabolic changes in nerve-fibers or their enhance- ment by stimulation, and while the recent results of Tashiro demon- strate a minute evolution of carbon dioxide from excised nerves, it cannot be regarded as proved that this metabolic activity is very closely associated with the conduction of the stimulus. It may, rather, be concerned with the maintenance of the nutrition or repair of the nerve, and the inability of nerve-fibers to display fatigue on repeated stimula- tion lends strong encouragement to this view, for evidently no material 1 A small number of coefficients exceeding 2 are attributed by Maxwell to experi- mental errors and are not included in the above averages. 428 PROCESSES INFERRED FROM INDIRECT OBSERVATTON is used up in consequence of their excitation. On the other hand, Carlson's experiments with the heart ganglion of Limulus and the above-cited experiments on the effects of heating the respiratory center show that chemical changes play a predominant role in the activities of Nerve Cells, and, as a matter of fact, the consumption of oxygen by the brain is very large, and it is the first tissue to suffer from lack of oxygen, indicating a very high level of metabolic activity in the cellular elements of the nervous system. The conducting fibers and the nerve- cells from which they issue stand therefore in sharp contrast to one another in respect to the metabolic foundations of their functional activity, and we are thus led to recall the fundamental difference between their susceptibilities to the various classes of chemical stimu- lants to which reference has been made in a previous chapter. Nerve- fibers are powerfully stimulated by salts which precipitate calcium, nerve-cells are insensitive to these reagents. Nerve-cells are stimu- lated by a variety of specific substances, by polyphenols and by Creatine, for example, to which nerve-fibers are indifferent. The phenomena of Muscular Contraction and the change which trans- forms the nervous impulse into a muscular stimulus at the myoneural junctions are, it would appear, conditioned in their speed by underlying chemical reactions. Thus Burnett has determined the influence of temperature upon the Latent Period of indirect muscular stimulation (i. e., through the intermediation of a motor-nerve) and finds that the period consumed in the transformation of the nervous into the muscular stimulus is halved or even more reduced by a rise of ten degrees in the temperature. Similarly the changes involved in the stimulation of Sensory Nerve Endings are determined by chemical factors, since T. E. Moore has shown that the temperature-coefficient of the reaction to cutaneous stimulation by heat is of the chemical magnitude. Corre- sponding with these facts we find that nerve-endings readily undergo fatigue. We have seen that the rate of the heart-beat is doubled or more than doubled by a rise of 10 and the same thing has been found to be true for other rhythmic muscular contractions. The rate of conduction of the Action-current in Muscles, however, appears, from the investigations of Lucas, to be a process analogous to the conduc- tion of a nervous impulse, comparatively little affected by temperature (coefficient from 1.45 to 1.65). It is a general characteristic of Photochemical Reactions, and a pecu- liarity which distinguishes them from all other types of chemical transformation, that they are practically unaffected by temperature, the temperature-coefficients being usually unity, and at any rate not in excess of the magnitudes commonly obtained in purely physical phenomena. This being the case it is a very significant fact that the temperature-coefficients of the phenomena induced by light in living organisms are usually high and distinctly of the order indicating the involvement of chemical reactions of the ordinary type. Thus the phototropic bending induced by light in Avena sativa has been shown INFLUENCE OF LIGHT UPON LIFE-PROCESSES 429 by de Vries to be increased from three to five times in velocity by a rise of 10 in temperature. Still more remarkable is the fact that the Assimilation of Carbon Dioxide by green plants in sunlight which underlies the Photosynthesis of Carbohydrates is also doubled or more than doubled by a rise of ten degrees in temperature; the following results are compiled from the measurements of Gabrielle Matthsei, the absorption measured being that of leaves of Prunus laurocerasus exposed to gaslight of constant intensity: Carbon dioxide Temperature-coefficient Temperature. assimilated. per 10 C. -6 0.2 28.7 1.75 2.40 10 4.2 2.12 20 8.9 1.76 30 15.7 1.81 37 23.8 0.23 40.5 14.9 Heat-injury already appears at 30, but below this temperature the coefficients clearly indicate that the rate of assimilation is not deter- mined by the photochemical process but by a reaction of the ordinary type. These results may be interpreted by supposing that the photo- chemical reaction (transformation of CO 2 and H 2 O into formaldehyde) is retarded by its product, and that the speed of photosynthesis is therefore determined by the rate at which this product is removed by a secondary reaction (condensation of formaldehyde into glucose). THE INFLUENCE OF LIGHT UPON LIFE-PROCESSES. Photosensitive Substances are of very widespread occurrence in living tissues. This is evidenced by the fact that the effects of light upon organisms are not by any means confined to the specialized cells which comprise the visual organs in the higher metazoa. The synthesis of Carbohydrates in plants is brought about by the action of sunlight upon vegetable tissues which contain Chlorophyll or some analogous pigment and are exposed to an atmosphere containing carbon dioxide, but quite independently of this, light additionally exerts an effect upon the proto- plasm of the cells of most plants, leading to a bending of sessile forms or an actual migration of motile forms toward or away from the source of light, a phenomenon known as Phototropism or Heliotropism. This phenomenon is also very generally displayed by animals, and the investigations of J. Loeb have demonstrated that the mechanism of heliotropism in animals and in plants is essentially the same, nor is it invariably associated, even in animals, with the possession of specific light-sensitive organs. For many of the unicellular forms of life sun- light is very definitely toxic, and this is true not only for pigmented but also for colorless cells. The most toxic portion of the spectrum lies in the ultraviolet region, a fact which bacteriologists have attempted 430 PROCESSES INFERRED FROM INDIRECT OBSERVATION to utilize for the sterilization of water and milk; a difficulty is created, however, by the deficient power of ultraviolet light to penetrate liquids, which necessitates the exposure of only thin layers to the toxic rays. The region of the spectrum which is most efficient in causing helio- tropic movements or curvature varies in different species of animals and plants. Among the plants the rays of the blue end of the spectrum are usually the most efficient. Thus Blaauw has determined the dura- tion of exposure to light derived from various parts of the carbon arc which is necessary to induce heliotropic curvature in the seedlings of Avena. The following are his results: Duration of illumination Position of the rays in in seconds. the spectrum. 6300 '.. .... : . . . . . 534 JUM 1200 510 " 120 499 15 491 " 5 487 " 4 478 " 3 . . . . . . . . . . ... ... ... 4 , ... 466 " 6 448 " The maximum effect is therefore obtained between 478 MM an d 466 ///z, that is, in the blue region of the spectrum. Among animal forms Loeb and Wasteneys have found that Eudendrium and Arenicola are similarly affected chiefly by the blue rays, while other animals, for example the crustaceans and insects are primarily affected by the rays lying en the border of the green and yellow. Even closely allied forms may, however, differ very decidedly in the part of the spectrum which is most efficient in eliciting heliotropic curvature. Thus Loeb and Wasteneys have observed that the green flagellate Euglena viridis is most affected by the blue rays lying between X = 470 and 480 ///*, while the closely allied flagellate Chalmydomonas pisiformis is especially sensitive to the yellow-green rays in the neighborhood of X = 534 w That the foundation of these light effects resides in a Photochemical Reaction which is induced within the organism is shown by the applica- bility of the Bunsen-Roscoe Law, which is generally characteristic of photochemical reactions and applies, for example, to the blackening of a sensitized photographic plate by exposure to light. This may be enunciated as follows: The chemical effect induced by light is pro- portional to the product of the intensity multiplied by the duration of illumination, or in symbols: E = Kit where E is the extent of photochemical transformation, i the intensity of the light, and t the duration of the illumination and K a propor- tionality-factor which is constant for the particular photochemical transformation under consideration. INFLUENCE OF LIGHT UPON LIFE-PROCESSES 431 The validity of the Bunsen-Roscoe law in the heliotropism of organ- isms has been established in a variety of investigations. Thus Blaauw has determined the time required to produce heliotropic curvature in the seedlings of Avena sativa by varying intensities of illumination with the following results : Duration of Candle-meters Candle-meters. illumination. X seconds. 26.3 20.6 21.9 18.6 19.1 16.2 17.2 18.3 19.7 22.4 23.9 21.6 24.8 27.5 24.2 21.8 16.9 18.9 18.0 24.7 20.5 22.8 19.0 19.8 16.4 26.5 It will be seen that the product of the intensity into the duration of the illumination approaches the constant value of 20, and indeed, when one considers the very great range of intensities employed and the inherent variability of living material, the degree of constancy observed is really astonishing. A remarkable instance of the applica- bility of this law to the Heliotropism of animals is afforded by the experiments of Loeb and Wasteneys, upon the polyps of Eudendrium. These hydranths are exceedingly variable in their response to light and it was accordingly necessary to make a great number of measurements and treat the results statistically. The polyps were exposed to three different intensities of light, a light of definite strength being stationed at three different distances, namely 25 cm., 37.5 cm. and 50 cm. from the organisms and they were exposed to the light for such periods as to render the product i X t a constant. Under these circumstances it was found that some among any group of polyps underwent helio- tropic curvature, while others did not. The percentage of bent polyps was determined in each case, and if the Bunsen-Roscoe law were valid it is evident that these percentages should be the same for all three intensities of light, i. e., the percentages undergoing bending at the distances 25, 37.5 and 50 cm. from the light should be the same or 1 : 1 ; 1, The actual ratios were determined for each of the possible 0.000439 ... 13 " 0.000609 . . . . ... 10 " 0.000855 . . . . . 6 " 0.001769. . . ... 3 " 0.002706. . . . ... . 100 minutes 004773 ' 60 " 01018 . 30 " 01640 . . . . 20 " 0249 . . . . . 15 " 0.0498 ... 8 0.0898 ... 4 0.6156 . . . . : 40 seconds 1.0998 ... 25 3.0281 ... 8 5 456 4 " 8.453 ... 2 18 94 1 " 45.05 ... 2/5 " 308.7 . . . 2/26 " 511.4 l/ 26 " 1255. . . .': v " 1902 . . . Vioo " 7905 . . . V0 " 13094. , . v - : . . . . . . . Vsoo " 26520. .: . -. . . . . . Viooo " 432 PROCESSES INFERRED FROM INDIRECT OBSERVATION pairs of distances and they differed more or less from the ideal value of unity, but the average of a large number of these ratios differed from unity only by an amount commensurable with the probable error of the average. The following are their results, the values enclosed in brackets being rejected by Chauvenet's criterion for the rejection of extreme variates. 1 Times of exposure in minutes. Ratio of percentage of hydranths bending toward light. 25cm. 37.5 cm. 50 cm. 25cm.; 37.5cm. 25 cm. ; 50 cm. 37.5cm.; 50cm. 15 60 1.50 - 20 80 .... 1.30 10 22.5 40 1.20 (3.08) (2.56) 10 22.5 40 0.94 1.47 1 . 55 10 22.5 40 1.57 (2.30) (2.43) 10 22.5 40 1.43 1.04 0.94 10 22.5 40 0.76 1.09 1.47 10 22.5 40 1.05 1.13 0.90 0.96 10 22.5 40 1.15 .... 0.99 7 15.75 28 0.84 0.62 0.74 7 15.75 28 1.70 0.49 0.58 7 15.75 28 0.85 1.25 1.35 7 15.75 28 (2.09) 0.99 1.08 7 15.75 28 1.14 1.15 0.55 7 15.75 28 0.44 0.92 0.44 7 15.75 28 1.52 0.80 0.61 7 15.74 28 0.59 0.36 0.70 7 15.75 28 0.48 1.07 0.31 7 15.75 28 1.00 0.48 1.80 7 15.75 28 0.69 1.09 0.81 7 15.75 28 1.26 0.85 1.09 7 15.75 28 0.86 1.38 0.85 7 15.75 28 0.70 1.07 1.59 7 15.75 28 0.77 1.25 7 15.75 28 0.60 \ Average* . 1 01 fl Q7 Qfl Probable err or* . . . ,. j. . \j i U . 7 / U . t7O 0.05 0.04 0.06 * The averages and probable errors given are those recalculated by the authors since the original article was published. It is a general law of Photochemical Action that only those rays are effective which are absorbed by the system in which the reaction occurs. Visible light-rays are not, as a general rule, selectively ab- sorbed by protoplasm and hence their action is usually confined to or exerted reflexly through specialized pigmented areas which constitute the receptive elements of optical sense-organs. White light which is not toxic for the majority of tissues may be rendered toxic, as L. Loeb has shown, by impregnating the tissue with certain dyes, particularly Eosin, which in such cases acts as the photochemical absorbent or sensitizer. Ultraviolet Light, however, is universally toxic even for 1 W. Chauvenet: A Manual of Spherical and Practical Astronomy, Philadelphia, 1891, vol. 2, p. 558. INFLUENCE OF LIGHT UPON LIFE-PROCESSES 433 colorless organisms, and since this toxicity presumably depends upon and is attributable to photochemical reactions, the question presents itself: To which constituent of the protoplasm are we to attribute the selective absorption of these rays which we may presume to be the necessary precedent to their photochemical activity? It was pointed out over forty years ago by Soret that the majority of proteins exhibit a well-marked absorption-band in the ultraviolet part of the spectrum. In seeking for the origin of this absorption- band Soret found that it is especially well exhibited by solutions of Tyrosine and therefore referred it to the tyrosine radical in the protein molecule. These observations have been extended by Kober, who has carried out a spectrographic examination of solutions of the various Amino-acids which are the end-results of protein hydrolysis and of certain Polypeptides. Kober has confirmed the existence of an absorp- tion-band in the ultra-violet in solutions of tyrosine, and finds that a similar band is exhibited by solutions of Phenylalanine. The other amino-acid constitutents of the protein molecule exhibit only general, i. e., non-selective absorption in the ultraviolet spectrum. The possibility is thus indicated that the tyrosine and phenylalanine radicals of the proteins constitute the optical sensitizers which render living cells susceptible to the toxic action of ultraviolet light. If this were the case, then passage of the light through solutions of proteins or the aromatic amino-acids should, by absorption of the toxic ray, to a greater or less extent, deprive the light of its toxicity for protoplasm. This possibility has been investigated by Harris and Hoyt, who have found that the passage of ultraviolet light through protein or peptone solutions partially detoxicates it, while passage through solutions of Cystine, Tyrosine or Amino -benzole Acid has a remarkable effect in shielding the organisms from injury. Other dissolved substances such as sugar, urea, alanine, glycocoll, etc., were found to be devoid of pro- tective power. Leucine undergoes decomposition when exposed to ultraviolet light and it exerts a certain measure of protection. The following are illustrative results, the light from a Cooper-Hewitt ultra- violet light being passed through the solution contained in a quartz beaker before reaching the organisms (Paramoecia) suspended in dis- tilled water below the beaker : Average determination- Solution, period, seconds. Water 130 1 . per cent, al anine 130 1 . per cent, glycocoll . 130 1 . per cent, aspartic aci d 130 1 . per cent, glutamic acid 135 1 . per cent, leucine . 250 . 5 per cent, tyrosine 420 1 . per cent, amino-benz-oic acid 2400 . 5 per cent. NaOH 150 1 . per cent. NaOH 170 1 . per cent, glutamic acid in 1 per cent. NaOH 200 1.0 per cent, cystine in 0.5 per cent. NaOH 1200 1 . per cent, tyrosine in 0. 2 per cent. NaOH . . . Unaffected after 40 minutes' exposure, 28 434 PROCESSES INFERRED FROM INDIRECT OBSERVATION The absorption of ultraviolet rays by tyrosine has been found by Kober to be markedly increased by an alkaline reaction and, as the above results show, the detoxication of the ultraviolet light by tyrosine solutions is also very greatly increased by an alkaline reaction. The results of Harris and Hoyt are thus in harmony with the view that the susceptibility of protoplasm to ultraviolet light is conditioned by the selective absorption and consequent "activation of the toxic rays by the aromatic ami no-acid radicals of the proteins. These results have a practical as well as a theoretical bearing, for they imply that fluids containing proteins would be much more difficult to sterilize with ultraviolet light than water, owing to the protective action of the proteins in the fluid through which the light has to pass before it impinges upon the protoplasm of the infecting organisms. THE STORAGE OF POTENTIAL ENERGY: THE PHOTO- SYNTHESIS OF CARBOHYDRATES. The leaves and other chlorophyll-containing organs of green plants absorb Carbon Dioxide from the atmosphere and simultaneously liberate an equal volume of oxygen. The carbon which is thus retained is built up into the tissues or reserve-materials of the plant, appearing chiefly in the form of Carbohydrates which accumulate very rapidly during active assimilation. The process of carbon dioxide assimilation by green plants takes place only in the light and in the presence of Chlorophyll or related pigments. Within certain limits the rate of absorption is proportionate to the intensity of the illumination of the leaf, and to the percentage of carbon dioxide in the atmosphere. Not all parts of the spectrum are equally efficient in promoting this process, the red rays between B and C causing the most rapid -assimilation while the activity of the rays between D and E on the Frauenhofer scale is a minimum, and there is a second maximum in the violet, beyond R This was first shown in a most ingenious manner by Engelmann. The Aerobic Bacteria require the presence of free oxygen to display motility. If some green algae and aerobic bacteria be imprisoned together under an air-tight cover- glass and kept in the dark, the free oxygen is soon consumed and the bacteria become motionless. If the cell is now exposed to light the algae decompose carbon dioxide, setting free oxygen, and the bacteria become motile again. Exposure of the cell in different parts of the spectrum yielded the above-quoted results. The assimilation of carbon dioxide by green plants is the foundation of the existence, not only of the plants themselves, but of the animal world. The radiant energy of the sun which is in this manner stored up in the tissues of the green plants, reappears at the other extremity of the life-process as the heat or muscular energy and mechanical work of an animal. A very striking peculiarity of living material also originates in this process, for while the components of protoplasm are, STORAGE OF POTENTIAL ENERGY 435 as a rule optically active, i. e., rotate the plane of polarized light to the* right or left, the products of laboratory-syntheses and those substances in nature which have never passed through the life-cycle (and some of those which have done so) are optically inactive. It is true that we can decompose a racemic and optically inactive mixture into optically active parts by utilizing the selective enzymatic activities of Yeasts, or, as Pasteur did, we may sort out large crystals by hand into two kinds possessing equal and opposite rotatory powers, but it will be observed that all of these processes involve the intrusion of a living agent. According to the view of Byk, optical activity originated in the earth through the circular polarization of light which occurs when light is reflected from the surface of the sea. If, on the other hand, we revert to Arrhenius' theory of the origin of life upon the earth, we may suppose that optical activity was transmitted to this planet by Bacterial Spores floating in interstellar space. However this may be, the phenomenon of optical activity is at present a distinguishing charac- teristic of the components of living matter, and it originates in the very first step in the life-cycle, for the carbohydrates which result from the photosynthetic activities of plants are optically asymmetrical. Notwithstanding the fact that the immediate connection between the assimilation of carbon dioxide by green plants and the appearance of carbohydrates has long been understood, the intermediate products which are formed in the process; the various stages which link the absorption of carbon dioxide to the appearance of starch or sugars in the tissues, have long been sought for in vain. The classical theory, proposed by Baeyer in 1 870, is that the carbon dioxide is first reduced to Formaldehyde CO 2 + H 2 O -> HCHO + O 2 and that the formaldehyde which is thus formed is subsequently con- densed to a hexose: 6HCHO = C 6 Hi 2 O 6 If this view is correct then we should expect to find formaldehyde among the constituents of the green plants when engaged in active assimilation. Very many attempts have been made to establish the presence of formaldehyde in the tissues of plants and they cannot yet be said to have yielded any very definite information. Several excep- tional difficulties attach to this investigation. In the first place it is certain that if formaldehyde occurs in green leaves at all it is never present except in very minute amounts. Indeed it is essential that this should be so, because formaldehyde is a very powerful protoplasmic poison and the accumulation of any amount in excess of a minute trace would result in the complete arrest of protoplasmic activities. Thus Elodea canadensis is cited as a form which is exceedingly resistant to the toxic action of formaldehyde, yet it will only withstand a 0.001 per cent, solution. 436 PROCESSES INFERRED FROM INDIRECT OBSERVATION We must expect to find formaldehyde in vegetable tissues, if it occurs therein at all, therefore, only in minute traces. Now although we possess very sensitive reagents for aldehydes, yet these do not as a general rule exclude the possibility of much more complex aldehydes than formaldehyde being present and yielding the reaction. Even Proteins and many other tissue-constituents will yield reactions indica- tive of an aldehyde-grouping. This would not perhaps constitute an insurmountable difficulty if it were not for the fact, as we have seen, that the aldehyde we are seeking to identify is at the most only present in minute traces. Another way of attacking the problem might appear to be feasible, namely that of extracting Chlorophyll from green plants and utilizing its light-activating properties to bring about the synthesis of for- maldehyde in laboratory-glassware, apart from the complications and secondary reactions which attend the process in living tissues. Numer- ous attempts to accomplish this have failed. According to Usher and Priestley, however, the source of failure has resided in the employment of comparatively thick layers of the chlorophyll solution. If we blow carbon dioxide through a test-tube or flask filled with chlorophyll and exposed to light, we cannot expect to observe much photosynthesis, because the most superficial layers of the chlorophyll solution will absorb all of the active light-rays and transmit to the underlying solution only those which are chemically inactive. In the living plant the chlorophyll is disposed quite differently. Here we observe that pigment is confined to exceedingly thin layers at the surfaces of a series of bodies known as the Chloroplasts in which active photosynthesis can be shown to be proceeding during illumination. Usher and Priestley have sought to imitate this architecture of the photosynthetic apparatus, by painting the surfaces of plates of gelatin with a thin layer of chlorophyll and then blowing carbon dioxide over them and exposing them to light. Under these conditions they state that a comparatively rapid disengagement of oxygen occurs, the surface film becoming wrinkled and distorted by the accumulation of bubbles of oxygen below it, while very evident quantities of formaldehyde are found in the underlying gelatin. The accumulation of formaldehyde in this case, as contrasted with its evanescence in the tissues of plants they refer to the absence of the enzymes necessary to accomplish the removal of the formaldehyde by condensation, which, in the plants, are present in the underlying substance of the chloroplasts. Against this experiment it has been urged by several investigators that the presence of formaldehyde in gelatin jellies is very difficult to establish, since most samples of gelatin themselves yield a very pronounced aldehyde- reaction. Usher and Priestley, however, state that the gelatin which they employed was free from aldehydes. On the other hand the syn- thesis of formaldehyde and other products from carbon dioxide and water has frequently been accomplished without the intermediation of chlorophyll by the use of the silent electric discharge, and by exposure STORAGE OF POTENTIAL ENERGY 437 to ultraviolet light or to sunlight in solutions containing salts of Uranium. We have seen that the rate of assimilation of Carbon Dioxide is governed, not primarily by the velocity of the photochemical reaction, birt by the velocity of a subsequent reaction which removes its products. This is shown by the fact that the temperature-coefficient of carbon- dioxide assimilation is of the usual chemical magnitude and not unity, as would be the case in a purely photochemical process. If the prod- uct of the photochemical reaction is in truth formaldehyde, as Baeyer's hypothesis assumes, then its accumulation would very evidently be injurious and we can readily understand how its removal, which presumably does not require the agency of illumination, would be an essential condition of the continuance of the reaction and would " set the pace" of the whole process. It is not certain, however, at exactly what stage of carbohydrate-synthesis the necessity for light ceases. Thus W. Loeb has obtained not only the formation but also the partial polymerization of formaldehyde with the silent electric discharge. The fact that starch-formation will go on in tubers and other plant- tissues which are not exposed to the light throws no light upon this question, for the starch in these instances is not formed from formalde- hyde but from hexoses or other comparatively complex carbon com- pounds. In regard to the nature of the earliest carbohydrate to arise in photosynthesis the most natural supposition would appear to be the formation of Glucose 6HCHO = CeHizOe or some other hexose, since this synthesis has actually been performed in the laboratory. As a matter of fact, however, there is much evi- dence tending to show that the first carbohydrate to be produced in photosynthesis is actually Cane-sugar (sucrose). This view, which was first put forward by Brown and Morris, has received very strong support from the investigations of Parkin. This observer employed for his experiments the leaves of the snowdrop, Galanthus nivalis, which are peculiar in that they do not form Starch during photosyn- thesis, so that the analyses of sugar-content are not complicated by the possible presence of sugars, Maltose or Glucose, derived from the hydrolysis of starch. As a matter of fact it was found by Parkin that the leaves of the snowdrop contain only three carbohydrates, namely Sucrose, Fructose (levulose) and Glucose. Of these the per- centages of hexose remain very constant throughout any given twenty- four hours, not increasing during the illumination of the day, nor decreasing during the night, while the percentage of sucrose rapidly increases during the day and decreases decidedly at night. Moreover the proportion of sucrose to the other sugars is greatest at the apical portions of leaves where assimilation is most active, and decreases toward the base. Two interpretations of this result, however, may 438 PROCESSES INFERRED FROM INDIRECT OBSERVATION be advanced. The cane-sugar may be in truth the first sugar to be synthesized, or, on the other hand, glucose may be the first sugar formed, levulose arising from it by a transformation which can be accomplished in laboratory-glassware, and the constancy of the hexose percentage may merely mean that hexose in excess of this amount is condensed to cane-sugar as rapidly as it is formed. It should be mentioned, however, that the formation of levulose from glucose by alkalies in laboratory-glassware is accompanied by the simultaneous formation of Mannose, which sugar is absent from foliage- leaves. On the other hand no laboratory-method of directly deriving cane-sugar from formaldehyde has yet been discovered. THE CONVERSION OF CHEMICAL INTO MECHANICAL ENERGY: THE CHEMICAL MECHANICS OF MUSCULAR CONTRACTION. We have seen that upon a normal mixed diet the necessary energy for the performance of muscular work is derived from the oxidation of Carbohydrates and that the final products of this oxidation are carbon dioxide and water, an intermediate stage of the combustion being the formation of Lactic Acid. So much we can ascertain by methods of direct analysis. If we desire, however, to complete the story of the energy-cycle which begins with photosynthesis in the plant, and cul- minates in the release of heat and mechanical work by the animal, purely analytical methods will not suffice and we are impelled to seek additional information by the method of inference from indirect observation. Our object is to ascertain the nature of the chemical machine which transforms the potential energy of carbohydrates into muscular work and heat. This problem divides itself into two parts, namely the question of the nature of the process of combustion and the question of the means of transforming the energy which combustion releases into mechanical work. In respect to the first of these questions, it has long been a familiar fact that when a muscle is repeatedly stimulated, either directly or indirectly through its motor-nerve, the first few contractions gradually and with considerable regularity increase in height until they reach a maximum for a given strength of stimulus. This phenomenon to which the name of "treppe" or the "Staircase Phenomenon" was given by Bowditch, has been the subject of considerable investigation and conjecture. Of a similar nature is the phenomenon of "Summation of Stimuli," whereby a stimulus of strength insufficient to give rise to a response when it is first applied, may be made, by repetition, to elicit a response. It is to Waller that we owe the suggestion that the "staircase" is, in reality, due to the increased production of carbon dioxide by the contractile or conducting tissue. He observed that small amounts of carbon dioxide augment the electrical response of nerve-fibers to CONVERSION OF CHEMICAL INTO MECHANICAL ENERGY 439 stimulation and that a short tetanization of the nerve produced a pre- cisely similar augmentation. Lee has extended this idea to muscular tissues and he has pointed out that the action of the products of muscular activity upon the performance of muscular work is two-fold producing in moderate quantities or for a short time a marked increase in the irritability and working-power of the muscle, while in larger quantities or after a longer period of action they produce a marked depression or "Fatigue*' of the muscle, ending by totally preventing the further release of muscular energy. The nature of the products which bring about these results has been established by Lee, who has found that perfusion of a muscle with a dilute solution of Lactic Acid or an acid phosphate increases its irritability and power to do work, while more concentrated solutions of the same substances diminish and finally abolish its irritability and contractility. Both of these sub- stances are known, by direct estimation, to accumulate in a muscle which is doing work. If we consider a muscle which is being tetanized by rapidly repeated stimuli, it is evident that the rate at which the muscle is doing work may be regarded as an expression of the rate at which the underlying chemical changes are taking place. During the initial or rising part of the curve of tetanus which is nearly always to be observed, the velocity of the underlying chemical changes must be increasing. During the period of maximal contraction while the recording-lever remains at a constant level it is evident that the rate of doing work and therefore the velocity of the underlying chemical change are practically constant, during the third, or descending part of the curve the velocity of the chemical changes is evidently decreasing. Similar considerations apply, of course, when the muscle, instead of being stimulated at extremely small intervals, is being stimulated at longer intervals. The chemical changes which underlie and determine muscular con- traction are of such a character, therefore, that one or more of the products which result, first accelerate and later retard the process. We are familiar with many chemical reactions of this type; they are reactions in which one of the products acts as a catalyzer to the process and are therefore designated Autocatalyzed Reactions. Thus in the hydrolysis of Cane-sugar by neutral boiling water small quantities of mucic acid are developed which greatly accelerate the inversion. The hydrolysis of Methyl Acetate by water results in the liberation of acetic acid which very greatly accelerates the hydrolysis. The hydrolysis of the Ricino- leic Acid in pulverized castor-oil beans proceeds at first very slowly, and then with great rapidity, the acid which is first liberated enhancing the activity of the lipase in the macerated tissues. Instances of auto- catalytic oxidations are afforded by the spontaneous oxidation of many Metals and organic compounds in the presence of oxygen at atmos- pheric temperature and pressure. It has long been observed that in the spontaneous oxidation of these substances they acquire the power of inducing oxidations in other substances which are not spontaneously 440 PROCESSES INFERRED FROM INDIRECT OBSERVATION oxidizable, and it has been shown that this action is due to the forma- tion of peroxides which catalyze oxidations, including the oxidation of the spontaneously oxidizable material itself. In the case of metals the process is ultimately brought to a close by the thickness of the covering of oxide which excludes the air. To preserve metals from spontaneous oxidation, therefore, one of two methods should be adopted: Either they should constantly be kept clean and polished to avoid the accumu- lation of catalyzers, or they should be allowed to become so com- pletely tarnished that air can no longer penetrate to the underlying metal. The intermediate policy of sporadic infrequent polishing leads to maximal loss of the metal by oxidation. Very many instances of autocatalysis are afforded by the spontaneous oxidation of fats and oils, and particularly by the oxidation of the "Drying Oils" which are employed in paints and varnishes. It is a general characteristic of the processes of Autocatalysis that they begin relatively slowly, progressively increase in velocity to a maximum, and then fall off in velocity again until the reaction finally ceases. The cessation of the reaction may be due to the exhaustion of the Substrate or material undergoing transformation, as for example in the hydrolysis of cane-sugar, or it may be due to the back-pressure of the accumulated products, as in the case of the hydrolysis of methyl acetate. In general the autocatalyzers, like other catalysts, accelerate the attainment of equilibrium from either direction. The underlying combustion which releases the heat and mechanical energy of muscular contraction is therefore an example of a large class of chemical transformations which produce their own catalyzers. Of the various stages of the process only a few are known, but among the known products lactic acid and carbon dioxide are capable of identifica- tion as direct or indirect catalyzers of the combustion. Our knowledge of the second phase of the problem which is presented by the genesis of muscular work and heat is still more fragmentary and much more conjectural. No machines of the ordinary type with the details of which we are familiar, such as those which operate by gaseous or liquid pressures and mechanical thrusts, will even approximate in characteristics and behavior to the motile mechanisms of living proto- plasm. The low and only very slightly fluctuating heat of combustion precludes any explanation attributable to alternate expansions and contractions due to heating and cooling. Engelmann, indeed, has proposed such an explanation!, based upon the supposition that intense heating of minute particles in the muscle-substance may occur in a number of circumscribed foci. He has pointed out that a number of Doubly Refracting Substances, such as catgut or India-rubber, have the unusual property of contracting when they are heated, and he assumes that the heat-energy of combustion in muscular tissue is directly trans- formed into mechanical work by transient intense heating of localized doubly refracting elements. Many objections have been urged against this hypothesis and they appear in our present state of knowledge to CONVERSION OF CHEMICAL INTO MECHANICAL ENERGY 441 be insurmountable. The objection, for instance, that living matter is destroyed at the height of temperature required was met by Engelmann by supposing that the elements so heated only form a very small pro- portion of the whole contractile tissue. If this be so, then they cannot be the doubly refractile elements which we perceive under the micro- scope, for these form a very large proportion of the whole. The foun- dation of Engelmann's analogy between muscular tissue and catgut or caoutchouc therefore falls to the ground. Furthermore even the small proportion of the structural elements of muscular tissue which Engelmann assumes to be subjected to heating, having been destroyed thereby, would have to be decomposed and the products excreted. Muscular work should therefore consume muscle-tissue and the nitrog- enous excretion should increase. This, however, on a normal mixed diet, does not occur. Again, the intense local heating which Engel- mann assumes implies difficulty in the distribution and dissipation of the heat which results in muscular combustion, yet the swift relaxation which succeeds normal muscular contraction implies just the reverse. A direct transformation of heat into work through the agency of expansion or contraction is therefore an improbable explanation of muscular contraction. Other observers have sought to attribute the phenomena of mus- cular contraction to the Swelling or shrinkages of semisolid elements through the imbibition or giving up of water, as a jelly absorbs water from or parts with it to the surrounding medium. The known proc- esses of this kind are, however, relatively slow and gradual in develop- ment, whereas muscular contraction and relaxation may in the muscles of the insect's wing alternate no less frequently than 300 times per second. The only physical displacements which are capable at the same time of such rapid alternation, of the performance of so much mechanical work in a non-rigid system, and of transforming so large a propor- tion of energy into mechanical work as a living muscle, are the dis- placements which result from changes in Surface-tension. These are excessively rapid because the forces involved are of great magnitude and the frictional resistances which oppose them may be, under favor- able conditions, very small. The amount of energy stored up in a fluid surface is very great and the release of this energy by chemical or resultant electrical changes affecting the tension of a large surface would suffice to permit the performance of a large quantity of me- chanical work. The maximal attainable Efficiency of a surface-tension engine as Brunner and Wolf have shown, is fifty per cent., i. e., the heat absorbed in extending the surface of water is equivalent to one- half of the mechanical work done in producing the surface-extension. This is also the maximal efficiency which has ever been observed in the performance of muscular work. The earliest theory to regard a muscle as a surface-tension engine was that proposed by Imbert who assumed that the individual Fibrils 442 PROCESSES INFERRED FROM INDIRECT OBSERVATION which are demonstrable in muscular tissue are long thin cylinders which are maintained in a condition of elongation by passive stretching. Under the influence of increase of surface-tension at the surface of contact of these tubules and their fluid contents Imbert assumes that the tubules become more spherical and therefore shorter, the simul- taneous shortening and swelling of a number of these elements leading to the contraction of the muscle. According to this hypothesis the work performed in muscular contraction is derived from changes in the surface energy of the fluid contained in the tubules. Bernstein, rather drastically assuming certain magnitudes for the tension and alterations thereof at the surfaces of these tubules, inferred that if the tubules consist of the visible muscle-fibrils then the surface afforded is not sufficient to account, on Imbert's hypothesis, for the amount of work performed in contraction. If, however, we suppose that the fibrils are broken up into a number of separate elements, for example into rows of ellipsoids which become spherical when the tension of their surfaces increases, then the surface presented would be sufficient to account for the observed release of mechanical energy. Now recent investigations by Schafer, McDougall and others on the details of the microscopic structure of muscle, have revealed the presence in the fibril, not exactly of the structure imagined by Bernstein, but one that for the purposes of Imbert's hypothesis is precisely equivalent to it. Schafer describes the contractile elements of the muscle-fiber as fine columns or Sarcostyles which are divided into segments or Sarcomeres by thin transverse discs, known as Krause's Membranes. Each sarcomere contains a relatively opaque portion, the Sarcous Element, while those portions adjacent to Krause's membrane are relatively transparent and seen to consist of a fluid material. The sarcous element itself is double and, if stretched, the two portions separate at a line which runs transversely across the opaque portion of the sarcomere (Hensen's line). On contraction the sarcous elements become shorter and thicker, absorbing the fluid which constitutes the Hyaloplasm or intervening transparent area between the sarcous elements and Krause's mem- brane. We may therefore picture the muscle-fibril as consisting of a series of discs formed by minute tubules packed together and com- municating with spaces separating the discs and filled with fluid (Fig. 25). Evidently such a structure as this conforms to every requirement imposed by Bernstein upon Imbert's hypothesis, and it is an exceedingly significant fact that the details of the structure which we have out- lined become clearer and more elaborate as we successively pass from the relatively sluggish and inert smooth muscles, or the striated mus- cles of amphibia, to the muscles of insects with their lightning-like rapidity of contraction and enormous power of performing work. This fact alone prevents us from entertaining any doubt that this elaborate structure is an essential part of the muscular mechanism, and the salient characteristic of this structure is the enormous surface of contact which it brings about between the fluid and the semisolid CONVERSION OF CHEMICAL INTO MECHANICAL ENERGY 443 elements of the tissue. We may therefore with considerable confi- dence infer that muscular tissue is a surface-tension engine which converts the energy released by the combustion of carbohydrates into heat and mechanical work. Several mechanisms are imaginable whereby the chemical changes which accompany muscular work might bring about alterations of surface tension at interfaces within the tissue. The Heat of Combus- tion of carbohydrates must of itself contribute to affect the tension and the changes of Electrical Potential which also accompany muscular contraction would likewise, as is shown by the analogy of Lipmann's FIG. 25. Wave of contraction passing over a leg-muscle fiber of water-beetle. (After Schafer.) capillary electrometer, affect the tension of interfaces in the tissue. In this connection one fact should be very particularly noted, and that is that either of these factors, and whether they are determinative or not they must contribute in some measure to the outcome, would lead, not to an increase of superficial tension, as imagined by Imbert and Bernstein, but to a decrease. We are therefore led to inquire whether, after all, the alteration in form of the sarcous elements in contraction may not be due to a decrease rather than to an increase in interfacial tension, for otherwise the thermal and electrical changes which accom- pany muscular contraction must actually diminish and inhibit con- traction and conflict with the main objective of the whole process. 444 PROCESSES INFERRED FROM INDIRECT OBSERVATION A mechanism whereby reduction of interfacial tension might bring about the shortening of sarcous elements is depicted in the accom- panying figure (Fig. 26). If we suppose the sarcous element to consist of an elastic tube dipping into and filled by the fluid hyaloplasm, then the tension of this fluid, pulling upon the elastic tubule will draw the walls inward and hence stretch them longitudinally. This condition of balanced tensions, capillary and elastic, may be supposed to be the normal resting state of muscle. If, now by heat, an electrical potential, or other means, the inward pull upon the tubule is released, this will have just the same effect that internal pressure would have upon an initially unstretched elastic tube it will expand and shorten, as a hose expands and shortens when water under a head or pressure is suddenly injected into it. Thus the ends of the sarcous elements will approach and, the total capacity of each element being increased, fluid Krause's Membrane Sarcous Element Fine Septa * Hansen's Line Krause's Membrane FIG. 26. Schematic diagram of a muscle-element. from the hyaloplasm will enter them. The sum of the effects of a multi- tude of such shortenings constitutes the contraction of a muscle-fibril. The conception of a motile mechanism as a surface-tension engine may readily be extended to include ameboid and ciliary motion as well as the phenomenon of Protoplasmic Streaming which is so frequently displayed in cells in which ameboid motion is constrained by viscosity or by rigid walls, as in many plant-cells. The genesis of movements analogous on the one hand to ameboid motion and on the other to protoplasmic streaming may be illustrated in a simple model as follows: f to a ten per cent, solution of camphor in benzole a little dye, for example Sudan III or Scharlach R be added, to render the outline of a drop visible against a colorless background, and small drops of this be placed upon the surface of clean water in a watch-glass, very rapid and energetic movements of the edges of the drops may be observed exactly similar in character to those presented by the surface of Ameba. CONVERSION OF CHEMICAL INTO MECHANICAL ENERGY 445 Even processes similar in form to Pseudopodia are thrown out and retracted. These movements are due to the changes in interfacial tension caused by unequal diffusion of the camphor from the benzole into the water. They may be slowed by adding some viscous fluid, e. g., olive oil to the benzol solution and finally, when about an equal volume of olive oil has been added, we no longer obtain ameboid movements but, instead, we observe an incessant streaming movement of the fluid within the drops, exactly resembling those seen within the protoplasm of a plant-cell such as Chara. If the streaming movements are not easily perceived owing to the transparency of the drop, the addition of a little finely powdered arrowroot starch will render them manifest, and impose a still more close resemblance to the actual appearance of streaming movements in protoplasm. REFERENCES. INFLUENCE OF TEMPERATURE UPON LIFE PROCESSES: Cohen: Physical Chemistry for Physicians and Biologists, trans, by Fischer, New York, 1903. Robertson: Biol. Bull., 1906, 10, p. 242. Arch. Internat. de PhysioL, 1908, 6, p. 388. Maxwell: Jour. Biol. Chem., 1907, 3, p. 359. Lucas: Jour. Physiol., 1908, 37, p. 112. The Conduction of the Nervous Impulse, London, 1917. Ganter: Pfliiger's Arch , 1912, 146, p. 185. Loeb: The Mechanistic Conception of Life, Chicago, 1912. The Organism as a Whole, New York, 1916. Kanitz: Tempera tur und Lebensvorgange, Berlin, 1915 (consult for literature). INFLUENCE OF LIGHT UPON LIFE PROCESSES: Kober: Jour. Biol. Chem., 1915, 22, p. 433. Harris and Hoyl: Science, N. S., 1917, 46, p. 318. Loeb: Forced Movements, Tropisms and Animal .Conduct, Philadelphia, 1918 (consult for literature). PHOTOSYNTHESIS : Palladin: Plant Physiology, trans, by Livingstone, Philadelphia, 1918. Jost: Plant Physiology , trans, by Gibson, Oxford, 1907. Czapek: Biochemie der Pflanzen. Jena, 1913. Blackman and Matthaei: Proc. Roy. Soc., London, 1905, B 76, p. 402. Usher and Priestley: Ibid., 1905-6, 77, p. 369; 1906, 78, p. 318; 1911-12, 84, p. 101. Loeb, W.: Zeit. f. Elektrochem., 1905, 11, p. 745. Parkin: Biochemical Jour., 1912, 6, p. 1. MUSCULAR CONTRACTION AND MOTILITY OF PROTOPLASM: Imbert: Archives d. Physiol., 5th ser. , 1897, 9, p. 289. McDougal: Jour of Anat. and Physiol., 1897, 31, pp. 410 and 539; 1898, 32, p. 187. Bernstein: Pfliiger's Arch., 1901, 85, p. 271. Robertson: Trans. Roy. Soc. of South Australia, 1905, 29, p. 1. Quar. Jour. Exp. Physiol., 1909, 2, p. 303. Science, N. S., 1912, 36, p. 446. Lillie: Am. Jour. Physiol., 1908, 22, p. 75. Maxwell and Rayleigh: Article on Capillary Action, llth ed., Encyclopedia Britannica, 5, p. 256. CHAPTER XIX. PROCESSES INFERRED FROM INDIRECT OBSERVATION: FERTILIZATION AND EARLY DEVELOPMENT. THE SUBSTITUTION OF CHEMICAL AGENCIES FOR NORMAL FERTILIZATION. IN the vast majority of animal forms the stimulus of fertilization by a spermatozoon of the same or a very closely related species is essential for the development of the egg. The fact, however, that Partheno- genesis, or development without fertilization may occur under excep- tional circumstances or in a limited number of forms, shows that the part played by the spermatozoon, in so far as it constitutes the stimulus to development, may be performed by other agents. The discovery of the exact nature of agents capable of giving rise to development of the egg was essential to the understanding of the phenomenon of Fertilization, for the spermatozoon, besides affording to the egg the initiatory impulse to development also acts as a bearer of hereditary factors and is, moreover, itself a living and a motile organism so that a great complexity of materials and factors gain entry into the egg with the introduction of the spermatozoon, and the disentanglement of these numerous variables was impossible until a clue to their nature had been obtained by means of experiments in which the single func- tion of fertilization was imitated by physicochemical means. The solution of this problem we owe to the investigations of J. Loeb who followed up the observation of T. H. Morgan and others that unfertilized eggs of various marine organisms may occasionally begin to segment without fertilization in sea-water, but that such eggs invariably die after a few divisions. In seeking to ascertain the origin of this abnormal phenomenon Loeb found that in the eggs of a sea-urchin, Arbacia, development could be induced by merely exposing them for a period to slightly Hypertonic Sea-water and then returning them to normal sea-water. The means employed to render the sea-water hypertonic was, within certain limits, imma- terial. Thus the Osmotic Pressure might be raised by spontaneous evaporation, or by the addition of one part by volume of 1\ normal sodium chloride solution to nine parts by volume of sea-water, or yet again Cane-sugar or Urea or some other physiologically inert substance might be employed for this purpose and with like success. It was even found possible to cause development of the eggs by immersing them in a pure cane-sugar solution only slightly exceeding sea-water SUBSTITUTION OF AGENCIES FOR FERTILIZATION 447 in its osmotic pressure. The increase of osmotic pressure required is not great. If Sodium Chloride be employed an increase of forty per cent, in the osmotic pressure of the sea- water suffices to initiate develop- ment after an exposure of two hours. If sugar or urea be employed even a slighter increase of osmotic pressure suffices to bring about a like effect, because these substances penetrate the egg with greater difficulty than the inorganic salts and hence exert a greater osmotic tension on the external surface of the egg. The requisite concen- tration of the medium varies, however, with the duration of the expos- ure, a weaker concentration being effective after a longer exposure. This is shown by the following experiment: To 50 c.c. portions of artificial sea- water (Van t'Hoff's Solution) rendered favorably alkaline by the addition of 2 c.c. of tenth normal sodium hydroxide were added 0, 2, 4, 8 and 16 c.c. of 2| normal potassium chloride solution. Unfertil- ized eggs of a Pacific Ocean sea-urchin (Strongylocentrotus purpuratus) were divided between these five solutions and samples removed after varying periods of exposure and placed in normal sea-water. The following were the results obtained: Increase in the osmotic pressure of the medium. Period of exposure, minutes. per cent. 16 per cent. 30 per cent. 55 per cent. 87 per cent. 45 .... No larvae No larvae No larvae No larvae Numerous larvae. 64 .... " " " Numerous larvae 89 . . .. .' " Numerous larvae 116 ..'..;. 114 . . The fertilization which resulted from this procedure failed, however, to furnish a perfectly faithful imitation of the phenomenon of natural fertilization. It is true that the eggs frequently developed into free- swimming larvae, but the larvae produced in this manner were sickly and abnormal and did not survive very long. The percentage of eggs which developed into larvae was variable and in some species, partic- ularly in the sea-urchin Strongylocentrotus franciscanus, few if any of the eggs could be induced to develop by this procedure. The larvae in all cases behaved abnormally; they did not rise to the top of the water and swim there as normal larvae do, but swam instead at the bottom of the vessel containing them, and finally, the most marked peculiarity of all was the failure of the eggs to form a Fertilization-membrane. If the eggs of a mature female sea-urchin be removed from the ovaries by shaking them out in sea- water and are then mixed with sperm similarly procured from the spermaries of a male, the sperma- tozoa will immediately be seen clustering around the eggs, presenting the appearance of striving to enter them. Within a very brief period, under normal conditions, a spermatozoon will succeed in effecting an entry, and this event is at once indicated by the appearance upon the 448 PROCESSES INFERRED FROM INDIRECT OBSERVATION surface of the egg of a number of irregular clear blister-like protuber- ances which rapidly increase in number and extent, finally covering the surface of the egg with a clear hyaline layer, which is designated the fertilization-membrane (Fig. 27). This was lacking in the artifi- cially fertilized egg. The imperfect character of the imitation of fertilization which was thus achieved led Loeb to form the supposition that the osmotic method of inducing fertilization only accomplished a part of the effects ini- tiated by the spermatozoon, which he inferred carried into the egg agencies not only capable of starting the processes initiated by the hypertonic sea-water but also processes which the osmotic method did not suffice to initiate. This supposition was confirmed by the discovery of a series of agents capable of inducing Membrane -forma- tion in the sea-urchin egg. 1. 2. FIG. 27. 1, unfertilized egg of the Sea-urchin (Strongylocentrotus purpuratus) sur- rounded by spermatozoa; 2, the same egg about two minutes later, after the entrance of the spermatozoon and the formation of the fertilization-membrane. (After Loeb.) It was found that if mature sea-urchin eggs were introduced for a few minutes into sea-water to which a small proportion of a certain sample of Ethyl Acetate had been added, and then returned to normal sea-water, all of the eggs promptly formed a fertilization-membrane differing in no perceptible degree from the membranes formed in normal fertili- zation. Other esters failed to yield any comparable result, and an examination of the ethyl acetate employed in the original experiment showed that it had undergone hydrolysis and contained free ethyl alcohol and acetic acid. This led to an investigation of the behavior of the eggs in sea- water containing added alcohols and acids and to the discovery that the effect originally obtained with impure ethyl acetate was due to the Acetic Acid which it contained. It was found that all of the monobasic fatty acids which are soluble in sea-water, namely formic acid, acetic acid, propionic acid, butyric acid, valerianic acid and so forth, will induce membrane formation in 100 per cent, of mature eggs if they are exposed for a brief period to the action of the sea- water containing the fatty acid. The formation of the membrane SUBSTITUTION OF AGENCIES FOR FERTILIZATION 449 does not occur until after the restoration of the egg to the normal sea-water. The eggs which have been treated in this manner may undergo a few divisions but they very rapidly die, more rapidly, in fact, than unfertil- ized eggs exposed to similar conditions of temperature, etc. The processes thus initiated therefore, still afford an incomplete analogy to natural fertilization. It was found, however, that by a combination of these two processes, membrane-formation and osmotic treatment, which are separately incomplete, a perfect imitation of fertilization is procured and a high percentage of the eggs, usually 100 per cent., can be induced to develop and produce normal larvae. The precise details of time, of exposure, concentration and so forth, in Loeb's improved method of Artificial Parthenogenesis necessarily vary slightly with the temperature, reaction of the sea-water and species of Echinoderm employed. The following are, however, the details of the method as utilized for the fertilization of the Pacific sea-urchin, Strongylocentrotus pur pur aim at a temperature in the neighborhood of 15 C. The eggs, after extraction from the ovaries and rinsing in filtered 1 sea-water are immersed in a mixture of 50 c.c. of sea-water and 2.8 c.c. of tenth normal Butyric Acid solution, and the mixture is gently agitated to prevent the eggs, which become sticky, from adhering to the bottom of the vessel. After about two minutes the eggs are collected by gentle rotation of the shallow flat-bottomed vessel and transferred by means of a pipette to normal sea-water. If the exposure has been rightly chosen it will be found that the eggs almost immediately form membranes. After allowing them to remain for some fifteen to twenty minutes in the normal sea-water they are again collected in the manner described and transferred to Hypertonic Sea-water, prepared by adding 8 c.c. of 2J molecular sodium chloride solution to 50 c.c. of sea- water. They are exposed to this addition for a period varying from fifteen to sixty minutes, the optimal exposure varying somewhat with the eggs from different females. The eggs are now returned to normal sea-water. Within about one hour the first cell-division will be observed to have occurred, at the end of forty- eight hours swimming gastrulse will have been produced, and about two days later plutei with well-developed skeletons. Artificial fertilization has been extended to a variety of forms other than the Echinoderms. In a number of Annelids development may be induced by preliminary treatment with a cytolytic agent followed by treatment with hypertonic sea-water. As a rule, however, in this group the fatty acids do not constitute sufficiently potent cytolytic agents, and saponins or, better still, mammalian blood serum must be employed. Among the Molluscs simple treatment with hypertonic sea- water frequently suffices, especially if it be rendered slightly hyper- 1 The sea- water must be filtered to remove spermatozoa which may possibly be suspended in it. 29 450 PROCESSES INFERRED FROM INDIRECT OBSERVATION alkaline, the alkali playing the part of the cytolytic or membrane- forming agent. In the eggs of frogs simple puncture with a fine needle suffices to induce parthenogenetic development, for what reason is not at present clearly understood, although we may fairly infer that it arises from the incidental admixture of certain constituents of the eggs which are normally separated from one another. Artificial partheno- genesis has also been induced in the eggs of plants (Fucus) by treating them with butyric acid, followed by hypertonic sea-water. The eggs of all forms which have been made to undergo develop- ment by artificial means yield normal embryos and their development differs in no wise from that of normally fertilized animals. The rearing of marine animals is an excessively laborious task, but Delage has had the courage to undertake it in the case of artificially fertilized sea- urchins and succeeded in maintaining them until sexual maturity. In the case of the frog several specimens arising from artificially fertilized eggs have been brought to sexual maturity by Loeb and Bancroft (see Fig. 28). FIG. 28. A parthenogenetic frog. (After Loeb.) THE NATURE OF THE AGENTS WHICH FORM FERTILIZATION- MEMBRANES. The monobasic acids of the fatty series are all capable of producing, as we have seen, the formation of membranes in the sea-urchin egg provided they are soluble in sea-water. Now this action might con- ceivably be due to the dissociation of Hydrogen Ions by the acids, or it might be due, on the other hand, to the anion or the undissociated molecule of the acid. The latter is the correct alternative, for although the highly dissociated mineral acids will induce Membrane-formation in a limited percentage of eggs, the requisite concentration of these NATURE OF AGENTS WHICH FORM MEMBRANES 451 acids is far greater than it is in the case of the fatty acids. In fact one- thousandth normal Butyric Acid is more efficient in inducing membrane- formation than twelfth normal Hydrochloric Acid ; yet hydrochloric acid of even this concentration does not injure the eggs in the periods of exposure requisite, because normal fertilization by sperm can still occur in 100 per cent, of the eggs treated in this manner, and weaker concentrations of hydrochloric acid, which are usually ineffective in causing membrane-formation, are, of course, even less toxic. A very convincing experiment devised by Loeb to illustrate this point consists in adding a little Sodium Butyrate to a solution of hydrochloric acid in sea-water which is otherwise incapable of causing membrane-for- mation. The mixture immediately becomes an effective membrane- forming agent, although its acidity, if anything, has been reduced. The introduction of the sodium butyrate leads to an interaction with the hydrochloric acid, setting free a little butyric acid which accomplishes the initial stage of fertilization. It had been observed in 1887 by O. and R. Hertwig.that if sea- urchin eggs be immersed in sea-water saturated with Chloroform and only a trace of this substance will dissolve in sea- water fertili- zation-membranes are formed. It had also been found by Herbst that Benzene, Toluene and Creosote have a similar action. In all these cases, however, the membrane-formation was found to be rapidly suc- ceeded by Cytolysis and the disintegration of the eggs, so that develop- ment, of course, did not occur. Loeb found however, that if the eggs be exposed only for very brief periods to these solutions and then transferred to normal sea-water a percentage of the eggs will form membranes without cytolysis and may subsequently be induced to develop by treatment with hypertonic sea-water. It is a curious, and as yet unexplained fact that whereas eggs immersed in butyric-acid sea-water do not form fertilization-membranes until they are trans- ferred to normal sea-water, eggs treated with sea-water containing benzene or amylene form membranes before they are removed from the mixture. The various reagents which were thus found effective in inducing membrane-formation have this in common, that they are all fat- solvents or highly soluble in fats and furthermore they are all in greater or less degree Hemolytic Agents, that is, substances which are capable of dissolving red blood-corpuscles. This fact drew attention to the possibility that other hemolytic agents might be capable of exerting a like effect upon the unfertilized egg of the sea-urchin. According to Koeppe, besides heat and alternating electric currents there are five distinct groups of chemical agents which are distinguished by their power of inducing hemolysis of red blood-corpuscles or, more generally, Cytolysis of all types of living cells. These are: 1. Certain specific substances, for example the series of glucosides comprising the Saponins and Solanins, or the Bile-salts. 2. A series of Fat-sol- vents such as benzene, ether or alcohol. 3. Distilled water, 4. Hydro- 452 PROCESSES INFERRED FROM INDIRECT OBSERVATION gen ions and 5. Hydroxyl ions. Successive experiments have shown that each of these groups of reagents may, by appropriate manipula- 6. 7. FIG. 29. Formation of fertilization-membrane and cytolysis of the sea-urchin egg on treatment with saponin. 1, appearance of egg when first exposed to saponin solu- tion at 9.07 A.M.; 2, 3, and 4, formation of fertilization-membrane. The stage of com- plete membrane-formation as depicted in 4 was reached at 9.15 A.M. (if at this stage the egg is withdrawn from the influence of the saponin it can develop). Cytolysis, 5, began at 9.20 A.M.; 6 and 7, advanced stages of cytolysis induced by saponin. (After Loeb,) tion, be made to cause membrane-formation in the unfertilized sea- urchin egg. The Saponins especially are extraordinarily efficient in inducing this phenomenon, membranes being formed in 100 per cent. NATURE OF AGENTS WHICH FORM MEMBRANES 453 of eggs by a dilution of one in a hundred thousand after an exposure of from 30 to 60 minutes; these membranes are formed in the solution itself without transference to normal sea-water. If, however, the action of the saponin solution upon the egg be permitted to continue, membrane-formation is rapidly succeeded by cytolysis and the egg disintegrates. This is illustrated in the preceding figure (Fig. 29), showing the successive effects of a saponin solution (8 drops of J per cent, saponin to five c.c. of sea-water) upon the unfertilized eggs of Strongylocentrotus. It is perfectly clear from these results that the formation of the fertilization membrane represents an initial stage of cytolysis. If, however, the eggs be removed from the saponin solution before manifest cytolysis has occurred, washed in sea-water a number of times to remove the last traces of saponin, and then treated with hypertonic sea-water, normal development occurs and a large propor- tion of the eggs develop into larvae. Precisely the same results are obtained with Bile-salts. There remains, however, another class of cytolytic agent which has yet to be considered in this connection, namely the tissue-fluids of unrelated species of animals. The Blood of the mammalia contains cytolytic substances which hemolyze and destroy foreign corpuscles and cells, but not those of the same species. This cytolytic power of blood and other tissue-fluids is greatly enhanced by previous immuni- zation with the foreign cells, but within the body at least it is exercised without previous immunization. It was found by Loeb that this class of cytolytic agents is also capable of causing membrane-formation in the sea-urchin egg. Not every unrelated species of animal, however, would furnish a tissue-fluid or extract capable of causing membrane- formation, in fact only a limited number of forms were found to do so. Loeb considers that this is due to the variable permeability of the eggs for different lysins, and the permeability of the eggs for a particular lysin also seems to vary somewhat in the eggs of different females. The first cytolytic agent of this kind to be discovered was that contained in the blood of certain marine worms, namely Dendrostoma, which calls forth membrane-formation in the sea-urchin egg even if it is diluted a thousand times or more with sea-water. Later investigations have shown that a number of other invertebrates yield tissue-fluids or extracts which will cause membrane-formation. Most interesting results were, however, obtained by Loeb with the blood of mammalia or birds. The blood-sera of mammals (oxen, sheep or pigs) which have been rendered isotonic with sea-water by the addition of sodium chloride will induce membrane-formation in sea-urchin eggs, but not invariably nor in all of the eggs derived from different females. Eggs from one and the same female will form membranes in some samples of blood and not in others, and again, one and the same sample of blood will form membranes in the eggs of some females but not in others. As in the case of the saponins the membranes are formed in the solution itself, 454 PROCESSES INFERRED FROM INDIRECT OBSERVATION and removal to normal sea-water is not essential, but, unlike the mem- brane-formation by saponins it is not followed, in undiluted blood at least, by subsequent cytolysis. As we shall see, however, dilution of the blood by sea-water enables the cytolytic effect to appear, complet- ing the analogy to the action of the saponins. As in all the instances previously considered, membrane-formation is succeeded by one or two cell-divisions and then by death and disintegration of the eggs, unless they are treated with hypertonic sea-water which enables them to develop and give rise to normal embryos. Loeb sought for the origin of the differing action of the various sera by endeavoring to modify it, and he found that preliminary heating of the sera greatly enhanced their ability to induce fertilization. Pre- liminary treatment of the eggs, however, was found to be even more effective. This treatment or "sensitization" of the eggs consists in exposing them for a brief period to an isotonic solution of a chloride of an Alkaline Earth, calcium, strontium or barium. Of the three, however, strontium is much the most effective; the efficiency of barium might possibly exceed even that of strontium if it were not for the fact that barium is also exceedingly toxic for the eggs, while strontium is almost harmless. After exposure to the Strontium Chloride and subsequent transference to isotonic blood-serum, membranes are formed in nearly every case upon 100 per cent, of the eggs. It has been shown by A. R. Moore that this sensitization by strontium is due, not to any irreversible change induced in the egg itself by strontium, but more probably to the actual presence of the strontium within the egg, for if the eggs after exposure to the strontium chloride solution be washed free of the solution by two or three changes of sea-water, their sensitiveness to blood-sera is lost. The membrane-forming substance in blood-serum was found by Loeb to be remarkably resistant to heat. Exposure of ox-serum to a temperature of 73 for half an hour, which leads to coagulation of the serum-proteins, does not destroy its fertilizing power. Heating the serum to 100 does, however, destroy the active substance. The blood of Dendrostoma still retains a proportion of its membrane-forming power, even after having been heated rapidly to boiling point; more prolonged boiling (2 to 3 minutes), however, destroys its activity. The thermostabile character of the active substance at once distin- guishes it from the Alexin or bactericidal substance which is present in mammalian blood-seia, for this is inactivated by heating to 56. The active substance in mammalian sera is not extracted by shaking the serum with Ether. If several volumes of Acetone are added to the serum, the precipitate which results, after drying, powdering and resolution in sea-water, retains the power of inducing membrane- formation. The substance is therefore precipitated by acetone. A very minute and intensely active fraction may be prepared from mammalian blood-sera by the following procedure. One hundred c.c. of ten per cent. Barium Chloride solution are added, with constant NATURE OF AGENTS WHICH FORM MEMBRANES 455 stirring, to each liter of serum. This mixture is kept in a cool place for forty-eight hours, when the supernatant fluid can be decanted. The residue is washed several times with large volmes of 2 per cent, barium chloride solution to remove all traces of serum. The precipitate is now stirred up with tenth-normal hydrochloric acid, warmed to 45 C. using 50 c.c. for the precipitate from each liter of serum. After stirring for an hour or more the mixture is centrifuged and to the clear fluid thus obtained an equal volume of tenth-normal sulphuric acid is added. This solution is allowed to stand at 40 C. for twenty-four hours and then thoroughly centrifuged to remove barium sulphate. To the clear fluid are added four or five volumes of acetone and the mixture is cooled for eight hours or more, at the end of which time the white flocculent precipitate has settled. It can then be collected on a hard- ened filter, washed with ether and dried over sulphuric acid. This preparation, which has been designated Oocytin has been carefully examined by G. W. Clark, who finds that successive samples differ in elementary composition, showing clearly that it is a mixture of two or more substances. It gives all the protein reactions but also yields on hydrolysis notable quantities of Hypoxanthine and a Pentose, but only a trace of phosphoric acid. These products correspond to those which would be yielded by the Nucleosides or glucosidal fractions derivable from the nucleic acids by partial hydrolysis. The presence of appre- ciable amounts of this glucoside in the preparation is of peculiar significance when the glucosidal structure of the Saponins, which are similarly potent in inducing membrane-formation, is borne in mind. The membrane-forming power of oocytin acting upon eggs sensi- tized by strontium chloride is very great, comparable in fact with that of the saponins. It will induce membrane-formation at a dilution of one in five hundred thousand. The sensitizing effect of strontium is clearly seen to lie in the fact that it precipitates the oocytin and so concentrates it within or upon the surface of the egg. In concentrated solutions eggs which have been freshly transferred from strontium chloride solution collect a dense precipitate at their periphery which may often mechanically prevent or delay the formation of the fertili- zation-membrane. That the activity of the preparation is not due to contamination by barium or other inorganic substances is shown by the fact that it is inactivated by heating for a few minutes to 80 Q. ; the temperature at which the activity of blood-serum itself is destroyed. From the spermatozoa of the sea-urchin, cytolyzed by distilled water, a similar fraction may be prepared having analogous potency in inducing membrane-formation in eggs which have been sensitized by immersion in strontium chloride solution. This material has also been found by Clark to yield notable amounts of a purine base and a pen- tose on hydrolysis. It would appear very probable therefore that membrane-formation in natural fertilization is brought about by the introduction into the egg, within the body of the spermatozoon, of a glucosidal cytolytic agent, which is related to the nucleosides. 456 PROCESSES INFERRED FROM INDIRECT OBSERVATION The action of oocytin upon the sea-urchin egg differs from that of isotonic mammalian blood serum in two respects; firstly in the fact that prolonged exposure of the eggs to oocytin solutions causes cytoly- sis and secondly in the fact that it is potent at a great number of differ- ent dilutions whereas the potency of isotonic serum to induce mem- brane-formation disappears upon dilution of the serum to one-half, or at all events one-fourth, by the addition of sea-water. This differ- ence in behavior is apparent, however, and not real. It is due to the inhibiting action of the proteins which are also present in the serum. If isotonic blood-serum be diluted by successive additions of sea- water the membrane-forming power is at first weakened and then disappears, but upon further dilution it reappears and then is retained to very high dilutions. The following are illustrative experiments: OX-SERUM SAMPLE III. Eggs sensitized by four minutes' immersion in f m. SrCl 2 Dilution of the Per cent, of membrane Per cent, of membrane isotonic formed in fifteen formed in fifty serum. minutes. minutes. 1 100 100 1/2 5 50 1/4 1/8 . ' Not observed 44 1/16 100 OX-SERUM SAMPLE V. Eggs sensitized by four minutes' immersion in f m. SrCl 2 . Dilution of the Per cent, of membrane Per cent, of membrane isotonic formed in fifteen formed in ninety serum. minutes. minutes. 1 68 100 (none cytolyzed) 1/2 80 86 (none cytolyzed) 1/4 (none cytolyzed) 1/8 16 ( 1 per cent, cytolyzed) 1/16 1 72 (10 per cent, cytolyzed) It is evident that when the membrane-forming power is regained in the higher dilutions the power of inducing cytolysis is also acquired, so that the action of the blood-serum now resembles that of saponin or oocytin in every respect. The failure of cytolysis to appear in undiluted serum is due to the inhibiting effect of the high concentration of Protein which it contains, and even membrane-formation may be inhibited if the concentration of serum-proteins is too high or if addi- tional protein be dissolved in the serum. Such sera will nevertheless induce membrane-formation and cytolysis if they are diluted, the inhib- iting effect of the proteins becoming negligible at a dilution of one in sixteen or one in thirty-two. Even membrane-formation by Butyric Acid or by spermatozoa may NATURE OF AGENTS WHICH FORM MEMBRANES 457 be inhibited by the addition of proteins to the sea- water. The following table shows the relative efficiency of various proteins in inhibiting membrane-formation : Highest observed Lowest observed concentration which concentration which permits membrane- prevents membrane- formation by formation by Protein. butyric acid. butyric acid. Mixed serum proteins 3.7 7.4 Gelatin .. . . . 1.0 2.0 "Insoluble" serum-globulin .... 0.3 0.6 Casein , ... 0.25 0.5 Ovomucoid. . ... . . . . . 0.125 0.25 It is a very striking fact that the order of effectiveness of these proteins in preventing the formation, of membranes is the reverse order of their ability to pass through a porcelain filter. It has been suggested by Loeb and von Knaffle-Lenz that the formation of the fer- tilization-membrane is accompanied by the entry of water into the egg. This is prevented or delayed by the presence of colloids in the sur- rounding medium because they cannot penetrate the egg and hence exert an osmotic pressure tending to withdraw water from the egg. For similar reasons cytolysis is also inhibited and it has also been stated by B. Moore that the action of Hemolytic Agents in liquefying blood-corpuscles is similarly inhibited by proteins. The normal concentration of protein in blood-serum lies between 7 and 8 per cent, and it will be seen that this lies in the margin of the concentration which inhibits membrane-formation by butyric acid (and also by sperm). Hence if the oocytin content of a sample of serum be low, or the concentration of serum-proteins a little above the average, it will fail to cause membrane-formation even in sensitized eggs. Heating the serum permits membrane-formation to occur because it results in coagulating and removing the proteins, and dilu- tion achieves the same result in a different way. At first, however, the effect of dilution in reducing the membrane-forming power of the serum more than compensates for the diminished inhibition by the proteins, so that dilution of serum to one-half or one-fourth usually deprives even an initially active serum of the power to induce mem- brane-formation. Even when the undiluted serum is sufficiently potent to overcome the inhibition of its proteins so far as to cause membrane-formation, the inhibition is nevertheless operative and finds expression in the prevention of the subsequent cytolysis. The concentration of protein in the medium bathing the eggs which is required to inhibit membrane-formation . affords a quantitative measure of the potency of the fertilizing agent. The more concentrated a solution of Saponin, for example, the greater the amount of Ovo- mucoid which must be added to it to prevent the formation of mem- branes. From this it is evident that the "charge" of membrane- forming agent which the spermatozoon carries into the egg must be less than that which is deposited upon sensitized eggs in an active serum 458 PROCESSES INFERRED FROM INDIRECT OBSERVATION which induces membrane-formation without previous dilution, for the concentration of proteins in normal undiluted serum is sufficient to inhibit the membrane-formation succeeding fertilization by sperma- tozoa. It is, however, possible to increase the "charge" of membrane- forming agent in spermatozoa by sensitizing them with strontium chloride solution and exposing them to blood-serum previous to fer- tilization. They thus accumulate the membrane-forming agent from the serum and carry it together with their own membrane-forming agent into the egg. The following experiment affords an illustration of this fact. Solutions of ovomucoid in sea-water were prepared con- taining 2, J, J and f per cent, of the protein, respectively and in 2 c.c. samples of each of these solutions were placed two drops of a thick suspension of the eggs of Strongylocentrotus purpuratus. The sperm from a male of the same species was divided into three portions. The one portion was untreated save by washing in sea- water. A second portion was immersed for four minutes in f m. strontium chloride and then for one minute in sea-water. The third portion was immersed for four minutes in f m. strontium chloride and then for four minutes in an isotonic undiluted blood serum. These three samples of sperm were then added to the eggs contained in the solutions of ovomucoid in sea-water described above. The concentrations of ovomucoid employed inhibited the formation of fertilization-membranes by the normal spermatozoa and by those which had merely been immersed in strontium chloride solution, but, except in the case of the strongest solution, they were unable to prevent the formation of membranes by the spermatozoa which had acquired an additional charge of cytolytic substance from blood serum : Concentration of ovomucoid solution. Per cent, membranes formed by untreated sperm after: Per cent, membranes formed by sperm washed in SrClz and then in sea- water after : Per cent, membranes formed by sperm washed in SrCla and then in serum after: 15 mins. 40 mins. 15 mins. 40 mins. 15 mins. 40 mins. 2 per cent. . 1/2 .... 1/4 .... 1/8 .... 5 7 8 8 20 30 18 30 40 18 It appears, therefore, that the cytolytic agent in mammalian blood- serum when introduced into the egg-cell together with the sperma- tozoon, brings about just the same effects as the cytolytic agent in the spermatozoon itself and the inference is thus rendered the more prob- able that these two agents are similar in character. THE EFFECT OF MEMBRANE-FORMING AGENTS UPON THE EGG. The essential feature of the process of Membrane-formation is its evidently close relationship to the phenomena of Cytolysis, or lique- faction and disintegration of the cell. In the view of Loeb it is cytolysis MEMBRANE-FORMING AGENTS 459 which is confined to the cortical layer of the egg. The formation of the membrane is accompanied by a very manifest increase in the volume of the egg which can only be accounted for by an imbibition of water. The cytolysis which succeeds membrane-formation is accompanied by a still greater swelling and imbibition of water. This has been attributed by Loeb and von Knaffle-Lenz to the partial liquefaction or destruction of an Emulsion-structure within the cell or at its periphery. We have seen (Chapter XIII) that protoplasm consists of an emulsion of lipoids in a protein medium and that this emulsion must be particularly concentrated at the surface of the protoplasm owing to the lowering of interfacial tension which is thus brought about. Any increase in the diameter of the lipoidal droplets in this superficial layer, or their coalescence, must lead to a corresponding increase in the width of the interstices between them, and hence to an enhanced permeability for water and salts. The fat-dissolving character of the majority of the cytolytic agents is thus the origin of the taking up of water by the egg which results in the physical phenomena of cytoly- sis or cell-liquefaction. Any other agent which will induce imbibition of water will, however, also bring about cytolysis. For example distilled water or Hypotonic Solutions bring about cytolysis because the excess of osmotic pressure within the egg forces water into the cell even through the normally narrow interstices of the cortical layer. Other agents may induce cytolysis by altering the solubility of the protein component of the emulsion, and hence cytolysis may be induced in certain cells by physiologically unbalanced salt solutions. Even membrane formation, as Lillie has observed, may be brought about in certain echinoderm eggs (Arbacia) by exposing them to pure solutions of certain Sodium Salts, and this effect is inhibited by an admixture of Calcium Chloride. The formation of the fertilization-membrane, therefore, which is the first step in the stimulation of development which constitutes fertilization, is essentially a partial and arrested cytolysis. The impor- tant question now presents itself, in what way does this partial cytolysis affect the chemical processes of the cell? One very decided effect of fertilization by spermatozoa is enhance- ment of Basal Metabolism, indicated by a greatly increased consump- tion of oxygen. The following measurements by Loeb and Wasteneys illustrate this fact: CONSUMPTION OF OXYGEN BY A GIVEN MASS OF ARBACIA EGGS. Mg. of oxygen consumed Time. per hour. Before fertilization , . . . .0.24 First hour after fertilization 0.94 1 Second hour after fertilization 0.80 Third hour after fertilization 0.87 Fourth hour after fertilization 0.91 Fifth hour after fertilization . . 1.05 1 This value is too high, owing to the presence of sperm which were washed away before the next determination was made. 460 PROCESSES INFERRED FROM INDIRECT OBSERVATION During the period occupied by the experiment the eggs had pro- ceeded to the thirty-two cell stage. The rate of oxidations does not reach its maximum instantaneously, but increases progressively. For example, Warburg in comparing the rates of oxidation in the 8-cell and 32-cell stages found that they were in the ratio of 4.2 to 6.8. Corresponding with these facts we find that deprivation of oxygen arrests the processes of development and prevents nuclear and cell- division. The same effect is brought about by Cyanides, which also arrest cellular oxidations and, in multicellular animals, act primarily by reducing tissue-respiration. It is possible to show, however, that other processes besides oxidations are initiated by fertilization, for when the fertilized eggs of Strongylocentrotus purpuratus are left in sea- water free from oxygen for twenty-four hours at 15 C. they will not develop during that time, but they will begin to develop at once if oxygen is admitted. It is found, however, that their development is no longer normal, since they form abnormal blastulse and never or rarely reach the gastrula stage (Loeb). If unfertilized eggs are kept for twenty-four hours without oxygen they remain uninjured, and upon the addition of sperm they develop normally and produce healthy plutei. The same result is obtained if development is arrested by potassium cyanide. It has been shown by Loeb in fact that not only is development arrested by deprivation of oxygen, but it is also, to some extent, reversed. Thus if development be initiated by membrane-formation with butyric acid in Arbaeia eggs, on restoring the eggs to normal sea- water they die within a few hours unless they are treated with hyper- tonic sea-water; moreover, they are no longer fertilizable by sperm. However, if, instead of transferring the eggs to normal sea-water, they are placed in sea-water containing sodium or potassium cyanide, or chloral hydrate, then after some hours they no longer die when they are returned to normal sea-water and, in fact, may now be fertilized by sperm. The fact that Chloral Hydrate and other narcotics, as well as cyanides, will arrest the development of fertilized eggs is a striking proof, in itself, that other chemical phenomena besides oxidation underlie development, for the narcotics, although they suppress or retard the processes of cell division and development do not perceptibly diminish the rate of oxidation in the egg. The acceleration of basal metabolism which occurs in fertilization, therefore, although essential to develop- ment, is not the only essential chemical transformation which underlies the process of development. The vast majority of the reactions which occur in living tissues are oxidations, reductions, or hydrolyses 1 and we may therefore consider it probable that Hydrolysis also occurs and performs an essential function in early development. It remains now to discuss the relative parts played by the two factors of fertilization, the one consisting in the partial cytolysis of the 1 Decarboxylization should perhaps be added to this list. Deaminization may with propriety be classed among the hydrolyses. MEMBRANE-FORMING AGENTS 461 egg and the other, which is also brought about by the spermatozoon and may be imitated by treating the eggs with hypertonic sea-water. In the first place, as regards the cytolytic effect, or Membrane-formation it has been found that the characteristic acceleration of Oxidations which is induced by complete fertilization is also induced by membrane- formation. Thus Warburg compared the rate of oxidations in unfer- tilized eggs and in eggs which had been fertilized by sperm, and he found that the consumption of oxygen in the eggs which had been fertilized was 10.5 times the consumption of oxygen in the unfertilized eggs. The same eggs after butyric acid treatment consumed 9.0 times as much oxygen as the unfertilized eggs; the effect of membrane- formation alone upon the basal metabolism was therefore very nearly equal to that of complete fertilization. These experiments were re- peated by Loeb and Wasteneys who, in another species of sea-urchin, found the ratio of oxygen-consumption in unfertilized and sperm- fertilized eggs to be 1 : 4.55, while two estimations of oxygen-consump- tion in the same unfertilized eggs after membrane-formation by butyric acid gave the values 1 : 4.72 and 1 : 4.28, indicating that the effect of membrane-formation is to raise the rate of oxidations to approximately the same height as the entrance of a spermatozoon. We have seen that membrane-formation is essentially a partial and arrested Cytolysis. That this is really the essential feature in the proc- ess and not merely an incidental phenomenon is shown by the fact that if cytolysis be pushed even further, by whatever agent it may be caused, the effect is to increase the consumption of oxygen by the egg and approximately in proportion to the degree of cytolysis which is induced. Cojnplete cytolysis of the egg of the sea-urchin can be caused by the addition of Saponin to the sea-water. Loeb and Wasteneys measured the rate of oxidations in a batch of unfertilized eggs in sea- water and they found. that they consumed 0.15 mg. of oxygen per hour at 15 C. The eggs were then cytolyzed with saponin and the amount of oxygen consumed per hour at 15 C. determined again. It was found to be 1.07 mg. The complete cytolysis of the eggs, therefore, increased the rate of oxidation 700 per cent., or rather more than fertilization itself. Cytolysis by hypotonic sea-water also causes an increase in oxidations. The second factor in artificial fertilization, the treatment with Hyper- tonic Sea-water, also increases the consumption of oxygen by the egg, but only to a relatively slight degree, and not at all if it succeeds mem- brane-formation whether induced by a spermatozoon or by butyric acid. Thus Loeb and Wasteneys obtained the following results with the unfertilized and otherwise untreated eggs of Strongylocentrotus purpuratus: Oxygen-consumption in ninety minutes, Solution. mgm. Normal sea-water 0.30 Hypertonic sea-water ( = 50 c.c. sea-water + 9 c.c. 2| m. NaCl + KC1 + CaCl 2 ) 0.67 Normal sea-water half an hour later 0.51 Normal sea- water twenty-one hours later 0.48 462 PROCESSES INFERRED FROM INDIRECT OBSERVATION The increase in oxygen-consumption is obviously much less than that caused by membrane-formation and it is, moreover, transitory, falling off with time after the exposure instead of increasing, as it does when the eggs are normally fertilized or treated with butyric acid. On the other hand in eggs in which membrane-formation has been induced by butyric acid or by the entry of a spermatozoon, no increase whatever and no important decrease in the rate of oxidations could be observed on treatment with hypertonic sea-water. The corrective effect of hypertonic solutions in preventing the death and disintegra- tion of the eggs which succeeds membrane-formation by cytolytic agents is therefore not to be sought in an effect upon oxidations. It may possibly reside in an effect upon underlying hydrolyses, for these, as we have seen, will bring about the destruction of the egg if they are permitted to go forward while the oxidations are retarded or prevented by lack of oxygen or by cyanides, and hence if they were dispropor- tionately rapid, even in eggs in which oxidation were proceeding they might be presumed to exert a like deleterious effect. However this may be, the action of the hypertonic solutions upon the egg is not reversible upon restoration to normal sea-water. The effect is to induce a permanent alteration of the egg which renders it able to withstand partial cytolysis (membrane-formation) without injury. Thus Loeb has shown that if the unfertilized eggs of Strongylocentrotus purpuratus be placed for from two to two and a half hours in hypertonic sea-water (50 c.c. sea-water + 8 c.c. 2| m. Ringer solution) they may be returned to normal sea-water and subsequent treatment with butyric acid, even forty-eight hours later, will induce, not merely membrane-formation, but full and normal development of the embryo. THE RELATIONSHIP OF PHOSPHOLIPINS TO THE SYNTHESIS OF NUCLEAR MATERIAL AND THE EFFECTS OF LECITHIN UPON EARLY DEVELOPMENT. The leading results of the early development of the embryo are, in the first place, the very great increase of cellular surface due to repeated subdivisions of the original egg-cell and in the second place an increase in the proportion of nuclear to cytoplasmic constituents. The earlier estimations of Boveri led him to the conclusion that the mass of nuclear material in the cells is doubled at each cell-division, but the more recent estimations of Conklin have tended to greatly reduce this estimate, the average nuclear growth during cleavage amounting, it appears, to not more than from five to nine per cent, for each cleavage that occurs. Nevertheless there is a definite increase in nuclear material during the formation of the multitude of new cells which comprises the Blastula-stage of the sea-urchin and since during this period of development no growth of cytoplasm occurs, the cyto- plasm of the new cells occupying collectively the same space as the original egg-cell, it is evident that a disproportion of nuclear to cyto- RELATIONSHIP OF PHOSPHOLIPINS TO SYNTHESIS 463 plasmic material must be established, a disproportion which subsequent development corrects. The Synthesis of Nuclear Material is a self-accelerated or autocatalyzed phenomenon. This follows from the fact that each successive cell division occupies about the same length of time as the preceding one, but the number of nuclei which results from the divisions is at each division twice as great as in the preceding one. The rate of production of nuclei therefore forms a geometrical progression in time intervals which constitute an arithmetical progression. The synthesis of nuclear material thus evidently accelerates the formation of fresh nuclear material- (Loeb.) The question now presents itself as to the origin of the materials from which the nuclei are synthesized. The most characteristic constituent of the nucleus is Nucleic Acid which is built up by the combination of purine bases, a carbohydrate radical and phosphoric acid. The derivation of the first two of these components from proteins and from carbohydrates previously present in the egg is readily conceivable but the question of the origin of the phosphoric acid component suggests several interesting possibilities. In the first place it is evidently not derived from the external medium which bathes the cells, for perfectly normal development will occur in Van t'Hoff 's Solution, which contains no phosphates. The phosphoric acid which is required for the synthesis of nucleins must therefore be derived from some constituent of the egg-cell. Two groups of constituents present themselves as abundant sources of phosphoric acid, namely inorganic phosphate and the Phospholipins, of which egg-lecithin may be taken as a type. Now we have no evidence whatever that nucleic acid can be synthesized directly from inorganic phosphates, but we have, on the contrary, a great deal of evidence which goes to show that phospholipins contribute in the synthesis of nuclear materials. Thus, Miescher has shown that during spermatogenesis in the salmon the " lecithin "-content of the tissues diminishes, Hoppe-Seyler has pointed out that the Lecithin-content of embryonic tissues is exceptionally high and Mesernitzky and Plimmer and Scott and others have shown that the lecithin-content of hen's eggs which is initially very high, progressively diminishes during the development of the embryo. That the same process occurs in the development of the sea-urchin egg has been shown by Robertson and Wasteneys, who estimated the pro- portion of alcohol-soluble phosphorus in eggs which had just been fertilized and again in eggs which had developed to blastulse and plutei. The following were the results obtained with the developing eggs of Strongylocentrotus purpuratus: Percentage of the total phosphorus present in alcohol-soluble forms. Stage of development. Experiment I. Experiment lI7 Fertilized eggs . . . . 39.5 46.5 Blastulse 36.5 38.8 Plutei 35.2 35.1 464 PROCESSES INFERRED FROM INDIRECT OBSERVATION In the first experiment the alcohol-soluble phosphorus (phospho- lipins) decreased by one-eighth, in the second by one-fourth, and this decrease was progressive. The experimental evidence from a diversity of forms therefore tends to establish a relationship between the disap- pearance of lecithin or other phospholipins and the synthesis of nuclear materials. This being the case, great importance attaches to the fact that lecithin, when added to the medium in which sea-urchin embryos are developing, strongly retards their development. The fertilization- membrane is dissolved by lecithin, 1 and hence if lecithin in sufficient concentration (0.15 per cent.) be added to sea- water containing recently fertilized eggs, the membranes are disintegrated and the cleavage-cells which have been formed fall apart, so that for merely mechanical reasons further development is an impossibility. If more dilute leci- thin solutions are employed this does not occur, but, at the same time, no effect upon the rate of development is observed. Very different results follow the exposure of the developing eggs to lecithin solutions, however, after the fertilization-membrane has in the normal course of development undergone rupture and liberated free-swimming blastulse. The following experiment is illustrative of the phenomena which are then observed: The eggs of a Strong ylocentrotus purpuratus female were divided into two portions. Both portions were placed in sea- water and fertilized with sperm. After twenty-four hours both lots of eggs had developed into free-swimming blastulse. One portion was now transferred to a mixture of fifty c.c. of sea- water and 5 c.c. of a 1.7 per cent, emulsion of egg-lecithin in sodium chloride solution for a period of twenty-four hours and then returned to normal sea- water. The other portion was left in normal sea-water. The following table shows the relative development of the two portions : Time after fertilization. Days. Portion 1 (controls). Portion 2. 1 Blastulse Blastulse (these were now trans- ferred to the lecithin mixture for twenty-four hours). 2 Gastrulae Blastulse (these were now trans- ferred to normal sea- water). 3 . . . . . . Gastrulae Blastulse. 4 . . . f . . . Gastrulse and early Blastulse. plutei 5 '.'. . . Fully developed Blastulse and 25 per cent, gas- plutei trulse. 6 Advanced plutei Early gastrulse with narrow unbranched intestine and large clear body-cavity. 7 . . . . Advanced plutei Unchanged. 8 ... . . . Advanced plutei The gastrulse are now retrograd- ing; the intestine has almost disappeared. ' Unchanged Unchanged. 1 We may infer from this that the periphery of the fertilization-membrane contains hpoidal constituents which are essential to the integrity of its structure. RELATIONSHIP OF PHOSPHOLIPINS TO SYNTHESIS 465 It is evident that the immersion of the blastulae for twenty-four hours in a 0.15 per cent, solution of lecithin enormously retards their develop- ment. Especially remarkable is the fact that after development has actually proceeded to the gastrula stage it shows a tendency to undergo reversion, retracing the course of development to the blastula stage. If purpuratus eggs are fertilized by sperm in more dilute solutions of lecithin in sea- water (0.003 per cent, to 0.015 per cent.) the fertilization- membranes are not dissolved sufficiently rapidly to affect development. In these solutions, as has been stated, development is not appreciably retarded until the blastula stage is reached, probably for the reason that colloids cannot traverse the fertilization membrane, and hence the lecithin cannot penetrate the cells of the embryo until the fertiliza- tion membrane has been ruptured. Thereafter development is very markedly retarded and the retardation is greater the greater the con- centration of the lecithin. The eggs are not injured by the lecithin, however, as they will ultimately develop to normal plutei if left in these solutions for a sufficient length of time. The action of Cholesterol is so very generally antagonistic to that of lecithin that one might anticipate tjiat it would, as in fact it does, antagonize the effects of lecithin upon the development of sea-urchin eggs. If cholesterol, suspended in a mixture of T ^ sodium oleate and sodium chloride be mixed with lecithin in equal proportions the retarding action of the lecithin upon the development of sea- urchin eggs is almost completely neutralized. The slight retardation which is observed in these mixtures may be due to the Sodium Oleate which is employed to keep the cholesterol in suspension, since sodium oleate is very toxic for sea-urchin eggs and embryos. Cholesterol itself, when added to sea-water, has no influence upon the rate of development of the eggs. The emulsions of cholesterol are, however, coagulated by the salts in sea-water and the cholesterol is completely thrown out of suspension in the form of coarse flocculi. Since the preparations of "lecithin" employed in these experiments simply consisted of the mixture of phospholipins which is thrown out of an ether extract of egg-yolks by the addition of acetone, it cannot be definitely decided whether the effects observed were in reality due to lecithin or possibly to some other Phospholipin present as an admix- ture in these preparations. The significant feature of these results lies, however, in the fact that if the phospholipins within the egg itself and in other developing tissues behave similarly to the "lecithin" from yolks of eggs, then their progressive disappearance during nuclear synthesis must result in a proportionate diminution of their retarding effect, so that the auto-acceleration of nuclear synthesis, alluded to above, may wholly or in part be due to the consumption of phospho- lipins which is incidental to the process; progressive removal of a retarder being, of course, equivalent in its effect to the progressive addition of a catalyzer. 30 466 PROCESSES INFERRED FROM INDIRECT OBSERVATION THE CHEMICAL MECHANICS OF CELL-DIVISION. The essential mechanical resultant of cell-division is the increase of the Protoplasmic Surfaces which is brought about. During the successive divisions which an egg-cell undergoes in developing to the blastula- stage the total area of the protoplasmic surfaces is enormously increased even in attaining the thirty-two cell stage, for example, the total proto- plasmic surface is increased by about three hundred per cent. Such an increase in a fluid surface necessarily implies either the performance of work by external forces or else a considerable reduction of Superficial Tension. In 1876 it was suggested by Butschli that cell-division is brought about through an increase of surface-tension, subsequent to nuclear division, in the equatorial region of the egg. He pointed out that substances diffusing from the nuclei or centrosomes must necessarily reach their highest concentration in the equatorial plane and hence assuming that these substances increase the surface-tension at the periphery of the egg, the most marked increase would occur in the equatorial region and, as a consequence, the surface-tension at the poles of the cell would be less than that at the equator. Such a conception of cell-division is, however, manifestly erroneous for an increase of interfacial tension at the equator, such as Butschli imagines to occur, implies an increase in the molecular attractive forces at the equator, and the fluids of the cell would not stream away from the region of high attraction but would, on the contrary, stream toward it. The result would be that the equatorial surface would tend to become highly curved, as areas of high tension in a fluid always do, and the surfaces at the poles would become relatively flattened. The result would be the formation of a flattened disc with a highly curved edge, the latter representing what was formerly the equatorial surface of the egg. Such a process obviously could not lead to cell-division. In fact, since the surfaces which Butschli imagines streaming from the nuclei or centrosomes are supposed by him to raise the surface-tension of the egg, their total effect could only be to diminish the surface of the egg relatively to its volume, if that were possible, and not to increase it, which is what the forces leading to cell-division actually accomplish. In fact no model' can be imagined in a fluid which will accomplish increase of surface by increase of superficial tension. Such a model can be devised in a non-fluid system, as, for example a rubber balloon subjected to compression by a rubber band around its equatorial circumference. The equator of the balloon would by this means be constricted and the single sphere would tend to divide into two, owing to the application of additional tension at its equator. This model is, however, in no way comparable to a fluid drop, for it is characteristic of the superficial tension of liquids that it is not altered by diminution or expansion of the surface, because it is really due to the unbalanced attraction of the underlying molecules of the liquid for each other. If a cleft is formed in a liquid drop the opposite walls of the cleft attract CHEMICAL MECHANICS OF CELL-DIVISION 467 one another and tend to close up the cleft again. If they fail to do so it can only be because the molecular attractions have been weakened, i. e., the surface-tensionTiiminished. In a rubber balloon there is no such attraction across the cleft; the tension is purely transverse and is not exerted perpendicularly to the surface as it is in a fluid, and so there is nothing to prevent a cleft from extending deeper and deeper into the equator of the rubber balloon provided the tension of the encircling band is reduced thereby to a greater extent than the tension of the balloon is increased. In 1895 it was suggested by Loeb that phenomena of Protoplasmic Streaming are what really lead to cell-division. He pointed out that in cell-division the protoplasm streams from the equator of the cell in opposite directions toward the two nuclei; the violence of these streaming movements, he suggested, brings about the mechanical separation of the two cells. The streaming of protoplasm from the equator toward the poles suggests that the phenomenon antecedent to cell-division is a diminu- tion of surface-tension in the equatorial region and not, as Biitschli suggests an increase. That such equatorial diminution in surface- tension will bring about the division of droplets into two is very readily shown by means of the following simple experiment : The formation of Soaps at the surface of oil-droplets, results as we have seen in Chapter XIII, in a diminution of the surface-tension of the droplets; if the formation of soap is local, that part of the surface upon which the soap is formed tends to spread. Since commercial olive oil almost invariably contains traces of Fatty Acid the result of bringing an alkali in contact with a drop of such oil will be the formation of soap at the points of contact. If, now, a drop of Olive Oil which is not too large (about 2 to 3 mm. in diameter) be floated on a layer of water, and a thread saturated with tenth normal alkali (NaOH or K0H) be brought gently into contact with a diameter of the drop, the almost immediate effect is the division of the drop into two. The phenomena accompany- ing this division are perfectly characteristic. Instantly the edges of the drop (the ends of the diameter along which the thread lies) recede from the thread, forming a notch at each end of the diameter, and violent streaming-motions occur at the surface away from the thread and toward the opposite poles of the drop. These streaming move- ments may be so violent as to rotate the droplets into which the drop divides through as much as 360. If the division does not occur too rapidly the streaming may result in the two droplets being connected by a thread of oil, which may be central or to one side, and it may then be clearly seen that the mechanism which brings about the snapping of this thread is the violent streaming in opposite directions which takes place in the drops. Phenomena almost exactly resembling those described by Loeb in dividing ova may readily be observed (Fig. 30). Frequently, also, processes resembling Pseudopodia are thrown out by the droplets in the act of their division. The segmentation of the drop is not due to mechanical division by 408 PROCESSES INFERRED FROM INDIRECT OBSERVATION the thread, for, in the first place, the streaming phenomena, etc., are obviously attributable to soap-formation, and, in the second place, the phenomena observed when a thread, un wetted save with water, is laid across the drop are quite different from those described above. The drop of oil adheres to the thread and forms an elongated ellipsoid, its long axis coinciding with the thread; in fact the drop of oil assumes somewhat the form which' the cell would assume were the phenomenon subsequent to nuclear division, as Biitschli imagines, an increase in surface-tension at the circumference of the equatorial layer. Similar phenomena may be obtained with submerged droplets, formed by adding chloroform to the oil to increase its specific gravity, or by droplets immersed in a column of salt solution of varying con- centration the lower layers being saturated, so that the drop floats midway without sinking or rising, only in this case stronger alkali must be used because the greater part of it is washed off the thread in passing it down to the drop through the upper layers of water or salt solution. 12 3 4 Fie. 30. Drawings of a case of cell-division in artificial parthenogenesis (sea-urchin egg) illustrating the underlying phenomenon of streaming. "The division began on one side (1) and the protoplasm then flowed in the direction of the arrows (2) in oppo- site directions toward the two nuclei. The connecting-piece becomes empty of proto- plasm and only the pigmented solid surface film is left (3) and finally this also disappears (4)." (After Loeb.) The action of alkalies is not confined to those mentioned above but, apparently, the division and accompanying phenomena can be brought about by means of threads dipped in all bases which form soaps with fatty acids. Thus tenth-normal potassium hydroxide or sodium hydrox- ide and a saturated solution of calcium hydroxide, all bring about the division, although the division when calcium hydroxide is used is less rapid than when tenth-normal sodium or potassium hydroxide are employed, because the concentration of a saturated solution of calcium hydroxide is only about twentieth normal. The division and accom- panying phenomena are also elicited in a marked degree by threads dipped in Choline. Not only the bases, but the soaps themselves bring about the division; thus if a thread smeared with Choline Oleate be laid across the diameter of a drop of olive oil, the division of the drop will occur, although more slowly than when choline itself is used. This shows that the action of these bases is due to the soap which is formed when they come into contact with the oil and not to hydroxyl ions. Now we have seen that the phosphoric acid component of the nucleic acid molecule is probably derived, during nuclear synthesis, from Lecithin or similar Phospholipins. The decomposition of lecithin for this purpose must lead to the setting free either of Choline itself FORMATION OF MONSTROSITIES 469 or of a soap of choline or some other nitrogenous base, formed by com- bination with the fatty-acid radicals of the phospholipin. Immediately following the division of the cell-nucleus into two, which precedes by a definite interval the division of the cell, we may suppose an active synthesis of nuclear materials to be occurring in the two nuclear regions. Hence, in these localities, provided that the above hypothesis be correct choline or some other nitrogenous base would be set free. If now, choline be liberated at both nuclei and diffuses from each nucleus equally in all directions its maximal concentration must obviously occur in the equatorial plane at right angles to the line joining the two nuclei. We have seen that choline, when applied to the diameter of a droplet of liquid immiscible with water (provided soap is formed) results in the division of 'the drop along that diameter. It is possible that choline, set free in nuclein synthesis, brings about, in a similar manner, the division of the cell, through the formation of soaps in the equatorial plane, either through combination with fatty acids in the cytoplasm, or else through its having been liberated in the neighborhood of the nuclei in combination with one or more of the oleic, stearic or palmitic acid groups of the lecithin molecule. It is not even necessary to presuppose an actual separation of the two nuclei; it is only necessary to suppose that the nuclein synthesis occurs with greater rapidity at opposite poles than elsewhere within the nucleus in order to understand how nuclear division may be brought about by essentially the same mechanism as that which brings about cell-division itself. ARTIFICIAL TWIN-FORMATION AND THE FORMATION OF MONSTROSITIES. In the normal development of the egg the early cleavage-cells, although distinct and separated from one another by a definite inter- face, nevertheless remain in close apposition to one another, So long as this is the case a single embryo develops. If, however, the first two cleavage-cells chance to fall apart and cease to remain in their normal closeness of apposition then each of the cells develops into a separate and complete embryo and twins are formed from a single egg; these are probably similar in origin to the "identical twins" which are occasion- ally encountered among higher animals and man. It has been found by Loeb that the separation of the first cleavage- cells may be brought about in over ninety per cent, of fertilized sea- urchin eggs, provided they are merely exposed, for some time after the first cell-division, to an artificial sea-water differing from normal sea-water in the lack of any one of the constituents Sodium, Potassium or Calcium. This change in the composition of the surrounding saline medium apparently so alters the consistency of the surfaces of the cleavage-cells that they no longer adhere to one another. It may be noted that as the fertilization membrane still surrounds both of the cleavage-cells and the composition of the external saline mixture can nevertheless affect the surfaces of the eggs, the fertilization membrane 470 PROCESSES INFERRED FROM INDIRECT OBSERVATION must be freely permeable for inorganic salts, although, as we have seen, it is not permeable for colloids. The two embryos develop side by side within the fertilization- membrane and form swimming blastulse. At the usual time the membrane bursts and sets the free-swimming embryos at liberty. They are smaller than normal embryos of the same age but otherwise differ in no respect from embryos which arise in the usual way. The opposite phenomenon, that of fusion of two egg-cells may also be brought about in a certain percentage of cases by treatment of the eggs with alkaline sea- water. This results in the production of gigantic embryos. Even at later stages of development similar fusions may be made to occur. Thus Stockard has found that fusion of the cells which subsequently give rise to the eyes of a fish embryo, Fundulus heteroclitus, may be caused by immersing the embryos at a certain stage of their development in sea-water containing an excess of Magnesium. The effect of this is to cause the development of fishes provided only with a single cyclopean eye. The origin of these ajid other like phe- nomena is to be sought in the influence which the composition of the surrounding medium exerts upon the consistency of the protein and lipoid emulsions within and at the surfaces of the cells. REFERENCES. GENERAL: Harvey: Science N. S., 1909, 30, p. 694. Jour, Exp. Zool., 1910, 8, p. 355. Biol. Bull., 1909-10, 18, p. 269 (consult for literature) ; 1914, 27, p. 237. McClendon: Science N. S., 1910, 32, pp. 122 and 317. Littie, F. R.: Jour. Exp. Zool., 1913, 14, p. 515. Loeb: Artificial Parthenogenesis and Fertilisation, Chicago, 1913. The Organ- ism as a Whole, New York, 1916. Godlewsk't: Physiologie der Zeugung, in Winterstein's Handbuch der Vergleichen- den Physiologie, Jena, 1914, Pt. 2, 3, p. 457. Lillie, R. S.: Jour. Biol. Chem., 1914, 17, p. 121. CYTOLYTIC AND MEMBRANE-FORMING AGENT IN BLOOD: Loeb, J.: University of California Pubs, in Physiol., 1907, 3, p. 57. Pfliiger's Arch, 1908, 124, p. 37. Arch. f. Entwicklungsmech., 1910, Pt. 2, 30, p. 44. Moore:' University of California Pubs, in Physiol., 1912, 4, p. 91. Robertson: Arch. f. Entwicklungsmechan., 1912-13, 35, p. 64; 1913, 37, p. 29. Jour. Biol. Chem., 1912 12, p. 163. Clark: Ibid., 1918, 35, p. 253. SYNTHESIS or NUCLEAR MATERIAL: Meischer: Histochemische und Physio! ogische Arbeiten, Leipzig, 1897. Loeb: Proc. 7th Int. Zool. Congress in Boston, 1907, Biol. Centr., 1910, 30, p. 437. Godlewski: Arch. f. Entwicklungsmech., 1908, 26, p. 278. Plimmcr and Scott: Trans. Chem. Soc., London, 1908, 93, p. 1700. Conklin: Jour. Exp. Zool., 1912, 12, p 1. Robertson and Wasteneys: Archiv. fur Entwicklungsmech., 1913, 37, p. 485. Robertson: Ibid., 1913, 37, p. 497. Browder: Univ. of Calif. Pubs. Physiol., 1915, 5, p. 1. CHEMICAL MECHANICS OF CELL-DIVISION: Robertson: Arch. f. Entwicklungsmech., 1909, 27, p. 29; 1911, 32, p. 308; 1912- 13), p. 692. McClendon: Am. Jour. Physiol., 1910, 27, p. 240. Arch. f. Entwicklungsmech., 1, 1912, 34, p. 263. FORMATION OF TWINS AND MONSTROSITIES: Stockard: Jour. Exp. Zool., 1907, 4, p. 165; 1909, 6, p. 286. Am. Jour. Anat., 1910, 10, p. 369. Loeb: Arch. f. Entwicklungsmech., 1909, 27, p. 119. Biol. Bull., 1905, 29, p. 50. McClendon: Am. Jour. Physiol., 1911-12. 29. p. 289. CHAPTER XX. PROCESSES INFERRED FROM INDIRECT OBSERVATION: GROWTH. GENERAL CHARACTERISTICS OF THE GROWTH-PROCESS. Regarded from the chemical point of view the growth of animals consists, essentially, in the transformation of simple, unorganised Foodstuffs, such as water, the inorganic salts, fats, carbohydrates, amino-acids, and so forth into new chemical entities which, collectively regarded, form the organised protoplasm of the animal tissues. Growth, therefore, involves the synthesis of a variety of chemical compounds in due proportion and succession to one another. This process obviously does not take place with uniform velocity throughout life. It is not at all unusual, for example, for an infant to grow, during the first months succeeding birth, at the rate of two pounds per month. Were this rate of growth maintained, then at twenty years of age we would weigh in the neighborhood of five hundred pounds. Nevertheless the process of growth is not one which undergoes a uniform retardation, diminishing in velocity by a uniform proportion per annum. On the contrary, the growth of children, and of animals, takes place in spurts, separated more or less distinctly from one another by periods of relatively languid growth. Thus the rate of growth in utero during the first half of gestation is so slow that prior to this period the weight of the human foetus is inappreciable in com- parison with that of the mother. This period of slow growth is suc- ceeded by the extraordinarily rapid accretion of tissue which charac- terises development duiing the months immediately prior to and succeeding delivery. A definite slackening of growth occurs, however, toward the end of the first year of extrauterine life, and this slowing down of growth is not an artefact, dependent upon weaning, since it occurs just as strikingly in bottle-fed infants. This resting period is succeeded by the relatively rapid growth of the third, fourth, and fifth years Another pause or slackening of growth succeeds this, to be followed by the energetic growth which accompanies adolescence. The growth of man, therefore, consists of periods of rapid and slow growth which alternate with one another, and if we plot the growth in any dimension, for example the growth in weight, on " coordinate paper" so that the weights are measured vertically and ages horizontally, we obtain a diagrammatic picture of the growth-process which is not a 472 PROCESSES INFERRED FROM INDIRECT OBSERVATION straight line, nor even a single curvilinear sweep, like the outline of a parabola or of the logarithmic curve which represents the progress of the ordinary type of chemical reaction. On the contrary, our diagram reveals distinct waves or large oscillations in the growth-process and resembles, as a matter of fact, the diagram which may be obtained by superimposing three S-shaped curves upon one another in such a manner that their adjacent extremities merge into one another. These waves or oscillations, or "Growth-cycles," as we may term them, are not accidental. They are easily distinguishable from the relatively slight irregularities or fluctuations of growth which every individual child or animal will display more or less frequently during its development. They are distinguishable from such accidental fluctuations because they occur at very nearly the same places in the growth-curve of every normal child, and in the average growth-diagram constructed from the data supplied by a large number of individuals, these large oscillations reveal themselves very distinctly, while the accidental and individual fluctuations cancel out and disappear in the average diagram because, in the long run, if we take a sufficient number and variety of individuals into account, just as many of these accidental fluctuations will be positive (i. e., supernormal in weight) as negative (i. e., subnormal in weight). But the large fluctuations, or Growth- cycles, remain unaffected in magnitude and position, and only appear more definitely in the diagram the greater the number of individuals which we measure or weigh. In the Growth of Man there are, in all, three distinguishable growth- cycles which are superimposed upon one another. Each cycle begins with a period of relatively slow growth, followed by a period of very rapid growth, and culminating, with the termination of the cycle, in a period of slackening growth again. In the case of the first two cycles this slackening of growth is followed by the fresh spurt or acceleration due to the succeeding cycle. In the case of the third or adolescent cycle of growth, the period of slackened growth-velocity insensibly merges into the period of relatively stationary development which we recognize as the adult condition. This developmental stasis may be interrupted, however, by the repair incident to the replacement of tissue which has been injured or destroyed, while even in the absence of such Regenerative Growth a vigorous and abnormal growth may occur, the growth, namely, of Malignant Tumors, which we may possibly interpret as constituting the superposition of a fourth, and physio- logically abnormal cycle of growth upon the third and normally final cycle in the development of man. Not only the growth of man, but also the growth of every mammal which as yet has been carefully investigated appears to consist of three more or less easily distinguishable cycles of growth. The growth of the Guinea-pig at first appeared to consist of only two difficultly distinguishable cycles, but the investigations of Read have shown that in this mammal the first growth-cycle is actually completed in utero, GENERAL CHARACTERISTICS OF GROWTH-PROCESS 473 instead of being interrupted when half-completed by birth, as it is in human beings. Corresponding to this we find that the guinea-pig is born at a much more advanced stage of development than man or the rat or mouse; their eyes are open, they have a full coat of hair, are able to choose and eat their own food and may be weaned altogether within a few days after delivery. The very general occurrence of three growth cycles in mammalian development renders very inviting the supposi- tion that they are referable to the existence of three Embryonic Layers, from one or other of which all the tissues of the adult are ultimately derived, but for this hypothesis there are as yet lacking the necessary experimental and anatomical proofs. In the accompanying figure (Fig. 31) are compared the growth- diagrams of human males of British birth and parentage and of male 60 KILOGRAMS WEEKS FIG. 31. Growth of human males. (Con- structed from the data obtained by the British Anthropometric Committee.) Growth of male white mice. white mice. The resemblance between the two curves, allowing for the difference of the time-units employed, is of a very striking character. The only notable difference lies in the relatively marked delay of the third, or adolescent growth-cycle in man as cpmpared with the mouse, the possible origin of which will be discussed subsequently. These Growth-cycles, so definitely situated in the curve of growth, and so invariable in their occurrence that they may be clearly recog- nised in the growth of mice no less than in the growth of man, must have some very definite physiological significance, and since, as we have seen, growth is essentially a chemical process resulting in the synthesis of living tissue from inanimate materials, these growth-cycles must have a chemical, no less than a physiological significance. The general similarity of the fundamental phenomena of growth in all living forms 474 PROCESSES INFERRED FROM INDIRECT OBSERVATION is strikingly revealed by the fact that the curves of growth obtained in Plants and even in the multiplication of Bacteria are essentially similar in character to those obtained in animals. As a rule, however, the growth of a plant or of a colony of bacteria displays evidence of only a single growth-cycle. Each of these growth-cycles is approximately symmetrical about its center, that is, on either side of the moment of most rapid growth; in other words the second half of the S-curve reproduces in the reverse order the characteristics of the first half. We have, then, in each growth-cycle considered by itself, a chemical process which begins relatively slowly, increases progressively in velocity until it is about half completed, and then slows off to its termination. The inquiry now immediately presents itself whether any chemical processes of Body-iueight. ^f^ Amount transformed FIG. 32 Comparison of the curve of growth of the white rat (constructed from data collected by Donaldson) with chemical reaction curves. this general character are known to occur elsewhere than in the build- ing up of tissue by a growing plant or animal? As a matter of fact, chemical transformations of this character are abundant and they are those in which one or more of the products catalyzes the further progress of the reaction. We have already in preceding chapters had occasion to dwell upon a number of chemical phenomena which occur in living tissues and elsewhere which belong to this category; it will merely be necessary therefore to refer in passing to the analogies afforded by the hydrolysis of castor-oil in the seeds of Ricinus, the hydrolysis of cane-sugar by boiling neutral water, the decomposition of methyl acetate by water, the oxidation of metals and of a variety of organic materials, and the chemical transformations which accompany and underlie the performance of muscular work. In all of these various GENERAL CHARACTERISTICS OF GROWTH -PROCESS 4?5 processes one or more of the products of the reaction is endowed with the property of facilitating the further progress of the reaction. Such transformations are designated Autocatalyzed Reactions (Fig. 32). The fact that each growth-cycle begins slowly and progressively increases in velocity until the moment of maximal growth-velocity is attained at the center of the cycle is sufficient in itself to show that the process of growth is autocatalyzed, whatever the mechanism of the self-acceleration may be. The resemblance of the process of growth to the transformations in an autocatalyzed reaction is not merely superficial, however, but extends even to quantitative details. It will be recollected that the relationship between the extent of transformation and the time in an ordinary Monomolecular Chemical Reaction is expressed by the equation: Velocity = k(a x) where "a x" is the amount of the original material which is as yet untransformed and "k" is a constant, specific for the particular reac- tions under consideration. The effect of catalyzers upon such a reac- tion is to multiply the value of "k" by a quantity which is proportional to the amount of catalyzer present. Now in an autocatalyzed reaction the amount of catalyzer which is present is proportional to the mass of the product of the reaction, that is, to "x." The equation for an Autocatalyzed Monomolecular Reaction becomes, therefore: Velocity = kx(a x) which, when integrated, yields the equation: Iog 10 - = ka(t - ti) where t is the time from the beginning of the measurements and ti is the time at which the reaction is half completed, i. e., the center of the autocatalytic curve. The applicability of this equation to the growth in numbers of Bacteria in a limited quantity of culture medium has been established by McKendrick. It is, however, not less applicable to relatively complex phenomena of Human Growth. The juvenile and adolescent cycles of growth in man are rather closely interfused, so that their separation into individual cycles is a difficult and uncertain matter. The infantile cycle, however, is rather definitely separated from the remainder of the human-growth curve by a rather long period or " pla- teau" of relatively slow growth. The Infantile Growth -cycle, therefore, at any rate for the first ten months succeeding birth, presents the relatively uncomplicated characteristics of a single cycle of growth to which the above equations may be applied. In the following compari- sons of the theoretical values calculated from the equation Iog 10 - = ka(t - ti) 476 PROCESSES INFERRED FROM INDIRECT OBSERVATION with the average values actually obtained from weighings of large numbers of infants the constants " a," " k" and " t," are calculated from all of the observations by the " method of least squares. In this way, for example, we find that the growth of British male infants born in South Australia, during the first nine months succeeding delivery, is expressed by the formula Iog 10 - = 0.136(t - 1.66) 341 .5 x time being reckoned in months from birth and weights in ounces avoir- dupois. In the following table the observed weights at the various ages are compared with those calculated from this formula: Age of infant in months. 1 2 3 4 5 6 7 8 The equation to the curve of growth for the first nine months of the extra-uterine life of South Australian females is found to be: SOUTH AUSTRALIAN MALES. Weight in ounces, int 3. Observed. 127 Calculated. 127 156 180 206 230 254 273 288 301 311 , . . . . . . 155 ...'*. . . . . 187 206 .'. . . . . . 224 . . .... ... 254 . . . . . ' 270 ; ... . . . .' 287 . . . . . , 4 300 311 and in the following table the observed weights at various ages are compared with those calculated from the formula: SOUTH AUSTRALIAN FEMALES. Age of infant in months. ... Weig ht in ounces. Observed. 121 Calculated. 121 142 164 187 209 230 249 267 282 295 1 ... 153 2 .... 168 3 .... 188 4 .... 209 5 . . 224 6 . . 253 7 ... 263 8 ... 270 9 ... 300 A similar comparison follows for British infants born in England: The equation to the infantile growth-cycle during the first nine months m males is represented by the formula: io 5 lo 0.127(t - 1.46) GENERAL CHARACTERISTICS OF GROWTH-PROCESS 477 BRITISH MALES. Weight in ounces. Age of infant in months. Observed. Calculated. 1 147 148 2 169 171 3 ........< 194 194 4 . . 219 216 5 234 235 6 252 252 7 269 266 8 276 277 9 . 283 287 The equation for the same period in females is represented by: logio = 0.106(t - 1.54) BRITISH FEMALES. Age of infant in months. 1 2 Weight in ounces. i. Observed. . . 143 Calculated. 146 160 180 165 184 202 218 202 218 . . 235 233 253 247 258 259 265 269 In all cases it will be seen that the agreement between the observed and the calculated weights is extremely close; in fact such consonance between the quantitative demands of a theoretical equation and the experimental estimations is not frequently obtained even in experi- ments conducted in laboratory-glassware. The probable reasons for the extreme regularity observed lie in the first place in the large number of measurements from which each average weight is computed and in the second place in the excellent conditions of thermostasis which the body of a warm-blooded animal affords. Even in such complex Metazoa as man, therefore, the process of growth in an individual growth-cycle appears to be determined and governed by the simple law which is characteristic of an Autocatalyzed Monomolecular Reaction. It will at once occur to the reader, however, that the process of growth, taken as a whole, cannot possibly be of this simplicity, for in the construction of the simplest of the multi- tudinous constituents of tissues a variety of parallel and successive chemical reactions must as a rule contribute to the result. The diversity of interdependent chemical phenomena involved in the building up of an organism so complicated as ourselves must be almost unimaginably great. How, then, can a reaction-formula characteristic of a single and uncomplicated transformation, peculiar only in produc- ing its own catalyzer, apply to the quantitative outcome of such a bewildering tissue of chemical events? 478 PROCESSES INFERRED FROM INDIRECT OBSERVATION The answer to this question is undoubtedly to be sought in the fact that in any system of interdependent chemical transformations the slowest reaction in the series governs the velocity of the whole. On the hither side of the slowest reaction all the raw materials for subsequent processes must accumulate and await the elaboration of the products which they utilize, while on the far side of the slowest reaction the subsequent processes are retarded to its pace by the consumption of their substrates. The slowest reaction in any chain of chemical processes is the Master-reaction which determines from moment to moment the quantitative relations of the product to the time. Now in the complex of events which constitutes growth not a single sig- nificant transformation is independent of the rest; each must evidently use some product of other transformations and contribute some product to get another series of processes. We can therefore understand how the whole phenomenon, notwithstanding its complexity and the multi- plicity of the chemical reactions involved in it, may nevertheless be governed, as to its quantitative outcome, by the rate at which a single reaction occurs. This reaction, as we have seen, is autocatalytic. We are thus led to inquire whether the growth-diagram, which is so similar in form to the curve which represents the progress of an auto- catalyzed chemical reaction, may properly be regarded as establishing the existence of Catalyzers of Growth which are numbered among the products of the growth-process, or Endogenous Catalyzers, as Hopkins has termed them, and also the existence of Impeding Factors, attribut- able either to the exhaustion of an essential constituent of the reaction, or to the accumulation of growth-products. The problem becomes somewhat clearer when we consider the simple case of Bacteria, growing on a limited amount of a given culture-medium. In this case, as McKendrick has shown, precisely analogous phenomena are exhibited to those which characterize the growth of higher organ- isms. The growth of the bacterial culture, measured by the total mass or number of bacteria produced at given time-intervals, is at first extremely slow; it increases in velocity, however, and at first almost in proportion to the number of bacteria produced. At a later stage growth is impeded and finally comes to a standstill when the density of the population of the culture-medium has attained a certain maximum. These phenomena are interpreted by McKendrick in the following manner: Each bacterium is capable of giving rise to a certain number of daughter-cells in a certain interval of time under constant nutritive conditions. This potentiality is transmitted to its offspring, so that were the nutritive constituents of the culture-medium inexhaustible, the velocity of reproduction would always be proportionate to the number of bacteria previously produced, or, in other words, the density of the bacterial population would increase in geometrical, while the time increased in arithmetical progression. In practice, however, the ability of the culture-medium to supply nutritive materials to the GENERAL CHARACTERISTICS OF GROWTH-PROCESS 479 bacteria is limited, and the rate of multiplication is slowed. McKen- drick infers, therefore, that the rate of multiplication is proportional to two factors; in the first place to the number of bacteria previously produced, and in the second to the concentration of the still-available foodstuffs. This leads to the equation: dx = kx(a x) at where "x" is the number of bacteria per unit-volume, "a x" is pro- portional to the concentration of available nutrients and "k" is a constant proportionality-factor. Integration of this differential equa- tion leads to the relationship log = ka(t - ti) a x where "x" is the number of bacteria per unit volume, a is the maximal density of population which is attainable in a given culture medium, "k" is a constant proportionality-factor and ti is the time at which the density of the bacterial population has attained half its maximum. 1 The relationship between the number or mass of bacteria produced and the time of incubation which is expressed in these equations is, however, identical with that which expresses the relationship between weight and age in any given growth-cycle of an animal or plant. It is also identical with the relationship between the mass of the products and the time in autocatalyzed chemical reactions, such as the hydrol- ysis of Methyl Acetate. The question therefore presents itself, whether the process of growth in a multicellular organism such as a mammal is comparable to an autocatalyzed chemical reaction, or whether McKen- drick's interpretation of the growth-curve of a bacterial population does not offer an alternative explanation of the facts. In other words two alternative possibilities would appear to exist : the one that the accelera- tive factor in growth is a chemical substance, as it is in autocatalyzed chemical reactions, the other that it is simply due to the multiplication of cells, each of which is possessed of like potentialities of reproduction. On closer analysis it will be seen, however, that these interpretations, at first sight alternative, are in reality identical. Reverting to the case afforded by the multiplication of bacteria in a limited amount of culture-medium, and looking to the beginning and end of the process, we see that the increase in bacterial population means essentially that the simple, unorganized constitutents of the culture-medium have been transformed into the substances composing the bacteria. Any acceleration experienced by the process must ultimately be due to the preceding synthesis, irrespective of the fact that the synthesis takes place in a heterogeneous system, i. e., in the separate particulate masses which form the individual bacteria. When 1 I have slightly, but unessentially, modified McKendrick's formulation of this relationship in order to make clearer the analogies which follow. 480 PROCESSES INFERRED FROM INDIRECT OBSERVATION we say that each bacterium has a like potentiality of reproduction we clearly express the fact that the synthesis of bacterial cell-substances which results in the production of a cell is a favoring condition for the production of new cells, in other words that some substance or sub- stances comprising the bacterium accelerate the production of new masses of bacterial substance. In ultimate terms, therefore, the two interpretations of the phenomenon are identical, the only essential difference between the more familiar cases of autocatalysis, such as the hydrolysis of methyl acetate, and the process of cell-multiplication, being the fact that in the latter process the reaction takes place in a heterogeneous chemical system, i. e., within the particulate masses comprising the cells. Yet the fact that a chemical reaction takes place in a heterogeneous medium does not imply that it is discontinuous. The production of calcium sulphate from a mixture of calcium hydrate and sulphuric acid is a continuous process despite the fact that the product is divided into particulate masses, which in this instance are crystals. On the other hand the instances of autocatalysis in heterogeneous systems are abundant in chemical literature, the oxidation of metals in contact with air being a familiar illustration of a group of autocata- lyzed reactions of this type. The Accelerative Factor in the process of growth is, therefore, a chemical substance or substances, or a chemical condition, which is strictly analogous to the accelerative factor in less complex auto- catalyzed phenomena. The autocatalytic character of the growth- process follows of necessity, in fact, from the fundamental characteristic which, more than any other, distinguishes living from non-living mate- rial, namely its potentiality of unlimited reproduction. When we assert that living cells all possess like potentiality of reproduction we merely state in morphological terminology that the production of living matter is a self-sustained or autocatalyzed phenomenon. Just as the produc- tion of living from inanimate matter is essentially a chemical process, so the acceleration of its production which is consequent upon the multi- plication of the particulate resultants of the process is, when viewed from the chemical standpoint, evidence that substances are produced in the creation of living matter which have the essential property of catalyzing its further manufacture. Regarding the possible nature of these endogenous catalyzers, we shall have something to say in a later part of this chapter. It remains to consider what may be the probable nature of the Inhibitive Factor which ultimately brings the process of growth to a standstill, which sets a limit to the normal dimensions of any given species of animal, and which predominates over the accelerative factor during the latter half of each growth-cycle. In the simpler instances of autocatalysis, as we have seen, the inhibitive factor may be, either the exhaustion of the materials undergoing transformation, or, on the other hand, the accumulation and consequent " back-pressure" of the products of the reaction, or both of these factors may play a part in GENERAL CHARACTERISTICS OF GROWTH-PROCESS 481 determining the magnitude of the inhibition. Either of these alter- natives would yield the time-relations expressed in the autocatalyzed reaction-formula, for the following reasons : In case the velocity of the reverse reaction is, at all stages of the transformation, negligible in comparison with that of the forward reac- tion, then the only inhibitive factor must be the exhaustion of the Substrate, or material undergoing transformation. The velocity of the process will be, as usual in chemical reactions, proportional to the mass of untransformed material and also to the mass of the catalyzer, that is, in these instances, to the mass of the products of the reactions. Desig- nating the mass of a product of the reaction at any moment by "x," and "a" the initial amount of the material undergoing transformation, this yields the relation : dx Velocity of transformation = = kx(a x) dt which is the formula characteristic of an autocatalyzed reaction. Coming, now, to the case in which the velocity of the reverse reaction is so considerable as to be comparable with that of the forward reaction, we will assume, in the first instance, that the materials undergoing transformation (or foodstuffs in growth) are inexhaustible, i. e., are constantly being renewed from the environment, so that the mass of material undergoing transformation is a constant which we may designate by the symbol of "A." The velocity of the forward reaction will then be, as in the above instance, proportionate to the mass of the catalyzer ( = product of the reaction, = "x") and also to the constant mass of substrate, that is, to "A." The velocity of the reverse reaction (breaking-down of the products of the reaction into the initial sub- stances again) will be proportional to the mass of the products ( = "x"), but also to the mass of the catalyzer ( = "x"), because in the majority of instances of "typical" catalysis the catalyzer accelerates both the forward and the reverse reactions in equal proportion. The velocity of the reverse reaction at any moment will therefore be proportionate to x 2 , and the net velocity of the process, being the difference between the velocities of the forward and the reverse reactions, will be given by : dx - = kl *A = UP in which "ki" and "k 2 " are the velocity-proportionality factors of the forward and reverse reactions respectively. Rearranging the terms of the equation this may be written: -^ k dt which is again identical with the ordinary formula of autocatalysis, with the exception that the constant "a," denoting the maximal attain- able value of "x" is now not the initial mass of material undergoing 31 482 PROCESSES INFERRED FROM INDIRECT OBSERVATION transformation, but the initial mass multiplied by the constant ratio of the velocity-constants of the forward and reverse reactions. In the case of the growth of Bacteria in a limited quantity of culture medium, McKendrick assumes that the inhibitive factor is simply the exhaustion of available foodstuffs, i. e., that it corresponds to the first of the alternative possibilities outlined above. In the growth of animals, however, it is difficult to see how the limited availability of Foodstuffs could be a deciding factor in the inhibition of normal growth, for the medium in which our cells actually live and grow is the lymph (or "tissue-fluid"), which is constantly supplied and renewed from the blood. Now the mechanisms of the body are, as we have seen, so devised that the composition of the blood is maintained in a condition of extraordinary uniformity. It is true that its content of the more particularly nutritional constituents fluctuates with the fluctuating absorption of nutrients from the alimentary canal, but these short- period fluctuations result in the long run in the mairtenance of a remarkably steady flow of nutrient materials to the tissues. The blood derives its nutrient constituents from the external environment and in fact contains them not merely in sufficient proportion to maintain an equilibrium of body-weight, but, even in adult animals, in considerable excess of the necessary minimum, the destruction of this excess consti- tuting the "Exogenous Metabolism" as contrasted with "Endogenous Metabolism," or irreducible minimum of nutrient-consumption incident to the maintenance of life. The medium in which our cells live, there- fore, is under normal dietetic conditions a medium of almost constant composition and, for the purposes of tissue-synthesis, it is inexhaustible since it is continually renewed. The Substrates of growth must there- fore be regarded as being of constant concentration and the inhibiting factor of growth must be sought elsewhere than in the exhaustion of available nutrients. On the other hand, if a portion of the tissues of an adult animal be injured or destroyed, the process of growth immediately recommences and is expressed in the phenomenon of Regeneration which, if mechanical factors do not impose an insuperable obstacle, continues until the complete restoration of the lost tissues has been accomplished. In other words, removal of the products of growth immediately reinaugu- rates the growth-process, just as the removal of the products of a "balanced" chemical reaction at equilibrium immediately reinitiates the forward reaction. We must infer, therefore, that in the growth of mammals, at least, it is the accumulation of the Products of Growth which normally inhibits the process and not the exhaustion of nutritive materials. In Plants the supply of nutritive materials to the cells is more fluctuating and dependent upon the environment, and here we may expect to find, and do actually find, a much more conspicuous part played by the supply of nutrients in determining the final attainable dimensions of the organism. Nevertheless plants of a given species, even under the most favorable nutritional conditions, do not exceed certain definable limits in their dimensions at maturity, and they GENERAL CHARACTERISTICS OF GROWTH-PROCESS 483 i display regeneration when portions of their tissues are removed. Even in the case of bacteria growing in a limited supply of culture- medium, there is evidence which tends to show that in many cases the accumulation of bacteria or bacterial products really sets the limit to their multiplication rather than the exhaustion of the nutrients in their culture medium. We infer, therefore, that the process of growth is governed by a series (in mammals usually three) of autocatalyzed chemical reactions in which the factor which determines the retardation and ultimate equilibrium of the process is the accumulation of the products, i. e., the growth itself. The constancy of the concentration of Growth-substrates in animals affords a readily intelligible explanation of the extraordinary simplicity of the quantitative relationship between growth and time, which, as we have seen, so frequently obtains. The relationship in question is that which characterises the progress of an autocatalyzed monomolecu- lar reaction, and even admitting the probability that a single chemical transformation may determine the speed and set the pace for the whole of the multitudinous variety of chemical processes involved in the growth of new protoplasm, yet it may seem strange that even this single reaction should be of so simple a character, more especially since,, as the construction of protoplasm involves synthesis of large out of relatively small molecules, we would expect any reaction involved in growth to be multimolecular. Now this may actually be the case, even in the Master-reaction which determines the quantitative outcome of all the growth-processes, for if the concentration of the substrates of growth remains undiminished by the growth which occurs, then any number of molecules of the substrates may participate in the synthesis which constitutes the governing reaction, without involving any depar- ture of the relationship between the time and extent of growth from that which is expressed in the monomolecular autocatalytic formula. If "n" molecules of the substrate combine to form one molecule of the product, then the velocity of the forward reaction will be given by: ' while that of the backward reaction will be given, as before, by *L _ k 2 x* dt hence the net effect, or actual growth, will be given by dx k xAn _ k x2 "dT which, rearranging the terms, becomes: dx = k dt 484 PROCESSES INFERRED FROM INDIRECT OBSERVATION which is again of the monomolecular form, save that the constant "a" in the formula is no longer proportional to the actual concentration of the substrates, but to the nth power of their concentration. That the backward reaction should be monomolecular is, of course, not a matter for surprise, since we may suppose that the majority of decom- positions which living tissue suffers consists in the interaction of a single molecule of some protoplasmic constituent either with water or with oxygen, the concentration of both of which substances is maintained automatically at an approximately constant level in the tissues. Thus the synthesis of a protein involves the interaction of many different amino-acid molecules, but its hydrolysis in dilute aqueous solution obeys the monomolecular formula, because only a single species of molecule, that of the protein itself, is undergoing appreciable change of mass or concentration in the process. Summarizing the general characteristics of the growth-process we may therefore state : 1. That the growth of man and of animals takes place in periods or cycles in which slow and rapid growth alternate, three of the cycles being usually appreciable in magnitude. 2. Each of the growth-cycles is the expression of an underlying self- accelerated chemical process. 3. The accelerating factor is some substance or group of substances produced during growth. 4. The supply of nutriment capable of transformation into living tissues may, in normal animals, be regarded as constant and undimin- ished by the process of growth itself. 5. The inhibiting factor, which ultimately brings the growth in any given cycle to a standstill, is the accumulation of the products of growth. 6. Removal of these products, as by local death or injury, or by general inanition, reinaugurates the process of growth, which continues until equilibrium is reattained. 7. The whole of the diverse processes which in the aggregate con- stitute growth are governed and determined in rate and magnitude by the specificially slowest essential process. 8. The forward reaction in the governing process may involve the interaction of many different molecules, but the reverse reaction appears, in many cases at least, to involve the decomposition of only a single species of molecule of variable mass or concentration. THE INFLUENCE OF RACE, SEX, AND ENVIRONMENT UPON THE GROWTH-PROCESS. The fact that the bodily dimensions of a given species of animal never exceed certain characteristic upper limits, no matter how favor- able the environmental conditions may be with respect to the abun- dance and variety of Nutrients, shows that the factors which inhibit the INFLUENCE OF RACE AND SEX ON GROWTH-PROCESS 485 growth in any given growth-cycle are primarily characteristic of the process itself and only in a minor degree dependent upon the dietary, provided it is in all respects sufficient. We have seen that the main inhibiting factor in growth arises from the accumulation of the products of growth and the enhanced rapidity of tissue-disintegration which ensues. The characteristic dimensions of an animal, therefore, and the same, to a less striking degree, is doubtless true of a plant, are determined mainly by the relative magnitude of the specific Velocity- constants of the forward and the opposed reactions. These are char- acteristic of the particular reactions which occur in a given race or sex, and are not influenced by the mere abundance or paucity of the dietary. That the bodily dimensions of an animal may be affected to a limited extent by the abundance of the Dietary is, however, a readily ascer- tainable fact. If the dietary be absolutely insufficient even to main- tain bodily heat and the output of work, the tissues are called upon to supply the energy-requirements, the animal loses weight and may ultimately die of inanition or of acute conditions supervening upon partial inanition. If the dietary insufficiency is less extreme than this, growth is nevertheless slowed, and the bodily dimensions attainable at maturity are smaller than is normal for the species. If, on the other hand, the diet is exceedingly abundant and other environmental conditions are exceptionally favorable, then the bodily dimensions at maturity may come to distinctly exceed the average, although the degree of supernormality which is attainable in this way is, of course, strictly limited. Mice, under no matter what favorable conditions of environment and abundance of food supplies, do not achieve the bodily dimensions of a guinea-pig or even of a rat. The supply of nutrients to the tissues is, as we have seen, determined primarily by the composition of the blood which, subject to short- period fluctuations, remains relatively constant throughout the growth and life of the animal. The "Nutrient Level" or concentration of growth-substrates in the blood is maintained by a dynamic equilib- rium which involves a variety of factors. On the one hand we have the availability of Foodstuffs in the external environment and the ability of the digestive apparatus to disintegrate them and to absorb the products of their disintegration. On the other hand we have the rate of utilization by the tissues and the equilibrium between the storage- capacities of the tissues for the various classes of foodstuffs, for poly- saccharides, fats, and amino-acids, and the concentration of these substances or their products in the blood and tissue-fluids. The height of the nutrient reservoir in the blood is thus governed by a balance between a certain rate of inflow and a certain rate of outflow. In addition to these factors, and in order to avoid an excessive accumula- tion of nutrient materials in the blood, an overflow is also provided in the phenomenon of Exogenous Metabolism, or the destruction of food- 486 PROCESSES INFERRED FROM INDIRECT OBSERVATION stuffs which have not yet come to comprise living matter, a process which, in the case of the amino-acids at all events, forms a very large proportion of the total metabolism of a normally nourished animal. If any of these several factors is decidedly altered in magnitude or velocity a more or less marked effect upon bodily weight will ensue. Thus if the rate of inflow be diminished beyond a certain point by an insufficient dietary the nutrient level sinks and growth is retarded, or, in the adult animal which has attained growth-equilibrium, the process of growth may be reversed and loss of tissue occur. The extent of this reversion is strictly limited in the more complex forms of metazoa by the necessity of maintaining certain mechanical conditions : the integrity of the skeleton, the functional ability of the digestive organs, the pulsa- tion of the heart, the integrity of a closed vascular system, the coordi- nating activities of the nervous system, and the continuance of respira- tory movements. If any of these suffer in so complex an organization as our own the whole must fail and death ensue. But in some less complex forms, as in the fresh water worm Planaria, starvation actually accomplishes Reversion of Growth until an embryonic stage of develop- ment is regained (Child). If, on the other hand, the rate of inflow of nutrients be maintained unaltered and the rate of outflow increased or diminished the rate of accretion of tissue must obviously be affected to a proportionate degree. In normal cases, since the rate of outflow or consumption of nutrients for tissue-building purposes is determined by the relative magnitudes of the specific velocity-constants of upbuilding and disintegration, the rate of outflow will vary in different species and not improbably in the two sexes of the same species, and to a certain extent in different individuals. The environment, on the contrary, provided the inflow of nutrients is maximal or at least sufficient, may be expected to play little if any part in determining the rate of outflow. The rate of overflow is also conditioned primarily by internal regula- tion, but we may observe the effects of its alteration in so far as the nutrient-level of the amino-acids is concerned, by the pronounced effects of hyper- or hypo-activity of the Thyroid upon the development of the tissues. The administration of thyroid extract leads to a very decisive increase in the rate of Deaminization of amino-acids, and in normal adults who have attained growth-equilibrium, this, which involves a fall of the nutrient-level, results in progressive loss of weight which may, if it affects essential tissues, result in dangerous or even fatal symptoms. The effects of hypo-activity are the opposite and the excessive accretions of tissue not being uniformly distributed, aberra- tions of growth occur which culminate in the condition of Myxedema. In amphibians excision of the thyroid, as Gudernatsch has very strikingly demonstrated, results in the arrest of Metamorphosis, possibly because the degeneration of certain tissues which is a necessary precedent of metamorphosis cannot occur. INFLUENCE OF RACE AND SEX ON GHOWTtt-PliOCESS 487 In the autocatalytic formula as applied to the process of growth: log = ka(t - ti) a x the constant "a" is proportional to some exponent of the concentration of growth-substrates, i. e., to the Nutrient-level. In any given species, therefore, we may expect to find that within certain limits its magnitude is affected by the environment and especially by the abundance or paucity of the dietary. The constant " k" on the contrary is expressive of the specific velocity of the process of tissue-disintegration, charac- teristic of the species,, probably of the sex, and peculiar even to a particular individual. Thus we may expect, in a given species, to find that its magnitude is unaffected by the environment, but dependent upon Sex and Race. We have seen that the autocatalytic formula applies to the first nine months of extra-uterine growth in infants and that the values of "a" and "k" may be computed from all of the observed weights at the various ages chosen for the comparison of the equation with the results of actual measurement. In the following table the values of "a" and "k" for British Infants born in England and in Australia respectively and for South German infants born in Frankfurt (from the data of Schmidt-Monnard) are compared: COMPARISON OF THE EFFECTS OF RACE AND ENVIRONMENT UPON THE PARAMETERS OF THE GROWTH-CURVE. Males. Females. Race and place of birth. a (ounces). kxlO 6 . a (ounces). kxlO 6 . British (born in England) . . 318 399 312 340 British (born in Australia) . : 341.5 398 350 317 South German ..... 315 451 290 537 It will be seen that the parameters or constants of the growth curve of infants are affected in the sense indicated by the above discussion by the factors of sex, race and environment. While the value of "a" is not greatly affected by sex or by dissimilarity of race, the values obtained in the similar environments of Frankfurt and London being very alike, it is greatly affected by dissimilarities in environment, as a comparison of the values of " a" in Australia and in Europe shows. On the other hand, "k" is comparatively unaffected by environment, being practically identical for British males, whether born in Australia or in England, and very nearly the same for British females born in these two environments, whereas it is profoundly affected in magnitude by sex and race, as indicated by the marked difference in the values of "k" for males and females and for South-German as compared with British infants. When it is remembered that these parameters have not been calcu- lated arbitrarily, but that they are computed by the method of least squares from all of the observations and therefore partake in some measure in the errors incident to the observations, it will be seen that 488 PROCESSES INFERRED FROM INDIRECT OBSERVATION the above data afford a very remarkable demonstration of the correct- ness of the view that growth is determined by an underlying auto- catalyzed chemical process. It is furthermore clear that the form of the curve of growth in normal infants is determined by two separate groups of factors. The one, analogous to the absolute mass of the reacting substances in a chemical reaction, being dependent upon the environment and probably largely influenced by the abundance or deficiency of the habitual dietary; while the other, analogous to the specific velocity of a chemical reaction, is relatively, if not absolutely, independent of environmental or nutritional conditions, and, being expressive of the nature of the growth-process itself as distinguished from the availability of the materials for growth, is distinctively modi- fied by race and sex. THE SUBSTRATES OF GROWTH. The substrates of growth, i. e., the material out of which living tissues are synthesized, are the Foodstuffs, namely oxygen, water, inorganic salts, carbohydrates, fats and proteins. In the period of biochemical research which immediately followed the fundamental discoveries of Liebig and Voit, the application of the laws of the conservation of matter and energy to the phenomena of growth and metabolism appeared to supply all of the necessary clues for the interpretation of the relationship of the foodstuffs to the maintenance of life. But with the increasing refinement of our knowledge of the intimate chemical structure of the foodstuffs themselves it has become increasingly appar- ent to us during the recent decades that it is not sufficient merely to supply an animal or a human being with a sufficiency of nitrogen, carbon and calories to replace his daily waste in order to maintain the equilibrium between waste and repair in his tissues, nor is it even sufficient to supply these desiderata in digestible and assimilable form; it is furthermore necessary to supply irreducible minima of specified atomic groupings or complexes of nitrogen, carbon, hydrogen and so forth which, it appears, are essential constituents of living matter, and yet are not synthesizable by animal tissues. Thus the Pyrrole grouping, for example (see Chapter XV), which is an essential building- stone of Hemoglobin, would appear to be as much an elementary requirement of animals as nitrogen or carbon itself, inasmuch as, according to Abderhalden, they are unable to synthesize it from other carbon or nitrogen complexes in the diet and, lacking it, are just as assuredly suffering starvation as if they were lacking one of the more elementary desiderata. The variety of these essential constituents of the diet with which we are acquainted is already very great and is unquestionably destined to grow with increasing scope and refinement of investigation. It is highly probable that many of the raw materials from which the various SUBSTRATES OF GROWTH 489 Internal Secretions are synthesized are dietary constituents of this essential type, for example the Iminazolyl-grouping, which in all probability forms an essential constituent of the active principles of both lobes of the pituitary body, the Catechol-grouping which is an essential complement of the molecule of Adrenalin, and the Indole radical which, from the observations of Kendall, would appear to be a component of the active principle of the thyroid, are examples which will serve to illustrate the essential importance of specific molecular groupings or arrangements of atoms, which, if not synthesizable by animal tissues, must necessarily form a part of the diet in order to maintain bodily equilibrium; and to a still greater extent, of course, in order to render normal growth a possibility. The Vitamines, which appear to be nitrogenous substances closely related to the Purines, are dietary constituents of this type. They are essential for growth, and even for the maintenance of bodily equi- librium, yet the amount required to maintain the weight of the body or to permit satisfactory growth is extremely minute. They evidently represent a group of non-synthesizable essential constituents of living matter which would appear not to be excessively complicated in struc- ture since they are usually obtainable in crystalline form and their relationship to the pyrimidines and the purines has frequently been established. Then, again, there are fatty constituents or substances soluble in Fats which are probably of a more complex character and which are equally essential elements of a complex dietary. According to the older view of metabolism, fats and carbohydrates were considered to be mutually replaceable in the dietary in isodynamic, i. e., equicalorific proportions. Provided the fats in the dietary be not too greatly dimin- ished this is still recognized to be true, but it has now been repeatedly shown that development and maintenance upon an absolutely fat-free diet is impossible, no matter what excess of carbohydrate may be furnished and, furthermore, that Vegetable Oils do not supply this deficiency. According to McCollum the essential constituents of the diet, in addition to the requisite mineral salts, amino-acids and calorific value in the form of fats or carbohydrates, fall into two groups, of which one is soluble in water, while the other is insoluble in water and is soluble in fats and in fat-solvents such as ether. It is immaterial from what source these constituents may be derived; provided merely that they are both present and the diet conforms to the other requirements outlined it will suffice to maintain life and permit growth. These substances have been provisionally designated by McCollum "Fat- soluble A" and "Water-soluble B"; we are as yet ignorant of their structure or affinities. But from their essentiality for growth, and even maintenance for any prolonged period, we may infer that they are Substrates or raw materials which are required in the manufacture of living tissue and cannot be synthesized by the tissues themselves. The clearest indication of the dependence of tissue-synthesis upon the 490 PROCESSES INFERRED FROM INDIRECT OBSERVATION presence of specific atomic groupings in the dietary is, however, afforded by the investigations of Hopkins and Willcock and of Osborne and Mendel upon the ability of various pure Proteins to supply the nitrogen- requirements of growth and maintenance. We have seen that the various protein constituents of the tissues and of the diet are built up out of varying permutations and combinations of a limited number (nineteen in all) .of Amino-acid radicals which are linked together in long chains. Now certain of these nineteen radicals are lacking in some of the proteins, and the administration of such proteins to growing animals as the sole source of nitrogen in the diet enables us to ascertain whether the amino-acid which is lacking is synthesizable by animal tissues, for synthesized it must be if normal tissues are to be produced by the animal and it is not procurable preformed in the diet. From the investigations cited it appears very probable that the only amino-acid radical which is synthesizable by animal tissues is Glycocoll, or Amino-acetic Acid. Of the remainder, it is probable that all must be present preformed in the diet in order to permit the accre- tion of living tissue; at all events this has been positively established for several of the amino-acid radicals, for example Lysine, Tryptophane, Tyrosine, and Cystine. The alcohol-soluble protein of maize, Zein, is lacking in glycocoll, tryptophane and lysine, and the investigations of Hopkins and Willcock and of Osborne and Mendel have shown that if Zein be the sole source of nitrogen in the diet, not only is accretion of fresh tissue impossible, but the maintenance of that already formed is also impossible, so that when supplied with abundance of nitrogen, carbon and salts in correct proportion, water and calories, the animal nevertheless dies of inani- tion. If tryptophane be added maintenance becomes possible, but not growth. On such a diet, or if supplied with Gliadin which lacks only glycocoll and lysine, a young animal lives but ceases to grow and maintains an infantile appearance, and full capacity to grow upon readmission of the lacking constituent to the diet, until what would normally be a "ripe old age" (Fig. 33). Upon addition of lysine as well as tryptophane, normal growth and maintenance are at once rendered possible, the glycocoll being synthesized by the animal itself. Evi- dently the Endogenous Metabolism, or waste incidental to and an essen- tial consequence of life, of the amino-acid lysine is reducible to zero, possibly because a limited supply of lysine may be utilized over and over again in the processes of waste and repair, while, on the contrary, the endogenous metabolism of tryptophane is not reducible to zero, possibly because it is employed, not only in the manufacture of tissue, but also of constituents of the body which undergo irreversible consumption. The result is, at all events, that an inevitable waste of tryptophane attends the maintenance of life, and in its absence from the diet, the tissues of animals being 'unable to synthesize it from other nitrogenous constituents of the diet, tissue-waste can no longer be accurately SUBSTRATES OF GROWTH 491 balanced by tissue-repair, and continuous loss of tissue on an otherwise abundant diet is the inevitable outcome. FIG. 33. A and B show the contrast between two rats of the same age, one of which, B, has been stunted by receiving a diet (protein-free milk and gliadin) , deficient in lysine. The lower two pictures afford a comparison between two rats of the same weight but widely differing in age. The older, stunted rat, B, has not lost the character- istic proportions of the younger animal, C. (After Under hill.) It has long been realized that Gelatin is not in itself an adequate protein for the maintenance of nitrogenous equilibrium, although it is a " sparer of protein/' i. e., can furnish a portion, but not the whole of the nitrogen in the diet. We now recognize that this is due to the absence of tyrosine and tryptophane from the molecule of this protein. 492 PROCESSES INFERRED FROM INDIRECT OBSERVATION Casein is an inadequate protein on account of its deficient content of cystine (Fig. 34). In milk this deficiency is supplied by Lactalbumin. In addition to the various dietary constituents which have been definitely ascertained to be essential and irreplaceable there are others which we can infer, from known data, to be equally essential. Thus Cholesterol has been shown by Gardner and his collaborators not to be synthesized by animal tissues; the cholesterol in the blood and tissues being proportionate to the cholesterol derivable from the dietary. Now cholesterol is an essential constituent of nervous tissues, and derivatives of cholesterol, such as the bile-acids, play an essential part in the bodily economy. A diet lacking in cholesterol, which is not at the same time lacking in other essential dietary constituents, is very -soo Mo Uo IfO llo too no 11,0 IVO no /CO to to to Each division - to day* Days Each division - 20 days FIG. 34. Curves of growth of rats on basal rations plus casein, showing effect of addition of cystiiie to an inadequate allowance of casein. (After Osborne and Mendel.) difficult to devise, so that direct proof of its essentiality in the dietary has not yet been adduced. Still we may infer with a fair degree of confidence that a certain minimal content of cholesterol in the diet is requisite, if not for maintenance, then at least for normal growth and development. From these various investigations it is clear that the elementary substrates of growth are in all probability very numerous and represent a variety of chemical genera, or at any rate, lipoidal substances, basic substances and amino-acids, superadded to the more elementary requirements of nitrogen, carbon and calories. It is furthermore evident that growth, like all other chemical trans- formations, is absolutely dependent upon its raw materials or sub- strates, and cannot occur in their absence. On the other hand, the RELATIONSHIP OF ENDOCRINE ORGANS TO GROWTH 493 capacity to grow, as the above-cited investigations of Osborne and Mendel reveal, is not determined by age but, as we have already con- cluded upon other grounds, by a lack of balance between the forward and opposed reactions of tissue-synthesis and tissue-degradation, so that upon admission of the necessary substrates, no matter what the age prior to the death or senescence of the animal may be, growth occurs and continues until equilibrium, or equality of the velocities of tissue-synthesis and tissue-degradation is attained. We thus reach once more, and from a totally different angle, the conclusion that the relatively stationary weight of an adult animal is determined by the accumulation of the Products of Growth, and not in any sense by the exhaustion of its Substrates. THE RELATIONSHIP OF THE ENDOCRINE ORGANS TO GROWTH. We have seen that the chemical processes which underlie the growth of animals are of such a nature that they produce their own catalyzers. But if this be so then we are immediately impelled to the conclusion that Catalyzers of Growth exist, i. e., substances which, perhaps in minute proportion, and certainly quite independently of their nutritive or substrate- value may profoundly modify the growth of living tissues. The question now arises whether any evidence other than evidence of this inferential kind is obtainable of the veritable existence of such endogenous catalyzers of growth? In the simpler undifferentiated organisms the catalysis of growth, in common with all the other vital processes, is doubtless a function of every cell, and each cell contains the necessary materials for the accel- eration of the production of living matter. In the higher and more differentiated organisms, on the other hand, it is not at all improbable that the function of growth-catalysis is, to a greater or less extent, delegated to special cell-groups or organs, just as the function of motility is delegated to muscle-cells, that of conductivity is especially displayed by nerve-fibers, and those involved in digestion are delegated to the alimentary canal and dependent organs. We are thus led to direct our attention to the possibility of the existence in the body of special cell-groups exercising to an exceptional degree the function of growth- catalysis. The profound significance of certain of the various Endocrine Organs or glands of internal secretion in the processes of growth immediately suggests that these are the special cell-groups to which the function of growth-catalysis is most particularly delegated. We know from abundant clinical experience that disorders of the thyroid, thymus, sexual glands and particularly of the anterior lobe of the pituitary body, are reflected in a profoundly disturbed development of the various tissues of the body, while the action of the secretions of the Corpora Lutea in stimulating the outgrowth of placentae from the wall 494 PROCESSES INFERRED FROM INDIRECT OBSERVATION of the uterus is a striking example of the intensity and specificity of growth-stimulation which may be brought about by agencies of this type. It is possible that not all of the organs of internal secretion which are capable of affecting and modifying the growth of animals do so by virtue of growth-catalyzers which they elaborate. Thus hyperactivity of the thyroid leads to generalized loss of body-weight owing to a marked increase of metabolism and particularly of nitrogenous metabolism, while hypo-activity leads to the peculiar maladjustments of development which characterize the condition of myxedema. These effects, however, are more probably due to a general action of the thyroid principle in accelerating Exogenous Metabolism and reducing the nutritional level in the tissue-fluids. They are effects which more probably concern the concentration of the available substrates of growth than the specific rapidity of their elaboration into protoplasm. The disproportionate growth of connective tissues which characterizes myxedema is more probably to be attributed to the absence of the normal competition with the cellular elements for a limited supply of substrates than to any specific stimulation ' of connective-tissue synthesis. The function of the Thymus in growth is obscure and its true signifi- cance may perhaps be rather that of a storehouse of substances, for example Nucleic Acids, which will be required in subsequent develop- ment than of a factory of growth-catalyzers. The relationship of the anterior lobe of the Pituitary Body to the processes of growth is, how- ever, clearer and more defined, and is of such a character as to en- courage the supposition that in the hypophysis we have one instance among others of an organ in which the function of growth-catalysis is concentrated and specialized. The relationship of the pituitary gland to certain remarkable disturb- ances of growth was first pointed out in 1888 by the French surgeon Pierre Marie, who drew attention to two types of anomalous growth which postmortem examination showed to be invariably associated with abnormalities of the hypophysis. These rare pathological condi- tions are Gigantism and Acromegaly. There are occasional individuals in whom, either before or during adolescence, the growth of the skeleton undergoes an extraordinary acceleration so that they attain such an abnormal stature as to attract universal attention. Such are the individuals who are occasionally exhibited as "giants" in shows and fairs (Fig. 35). A closer inspection of these cases usually reveals other abnormalities which, in the adult at all events may be of two opposite types. The skin may be thin, transparent and hairless, the extremities small, muscular energy deficient, the genitals imperfectly developed, and,, according to Gushing, a decided intolerance for sugar is usually also present. On the other hand cases may be encountered in which the reverse of these charac- teristics may be noted, the skin is thick, coarse and hairy, the extremi* RELATIONSHIP OF ENDOCRINE ORGANS TO GROWTH 495 I FIG. 3,5. Preadolescent hyperpituitarism resulting in gigantism. Height, 8 ft. 3 ii weight, 275 pounds. (After Gushing.) 496 PROCESSES INFERRED FROM INDIRECT OBSERVATION ties are more or less enlarged and the development of the sexual organs may be exaggerated. These latter symptoms are, however, more commonly displayed in the second type of anomalous development of hypophyseal origin, that afforded by the instances of Acromegaly. In these individuals the symptoms do not usually supervene until maturity has been attained and the epiphyses of the bones have hardened so that growth in length is no longer possible. The extremities of the bones become enlarged so that the phalanges of the fingers, for example, are FIG. 36. Acromegalic gigantism. Height, 6 ft. 1 in.; weight, 247 pounds. (After Gushing.) spatula-shaped. The bodily weight becomes excessive, so that these individuals also, especially if above the average in stature, may from time to time be exhibited as giants. The features are coarsened and thickened and there is an extraordinary development of epithelial tissue and of epithelial appendages. The development of hair all over the body may be so excessive as to lend to the individual, especially when conjoined with thickened and distorted features and massive development of the* jaw, a truly simian appearance (Fig. 36). Close RELATIONSHIP OF ENDOCRINE ORGANS TO GROWTH 497 examination usually reveals disturbances of vision resulting in con- traction of the visual field. The sugar tolerance may be abnormally high or abnormally low. Violent headaches and periods of uncon- sciousness or mental confusion are frequently experienced. The ultimate fate of these cases is usually heralded by loss of muscular power and a train of symptoms which invite the supposition that the fundamental condition from which the original abnormalities arose tM FIG. 37. Dystrophia-adiposo-genitalis. Age, fifteen years; gain of 124 pounds in fourteen months. (After Gushing.) has become reversed. Examination of the skull by means of the a>ray usually results, both in these cases and in the instances of gigantism, in the discovery of a decided enlargement of the sella turcica, or bony cavity in which the pituitary body is enclosed. Post- mortem examination usually reveals a tumor in the neighborhood of the pituitary body, either a sarcoma of the gland itself or a tumor exterior to the gland but pressing upon it. It was pointed out by Marie that when the hypophyseal disturbance 32 498 PROCESSES INFERRED FROM INDIRECT OBSERVATION begins before adolescence the effect is to produce gigantism, while if the disturbance supervenes after the attainment of maturity acromegaly is the result. He regarded the two conditions as differing aspects of one and the same disease, of which the symptoms were in both instances attributable to hyperactivity of the hypophysis followed ultimately by its destruction. Subsequent investigators, and especially Gushing, have confirmed this view by more extended observation, but they have also added a third type of pituitary disturbance, which is designated Frohlich's disease, or Dyspituitarism. These cases may occur in childhood (Fig. 37) or in adults. They are characterised by exten- FIG. 38. Fat undersized animal on left has undergone partial hypophysectomy. Animal on the right is a normal animal of same sex and litter. (After Gushing.) sive deposits of subcutaneous fat, the skin is thin, transparent, and hairless, and the sexual organs and functions are usually undeveloped. Muscular energy is at a very low level, the intelligence is usually normal but slow These cases have in some instances been markedly alleviated by the administration of pituitary tissue or of pituitary and thyroid tissue or extracts combined, and they apparently arise from deficient tivity of the hypophysis without the preliminary stimulation which is esponsible tor the characteristic symptoms of gigantism or acromegal v. Experiments upon animals have shown us that while in mammals excision of the posterior lobe or pars nervpsa of the pituitary body mav be endured, complete excision of both lobes of the gland is fa.taJ, RELATIONSHIP OF ENDOCRINE ORGANS TO GROWTH 499 tial excision leads to underdevelopment and particularly to retarded development of the bones (Fig. 38). In amphibians complete removal of both parts of the hypophysis is possible at a very early stage of development and Smith has shown that in hypophysectomized tad- poles development and Metamorphosis are very strikingly retarded in comparison with the normals, while the skin remains unpigmented and the tadpoles have the appearance of albinos. The albinism, but not the defective development, may be cured or prevented by the adminis- tration of posterior-lobe extract. Feeding experiments in which pituitary tissue is administered to normal animals have yielded uniform, but by no means striking results. The Posterior-lobe tissue leads to loss of weight and intestinal disturbances which are not attributable to or indicative of any effect upon growth. The administration of Anterior-lobe tissue to rats has been observed by Aldrich and by Schafer to cause retardation of early growth, followed, in Schafer 's experiments, by a secondary accelera- tion. Wulzen and Maxwell, working with fowls, likewise obtained retar- r CRAMS, Normal IVeeKs 4 10 ZO 30 40 60 FIG. 39. Comparison of the growth-curves of normal and of pituitary-fed female white mice. dation followed by acceleration and the same effect has been observed in mice (Fig. 39). The uniform testimony afforded by all of these experiments is therefore that the administration of anterior-lobe tissue causes initial retardation and a secondary acceleration of growth, but both of these effects are slight. The inconspicuous character of these results is probably to be attributed to the fact that of all the tissues of the body, the Anterior Lobe of the pituitary gland is the one most richly supplied with blood. The circulation is in fact extraordinarily efficient and we may infer that the active product or products of the gland leave it very rapidly and do not accumulate therein. Hence the dosage of the active mate- rial which happens to be present in the gland at the moment of death of an animal may represent but a fraction of the quantity which is manu- factured and discharged in the course* of a day. When we administer pituitary tissue we are seeking to imitate or accentuate by a single daily administration of merely residual material, the action of a gland which is engaged every moment of the day in manufacturing and discharging the substance which influences the growth of tissues; we cannot, therefore, look for large results. As we shall see, much more decisive effects can be elicited by the administration of a con- 500 PROCESSES INFERRED FROM INDIRECT OBSERVATION centrated extract of the tissue, representing a much larger dose of the fresh tissue than would be practicable to employ. The Posterior Lobe of the pituitary is but poorly supplied with bloodvessels and hence the active material which it elaborates accumulates in the tissue and very minute doses of posterior-lobe tissue or extract are capable of eliciting the characteristic effects of Pituitrin upon smooth muscular tissue. The Pineal Gland is stated by McCord to have a decisive influence upon the growth of the Secondary Sexual Characters. Tumors of the pineal gland have not infrequently been described, and are usually associated in children with extraordinary precocity of sexual develop- ment. Either, therefore, the pineal gland elaborates a principle which directly and specifically accelerates the growth of the secondary sexual characters, or else it operates indirectly, by stimulating the interstitial cells of the ovary or testes. The relationship of the Nervous Tissues to the growth of the whole organism is one which can by no means be overlooked in this connec- tion. It is, indeed, not at all improbable that the nervous system performs the dual role of a conducting and coordinating mechanism and a factory of endogenous catalyzers of growth. As we shall see, the growth-catalyzers of which we have positive knowledge, Cholesterol, Lecithin and Tethelin, are all lipoidal in character and these substances, or substances related to them, are exceedingly abundant in nervous tissues. We cannot suppose that the substances which contribute to the building up of nervous tissues or result from their degeneration are not abundant in the circulating fluids in proportion to the development of the nervous tissues or the ratio of their mass to that of the whole body, and several of them we know to exert, and others we may reasonably suspect of exerting, effects analogous to catalysis upon the growth of other tissues. The development of the nervous system may thus be instrumental in determining the development of the whole body. THE METABOLIC RATE AND THE PARTITION OF NUTRIENTS. The loss of weight which occurs in Starvation is by no means uni- formly distributed throughout the body. The following table displays the loss of substance, in percentages of the normal weight, of the various tissues of cats after death from inanition : Loss of weight, Tissue or organ. per cent. Fat 97 Spleen .67 Liver .54 Testes :..;... 40 Muscles 31 Kidneys 26 Skin . ' 21 Intestine 18 Lungs . ! . . 18 Pancreas ..,%..:..... 17 Bones ........ 14 Heart . ... . .'. ........... 3 Central nervous system . METABOLIC RATE AND PARTITION OF NUTRIENTS 501 It will be observed that those organs which are most essential to the preservation of existence are those which suffer least extensively from the unbalanced tissue-degradation which results from the fall of the Nutrient-level consequent upon deprivation of food. This must be due to some definite peculiarity of the metabolism of those tissues which so especially maintain their weight under these adverse circumstances. The nature of this peculiarity may be inferred from the fact that the speed of metabolism is exceptionally great in just those tissues, the Heart and Nervous System, which most successfully resist the disinte- gration-effects of inanition. Thus the heart is constantly transforming large amounts of potential energy into mechanical work, the mainte- nance of life in the higher Metazoa depends in fact upon its doing so, and yet it carries within itself an extraordinarily small reserve of energy-yielding materials. The Glycogen-content of the muscular tissues of the heart, instead of being exceptionally high, is, as a matter of fact, exceptionally low. The heart must thus depend for the maintenance of its exertions upon the direct and constant withdrawal of nutrient materials from the circulating fluids. In so doing it is forced to compete with all the other tissues of the body and yet it does so with so much success that whereas the majority of the other tissues lose a very considerable part of their weight, the heart maintains the integrity of its substance until death is imminent. This implies that the rate of utilization of nutrients by the heart must greatly exceed that of the other tissues, so that the foodstuffs are appropriated in advance of the ability of other tissues to do so. The high Metabolic Rate of the central nervous system may be inferred from the fact that its consumption of oxygen is exceptionally great. The first effect of deprivation of oxygen is to arrest the higher activities of the central nervous system and those substances which paralyze the oxidizing enzymes, such as the Cyanides, arrest the activities of the central nervous system before any other tissue is affected to a com- parable degree. The intensity of Oxidations in the central nervous system testifies to the rapidity of the destruction of its constituents. The fact that it maintains its integrity even in starvation, therefore, implies a proportionate rapidity of reconstruction. The synthesis of the various tissues of the body from the foodstuffs which are contained in the circulating fluids may be regarded as a multitude of parallel reactions, all consuming similar substrates although not in identical amounts and proportions. Now in any group of Parallel Reactions, that is, of reactions which are occurring simul- taneously and consuming the same raw materials, each substrate which enters into the reactions is shared between them in proportion to the velocity with which they occur. The various reactions proceed at their own independent rates and if the quantity of materials available for transformation were unlimited, each reaction, or the synthesis of each particular kind and type of tissue, would go forward at the same speed as it would if the other tissue-syntheses were not occurring simul- 502 PROCESSES INFERRED FROM INDIRECT OBSERVATION taiK'ously. The quantity of available substrates or Nutrient -level of the tissue-fluids is, however, not unlimited but adjusted, as we have seen, by a dynamic equilibrium, to the average needs of the body as a whole. In the competition for these materials, therefore, the most specifically rapid syntheses will have a decided advantage over the specifically slower syntheses, and when the nutrient-level sinks below the normal, as in starvation, the more rapidly metabolizing tissues will maintain their integrity for relatively prolonged periods at the expense of the more slowly metabolizing tissues. If we now turn to the question of the origin of the varying metabolic rate of different tissues, we can only infer that the rapidly metabolizing tissues produce Endogenous Catalyzers of growth which are either more efficient accelerators than those which are produced by other tissues or else are produced in greater amount. We may thus clearly look to the nervous system and the tissues of the heart as the origin of very powerful or abundant catalyzers of growth. Since the majority of catalyzers, and probably the growth-catalyzers also, 1 accelerate both the forward and the backward reaction, both the anabolism and the catabolism of such tissues are exceptionally rapid. Since the effect of starvation is to favor the rapidly metabolizing tissues at the expense of those of slower metabolic rate the result must be to increase the proportion of rapidly metabolizing tissues in an animal and the production of growth-catalyzers per kilo of body-weight. Corresponding with this fact Osborne and Mendel found that a period of starvation greatly improves the subsequent utilization of foodstuffs, so that in a growing rat the total growth attained in a period of starva- tion followed by a period of feeding may exceed that attained by normal animals in a like period of time. A second period of starvation even enhances this effect. The same effect may often be noted in infants as a result of a period of subnutrition or of a lowered nutritional level due to the enhanced exogenous metabolism in fevers. From quite another avenue of experimental investigation the conclu- sion may also be drawn that a period of starvation increases the pro- portion of vigorously metabolizing tissues in the body. Embryonic Tissues and rapidly growing tissues generally have been shown by many observers, and particularly by Cramer, to contain a high proportion of Water, while those which metabolize most slowly and suffer most in any severe competition for nutrients contain a relatively low proportion of water. The nervous system, for example, contains an exceptionally high percentage of water. Now Aron has shown that a period of starva- tion or subnutrition leads both in children and in animals to a greater loss of nitrogen and calories than would normally be equivalent to the loss of body-weight; in other words the tissues are becoming progres- sively more dilute and of less calorific value. We are led again in this connection to recall the important observa- 1 We may infer this from the symmetry of the curve of growth. CATALYZERS OF GROWTH 503 tion of Child that starving planarians undergo retrogression to a relatively embryonic eharaeter. Child accounts for this Rejuvenescence by the sweeping out from the cell of accumulations of colloidal sub- stances which impede the cell-activities and are consumed in starvation for purposes of furnishing energy. The nature of the impediment constituted by these substances is, however, by no means clear; but it may very conceivably be possible that a high proportion of water is essential to the production of growth-catalyzers in abundance. The relative rejuvenescence of metazoa by starvation is, however, more probably to be attributed to the ascendency in mass and numbers acquired by the tissues which are normally possessed of a high metabolic rate, which enables them, when food is readmitted, to push forward all of the processes of growth, including the growth of slowly metabolizing and water-poor tissues, with unusual energy. CATALYZERS OF GROWTH. If a catalyzer is of the "typical" variety and is not in any degree consumed during the reaction which it accelerates, then it necessarily follows that it cannot alter the final Equilibrium of the reaction, for a shift in chemical equilibrium means, generally speaking, that heat is either produced or absorbed and the equivalent in work or heat must be supplied by agencies external to the reaction itself, or by spme other collateral chemical reaction. Since the catalyzer introduces no condition not implied in its presence, the energy-change involved in a shift of equilibrium would of necessity be equated by a change in the energy-content of the catalyzer which could only be supplied by its chemical transformation, i. e., by consuming it. It follows, of course, that a catalyzer cannot initiate a chemical reaction which is not already proceeding, however slowly, in its absence. If Endogenous Catalyzers of growth really exist, therefore, we should expect them to display the following characteristics, distinguishing them more or less clearly from the growth-substrates : 1. Since these catalyzers are not the only, nor necessarily quan- titatively important constituents of the tissues which are the sum of the products of growth, it follows that the effect of catalyzers of growth may be totally disproportionate to their nutritive (i. e., calorific) value. 2. The ultimate growth attained by two groups of animals under the influence of unequal amounts of the catalyzer may be expected to tend toward equality, since the ultimate station of equilibrium of a reaction is unaffected by a catalyzer, although the velocity with which equi- librium is attained may be profoundly affected. This tendency is, however, limited by three groups of factors, namely (a) the mechanical delay or prevention of growth which may be imposed upon an animal by the formation of a skeleton or of a circulatory or respiratory system of limited dimensions, (6) By the unequal effect of catalyzers upon 504 PROCESSES INFERRED FROM INDIRECT OBSERVATION different types of tissue, leading, as we shall see, to the favoring of tissues of high metabolic rate, other tissues being retarded in their growth by the successful competition of the favored tissues, (c) By the onset of senescence, which ultimately terminates and prevents the full fruition of the growth-process. 3. Growth may take place in the absence of catalyzers added to the diet, since they are produced by the growing tissues themselves or by organs to which this particular function has wholly or partially been delegated. The growth-catalyzers are therefore not essential dietary constituents in the sense in which the growth-substrates are essential. 4. Growth-catalyzers may be expected to appreciably influence the rate of growth even when superadded to an already varied and abun- dant diet, whereas, in normal animals, -provided all of the growth- substrates be present in the dietary in abundance the addition of a particular substrate in excess merely leads to enhanced exogenous metabolism of that foodstuff and not to enhanced utilization for tissue- building. 5. There is no reason to assume that the growth-catalyzer for any one group of tissues, is necessarily identical with that for any other. On the contrary we have evidence, as in the effect of the interstitial cells of the testes or ovaries upon the growth of secondary sexual charac- ters, and of the secretions of the corpora lutea upon the development of the placenta, that growth-catalyzers may exist which are specific for individual tissues. Growth-substrates, on the contrary, facilitate growth as a whole, and although at a low nutrient-level the high metabolic rate of certain tissues may enable them to appropriate the lion's share of the foodstuffs, yet under normal conditions all tissues are similarly affected in differing degrees by the various growth- substrates. 6. Growth-catalyzers will be unable to initiate new growths, just as other catalyzers are unable to initiate the reactions which they accelerate. Several substances have been discovered to influence the rate of growth of animals and of individual tissues when administered in dosages which are devoid of nutritive significance and which correspond in all of the particulars enumerated above with the anticipated proper- ties of growth-catalyzers. Thus if Cholesterol be administered either by mouth or subcutaneously to animals which have been previously inoculated with pieces of Carcinoma-tissue, the growth of the tumor .s enormously accelerated and out of all proportion to the nutritive value which the minute dosage of cholesterol which is requisite might be supposed to have, if we did not know that as a matter of fact the greater proportion of administered cholesterol is excreted unchanged. Not only is the rate of growth, of the primary tumor, as estimated by s increase of diameter, increased by one or two hundred per cent., the growth of Metastases or offshoots of the tunior in distant organs and the percentage of animals displaying metastases are very CATALYZERS OF GROWTH 505 remarkably increased. Sweet, Corson-White and Saxon had a strain of carcinoma which had never been known in their experience to yield metastases in rats. They administered cholesterol by mouth to a large number of rats inoculated with this tumor and obtained metastases in over ninety per cent, of the animals. It has also been shown by Browder that cholesterol has a remarkable influence upon the rate of multiplication of the infusorian Paramecium, increasing the number of generations produced in a given period by several hundred per cent. If cholesterol be administered to young mice in dosages of 40 mgm. per day, however, a result is obtained which is at first sight rather surprising, for the growth of the animals, instead of being accelerated, is very markedly retarded during the early weeks of the third growth- cycle (fifth to fifteenth week) and subsequently undergoes a secondary acceleration which, however, never makes up for the ground lost during d 70 80 90 100 110 120 130 140 160 WEEKS 5 10 15 20 25 30 40 50 FIG. 40. Influence of cholesterol upon the growth of male white mice. Dosage, 40 mgms. per day. The vertical cross-mark indicates average duration of life. the period of initial retardation (Fig. 40). Now when cholesterol is administered in unusual amounts to animals the excretory mechanisms prove insufficient and large deposits are formed in a variety of organs, particularly the liver, spleen and suprarenal capsules, and it might be imagined that this or some other deleterious effect of cholesterol, superadded to its effect upon growth is responsible for the retardation of the growth in weight of animals to which its administration leads. This, however, is not the case, for this effect of cholesterol is merely a particular instance of the general action of growth-catalyzers upon the adolescent growth of animals. It will be recollected that the administration of the tissue of the Anterior Lobe of the Pituitary Body to growing animals produces a like unexpected result, namely a retardation of the early adolescent growth followed by a secondary acceleration. Now hypophyseal tissue, when .")()() 1>R()CESSE8 INFERRED FROM INDIRECT OBSERVATION emulsified and administered by hypodermic injection, brings about an acceleration of the growth of inoculated carcinoma in rats which is just as marked as that which is caused by cholesterol. By extraction with alcohol and subsequent precipitation with ether a substance is obtained from the dried tissue of the anterior lobe of the pituitary body which has been designated Tethelin. This substance is evidently a lipoid, for it yields fatty acids on hydrolysis, but it is a lipoid of very exceptional physical and chemical characteristics. It is soluble in water, alcohol or ether, but insoluble in a mixture of certain definite pro- portions of alcohol and ether. It is present in ox-glands to the extent of about 0.7 per cent, of the fresh anterior-lobe tissue. The adminis- tration of four milligrams of this substance per day to mice from five weeks of age onward produces a most decisive change in the velocity and time-relations of growth. The effect is similar in kind to that of d 70 80 WEEKS FIG. 41. Influence of tethelin upon the growth of male white mice. The vertical cross-mark indicates average duration of life. the administration of pituitary tissue itself, that is, initial retardation followed by acceleration, but both effects are exaggerated so greatly as to involve total distortion of the curve of growth, the second growth- cycle appearing to be prolonged while the third or adolescent cycle is abbreviated and accelerated* (Fig. 41). The quantitative difference between the growth-effects obtained with tethelin and observed in anterior-lobe tissue administration are attributable to the difference m the dosage of tethelin which is received in the two cases. It is not practicable, for example, to administer much more than a twelfth of a fresh ox-gland per day to mice, because the quantity of meat consumed would otherwise constitute an important abnormality in the diet. Phis amount of pituitary tissue, however, contains only between eight and nine-tenths of a milligram of tethelin, or one-fifth the amount administered in the experiments cited above. CATALYZERS OF GROWTH 507 The influence of tethelin upon the growth of mice is therefore similar to the effect of administering cholesterol, save that results are attained by administration of tethelin with a tenth of the dosage that would be requisite in the case of cholesterol. It is very significant, therefore, that the action of tethelin upon inoculated Carcinoma in rats again reproduces the effects of cholesterol (Fig. 42.) Even more striking than its effect upon the growth in weight of the animals is, however, the effect of tethelin upon the general contour and appearance of mice to which it has been administered continuously. The tethelin-fed animals are remarkably robust and compact in build. Weight for weight they are smaller and size for size much heavier than normal animals. The contours of their surface are more rounded and fully adult animals retain a youthful appearance which is soon lost in 80- 60- 4-0 20- Per cent Increase over Diameter at 21 days Days after inoculation 21 23 25 28 30 FIG. 42. The acceleration of the growth of carcinomata (in rats) by hypodermic administrations of tethelin. normal animals. The coats of the males, even at fourteen, months of age, retain the glossy, silky appearance of the coats of young animals or of females, while six months or more prior to this age the coats of normal males are already shaggy, staring, and discolored. These differences are clearly displayed in the accompanying photograph, in which a normal and a tethelin-fed male of the same age (one year) and of the same weight (28.0 gm.) are compared (Fig. 43). The normal animal on the left has a shaggy, staring and discolored coat, while the tethelin-fed animal has a smooth, glossy and pure white coat. The normal animal is irregular in outline and loosely built, while the contour of the tethelin-fed animal is rounded and its build is compact. In each of these three instances of growth-catalysis, therefore, we meet with the apparently contradictory fact that while the growth of a 508 PROCESSES INFERRED FROM INDIRECT OBSERVATION neoplasm (carcinoma) is accelerated by the catalyzer, the growth of young animals prior to sexual maturity is retarded. It might be imagined that this constituted evidence of a fundamental difference between the metabolism of malignant tissue and that of normal tissue. This inference would not be justified, however, because in the first place no other evidence of a fundamental difference between the growth of malignant and of normal tissues has ever been advanced and, in the second place, the accelerative action of these catalyzers upon growth is not by any means confined to the growth of malignant tissues. Thus cholesterol accelerates, as we have seen, the division-rate in FIG. 43. Comparison of a normal (left) and a tethelin-fed (right) male white mouse, both one year old and 28 grams in weight. Note the smooth coat and compact form of the tethelin-fed mouse as contrasted with the loose form and rough coat of the normal animal. Paramecia. Our clinical experience abundantly confirms the fact that hyperactivity of the pituitary body leads to abnormally rapid develop- ment of bony and Epithelial Tissues and, finally, tethelin markedly accelerates the regeneration of epithelium lost by injury and the regain of weight lost during a period of inanition after the readmission of food. The action of tethelin in hastening the repair of epithelial lesions is so decided that it has been proposed as a means of accelerating the repair of slowly-healing wounds, such as the leg-ulcers which may result from varicose veins. We have the apparently opposed facts, therefore, that cholesterol and tethelin definitely accelerate the growth of certain types of tissue, CATALYZERS OF GROWTH 509 while the growth of the entire animal is retarded. Evidently, there- fore, there are in the body certain other and relatively bulky tissues of which the growth is directly or indirectly retarded by tethelin. The most probable reason for this retardation lies in the varying Metabolic Rates of the different tissues of the body and their consequent differing success in the competition for nutrients. There are, broadly speaking, two easily distinguishable groups of tissues in the animal body which differ fundamentally in function and metabolism. These are on the one hand the Parenchymatous Tissues, which are essentially cellular, self-maintaining cells derived from the ectoderm and entoderm of the three embryonic layers and on the other hand a variety of tissues which originate mainly but not exclusively from the mesoderm and constitute the Sclerenchyma or tissues of primarily structural or archi- tectural significance. These latter tissues are dependent. They can only arise through the activities of nucleated living cells, of which they constitute outgrowths, secretions, or products of retrogressive change. Of this character, for example, are the various fibrous tissues, the elastic and calcified tissues, and the ligaments, tendons and other structures which bind together and support the tissues of more varied and complex function. The sclerous tissues have a low Metabolic Rate, are among those which lose most heavily in the competition for a sub- normal supply of nutrients and, since they are as a rule devoid of the pow r er of multiplication or even of repair without the intervention and assistance of other cells, we may legitimately infer that they do not produce, as the parenchymatous tissues do, Endogenous Catalyzers which accelerate their synthesis and degradation. In fact since their synthesis is accomplished by other cells there would be no particular purpose served by their doing so. Thus the horny cells of superficial epidermis, which have lost the power of reproduction and growth in the course of the degenerative changes which have resulted in their trans- formation into Keratin, are renewed from time to time by the multi- plication of the cells of the Malpighian layer of the deeper epidermis. Cartilage and bone are similarly formed from cellular tissues and the fibrous tissues are excretions or transformation-products of the Fibro- blasts from which they originate. Even the muscular tissues may in like manner originate from special cells which have retained the potentiality of reproduction. But if these tissues do not produce endogenous catalyzers and in many cases cannot form the material of which they are composed, it is evident that growth-catalyzers from other sources can only affect their development in the indirect fashion of promoting the growth or multiplication of the cells or other tissues from which they arise. A catalyzer of growth may accelerate the formation of parenchyma- tous tissues, but its exceptional abundance or potency may actually retard the growth of the tissues which are not directly affected by it, through the deflection of nutrients to the parenchymatous elements. An important proportion of the total increment in weight of an animal 510 PROCESSES INFERRED FROM INDIRECT OBSERVATION during the adolescent growth-cycle is the formation of Connective Tissues 1 and if the development of certain of these be retarded in the manner indicated, it may readily be understood how the rate of growth of the animal as a whole, estimated by its weight, is retarded although the growth of its parenchymatous tissues may be considerably acceler- ated. That this is probably the correct interpretation of the facts is furthermore shown by the effect of discontinuing the administration of tethelin to mice after the initial retardation of growth has become well marked. The secondary acceleration of growth which succeeds the retardation in animals which received tethelin, cholesterol or pituitary tissue throughout their lives is, in this event very much enhanced, so that the effect of the initial retardation of growth is not only fully FIG. 44. Showing the effect of a brief period (five weeks) of administration of tethe- lin upon the subsequent growth of mice. Animal on the left (31 grams) is the average tethelin-treated animal at five hundred days. On the right (25 grams) an average normal animal of same age. compensated, but a supernormal accretion of weight occurs, carrying the animals far beyond the average of normal animals of the same age. This is strikingly shown in the preceding photograph (Fig. 44), in which a female mouse of average normal weight at five hundred days of age = 25 grams) is compared with a female representing the average weight = 33 grams) of animals which had received four milligrams of Tethelin daily from the fifth to the thirteenth week of age; the administrations being then discontinued. The remarkable overgrowth which is thus" attained is evident even in the average animal displayed in the photo- 1 Tims BischolT (Volt's Handbuch der Physiologic, Bd. 0, p. .511) finds that the muscular, skeletal and fatty tissues comprise 76 per cent, of the weight of the adult W AttJt 8 ' Cent '. f the W6ight f the newborn - Rubner estimates that a man 08 contains 37.8 kilos of cell mass of which 40 per cent, is muscular tissue CATALYZERS OF GROWTH 511 graph, but one-eighth of the animals so treated actually attained weights in excess of forty grams, a weight which, it may be stated, no normal female mouse ever attains. This remarkable overgrowth is probably attributable to the preceding development of parenchymatous tissues. The removal of the stimulus which enabled them to predominate in the struggle for nutrients gives the sclerous tissues the opportunity to develop, and the reattainment of normal proportionality between the sclerenchyma and parenchyma finally enables the stimulation of growth which has actually occurred to find expression in the super- normal weight of the animal as a whole. The occurrence of Acromegaly in man may actually indicate therefore, not a present hyperactivity of the hypophysis, but a preceding hyperactivity, succeeded, before the onset of the acromegalic symptoms, by a normal or even subnormal activity of the gland. It is a noteworthy fact that although the administration of Choles- terol or Tethelin to normal animals which have been inoculated with Carcinoma leads to acceleration of the growth of the neoplasm, yet it has so far proved impossible, despite many trials, to induce the spon- taneous development of tumors in animals by the administration of these substances. The percentage of mice which develop carcinoma is the same in animals which have received cholesterol or tethelin for the greater part of their lives as it is in normal animals. In other words these substances, like the catalyzers with which we are familiar in other chemical transformations, are unable to initiate the reaction which they accelerate. 1 Moreover the spontaneous development of carcinoma is even greatly delayed and the growth of the neoplasm when it has arisen is very much slowed by the continuous administra- tion of tethelin to animals. It would appear that the continuous administration of tethelin results in such a disproportionate develop- ment of parenchymatous tissues that they are enabled to compete successfully with the neoplasm for the nutrients in the tissue-fluids, whereas in the normal animal the neoplasm shares with the limited proportion of parenchyma the advantages of enhanced catalysis of the growth-processes. Carcinoma is essentially a disease of old age and the investigations of Wacker have shown that the cholesterol-content of the subcutaneous fats is exceptionally high in elderly people and in persons afflicted with carcinoma. Luden has also found that cholesterol is exceptionally abundant in the blood of individuals suffering from carcinoma, w r hile the oxidation-products of cholesterol which yield Lifschiitz's reac- tion without preliminary treatment with oxidizing-agents, which are abundant in normal blood, are absent or scanty in the blood of carci- nomatous individuals. 1 Erdmarm has described an innoculable tumor which was produced by the inocu- lation of foreign non-malignant tissue followed by an induced inflammatory reaction ; u H! administration of tethelin, but. tethelin ajqne was ineffective. 512 PROCESSES INFERRED FROM INDIRECT OBSERVATION OLD AGE AND SENESCENCE. The leading characteristic of old age is the low average Metabolic Rate of the tissues. From maturity to old age the calorific output steadily diminishes, the total reduction, according to Du Bois, being about thirteen per cent, by eighty years of age in men. This dimin- ished metabolism, if it is not accompanied by a corresponding diminu- tion of intake, may lead to the formation of extensive deposits of fat and the Obesity which occurs in a certain percentage of elderly indi- viduals. In general, however, the decreased metabolic rate is accom- panied by a progressive loss of body-weight. In man the senescent loss of body-weight begins relatively early, but proceeds very slowly, so that it only becomes notable at an age in excess of the mean duration 9 1C in 5 10 15 20 25 30 40 50 60 70 80 00 100 110 120 130 140 150 WEEKS FIG. 45. Growth curve of normal female white mice from four weeks until death of the last surviving animal. The vertical cross-mark indicates average duration of life. of life. In the mouse, on the contrary, the Senescent Loss of Weight is relatively sudden and rapid and is quite marked before the mean duration of life is attained. This is illustrated by the accompanying curve (Fig. 45), which displays the growth and senescence of female white mice from four weeks until the termination of the observations by the death of the last surviving animals. Deaths from epidemic infec- tion were excluded by the technique of the experiments. The terminal fluctuations of the curve are due to the irregularly occurring deaths of animals in which the process of senescence has been most rapid and which have lost most weight. The survivors therefore represent an earlier or less complete stage of senescence than those which have died, and each group of late deaths is consequently accompanied by a rise in the weight-curve of the survivors. Each rise, however, is succeeded by a fall, which is even more rapid than the preceding one, indicating OLD AGE AND SENESCENCE 513 that the process of senescence is in reality continuous, and, moreover, that it proceeds with a regularly increasing velocity which depends upon the age rather than upon the weight of the animals. The same characteristics are displayed by the curves in Fig. 41 on p. 506. There is no particular reason, implied in the nature of an auto- catalytic process, why the mass of its product should diminish. In fact, the station of Equilibrium in a purely autocatalytic process, un- complicated by side reaction, is asymptotically approached and never actually attained, so that the total mass of product, so far from decreas- ing at the apparent close of the reactions, is actuallly increasing at an infinitesimal rate. The process of growth, however, although it is autocatalyzed, does not conform to this particular characteristic of autocatalytic reactions and, a maximum yield of product having been attained, the tissues slowly disintegrate, even gathering speed as time proceeds, until, if no other factor intrudes to terminate life, Senile Atrophy of the tissues leads to irreparable weakening of some essential organ. A variety of hypotheses have been advanced to account for the phenomena of senescence which, even if all other dangers of life could be surmounted, would set an inevitable term to existence. A very natural supposition is that proposed by Biitschli, that death is due to the exhaustion of a certain substance the "life ferment" which is gradually used up during life. We cannot disassociate senescent atrophy from senescent death, however, since the death of aged indi- viduals is obviously determined by the progressive atrophy or degener- ation of essential tissues. Now senescent atrophy is attributable to the inability of the tissues to maintain their weight and we must therefore, in the terms of Biitschli's hypothesis, suppose that the gradual con- sumption of an essential substance which was originally contained in the germ-cells and can be manufactured only by them, has deprived the tissues of the power to form new protoplasm. Now this is not the case, for even in old age, injury, or removal of the Products of Growth, will institute vigorous Regeneration and repair. The capacity to grow is not lost or even impaired by age. Thus Osborne and Mendel have maintained rats in an infantile stage of development by depriving them of the single amino-acid Lysine. But upon readmission of lysine to the diet, even at an age exceeding the average normal duration of life (700 days), growth is immediately inaugurated, at the same speed that it would, in the normal course of events, have taken place in a normally fed animal of similar weight and stage of development. The retardation of growth by the accumulation of the products of growth is therefore one of the important factors in determining the inability of the adult tissues to maintain their weight in aged animals. It is not the only factor, however, because in that case, as we have seen, indefinitely prolonged equilibrium and not decline would be the resultant. A modification of Butschli's hypothesis is that proposed by Rubner, namely, that the protoplasm of an animal is able to sustain a limited number of molecular transformations and no more. Thus he points 33 514 PROCESSES INFERRED FROM INDIRECT OBSERVATION out that the total calorific output of a variety of animals from birth to old age is approximately the same, a striking exception, being, however, afforded by man: TOTAL CONSUMPTION OF CALORIES PER KILOGRAM OF BODY^-WEIGHT. Man 725,770 Horse 169,900 Cow 141,090 Dog 163,900 Cat 223,800 Guinea-pig 265,500 The instances are, however, not very numerous and if one marked exception to the "rule" occurs among such a small number of cases, other exceptions will doubtless be encountered. Indeed we may with more probability attribute the exceptional position of man in this small group to the much larger proportion of Nervous Tissues; tissues, that is, of high metabolic rate, which his body contains in comparison with the other animals enumerated. His duration of life is also, and possibly for the same reason, exceptionally great. Quite a different type of hypothesis to the foregoing is that pro- posed by Metchnikoff, who attributes senescence in part to the aber- rant activities of Phacocytes and in part to the absorption of toxic substances which are products of bacterial decomposition in the lower intestine. While there can be little doubt that some of the tissue- changes which are characteristic of old age, such as sclerosis, vascular lesions and so forth may be hastened or even brought about by repeated administrations of basic substances, such as Adrenaline or Tyramine which may be derived from amino-acids by Decarboxylation, yet as a general hypothesis of senescence this is too specific, too limited in its scope and applicability, to account for the phenomenon in the multi- tude of the forms of life which exhibit it. In fact, Metchnikoff did not advance his hypothesis as an explanation of "natural" old age, although he is commonly accredited with having done so, but as an explanation of what he considered to be the "premature" senescence of human beings, and, as such, it is a hypothesis which deserves very serious consideration. The effects produced by basic Nitrogenous Poisons related to the amino-acids are, however, confined to certain tissues and especially the circulatory and renal systems, while the effects of senescent atrophy modify in greater or less degree every tissue in the body. Organisms in which the structural changes pro- ducible by poisons of this character could not constitute an irreparable injury nevertheless display senescence and its necessary outcome, "natural death." The unicellular animals and certain unorganized types of living tissue, such as cancer-tissue, are, as Wiessmann and Loeb have espe- cially emphasized, actually or potentially immortal. 1 The Unicellular Those forms which undergo periodical conjugation may also exhibit senescence, which, however, may very possibly be due to causes analogous to those described below which lead to senescence in the metazoa. Cf., G. N. Calkins: Proc. Soc. Exper. Biol. and Med.,'1919, 16, p. 57, OLD AGE AND SENESCENCE 515 Organisms subdivide, and the daughter-cells which thus arise each contain the protoplasm of the parent-cell which is thus perpetuated indefinitely. No slackening of the process of reproduction occurs unless the supply of nutrients fails. Even in those forms such as the Infusoria, in which conjugation of two cells occasionally occurs, this is not generally essential to the maintenance of the indefinite repro- ducibility of the original protoplasm. In the growth of Cancer only the failure of the tissues of the host to support the parasitic tissue sets a term to its existence. If the tissue be transplanted from time to time into a fresh host it is propagated indefinitely. The failure of nutrients is again the only factor which limits indefinite reproduction. The mortality of higher organisms is therefore a consequence of their complexity, and a very probable explanation lies in the sub- division and delegation of functions and powers which renders this complexity possible. There is a very noticeable alteration in the relative proportions of the different types of tissue in the body with advancing age. As Metchnikoff has expressed it: "Old age is char- acterized by a conflict between the finer and more complicated ele- ments and the simple or more primitive elements of the organisms, a conflict that ends to the advantage of the latter. The picture is always the same atrophy of the more highly differentiated elements and their replacement by an overgrowth of connective tissue." In other words Sclerous Tissues acquire a dominance over the Parenchy- matous Tissues which are the most important or perhaps exclusive source of the endogenous catalyzers of growth. The senescent decay of the body may, in fact, be attributable to the increasing mass of dependent tissues with which nutrients must be shared and for the production and repair of which catalyzers must be provided. So long as the velocity of the forward reaction of growth predominates sufficiently over that of the backward reaction, the impulse to growth secures the continued accretion of tissue. Part of this tissue assists in the production of catalyzers, but part, that part constituted by the tissues of structural rather than functional significance, merely draws away nutrients from the tissues which produce the endogenous catalyzers. This has the effect, so far as the self-maintaining tissues are concerned, of progressive reduction of the Nutrient-le-vel, or diminution of the value of a a" in the autocatalytic equation. The value of a a," however, determines the ultimate or equilibrium-weight of the animal and as it sinks so must the weight of the animal diminish, the parenchymatous tissues being directly and the sclerous tissues only indirectly affected. Hence the proportion of sclerous to parenchymatous tissues is further enhanced and the process of senescence itself partakes of the autocatalytic character. It should be especially noted in this connection that the cost of production of Sclerous Tissues is not to-be estimated merely in terms of their mass. They are "expensive" tissues to manufacture in com- parison with the parenchymatous tissues. Not only are they poorer in 516 PROCESSES INFERRED FROM INDIRECT OBSERVATION water and therefore richer in organic materials than the parenchy- matous tissues, but the Proteins which they contain are of very abnor- mal composition, a composition which is specific for each type of sclerous tissue. They are incomplete proteins, containing certain ammo-acid radicals in exceptional abundance, while others which usually occur in proteins of cellular origin are lacking or present in unusually small amounts. To manufacture one molecule of a protein of this abnormal character several molecules of the ordinary types of protein must be sacrificed, just as several buildings constructed of wood, stone and brick must be sacrificed to obtain the materials wherewith to construct a similar building entirely of stone or of brick. Hence the drain upon the nutrient-level in the circulating fluids which is brought about by the sclerous tissues is far more than proportionate to their mass. We have seen that the administration of Growth-catalyzers must favor the development of parenchymatous as opposed to sclerous tissues. Corresponding with this view and with the views expressed above concerning the origin of senescence, we find that the continuous administration of Tethelin to mice, from the fifth week of age onward, or even its intermittent administration for several brief periods, leads to a remarkable prolongation of the average Duration of Life. Thus the duration of life of normal white mice was found in the particular stock employed to be 767 days for males and 719 days for females within a probable error of somewhat less than one month. Males which had received 4 mgm. of tethelin daily throughout their lives attained an average age of 866 days before death, while females inter- mittently receiving the same dosage attained an average age of 800 days. This would be equivalent to a prolongation of from ten to fifteen years in the average duration of life in man. Pituitary (anterior lobe) tissue, cholesterol, and lecithin alike failed to influence the duration of life, the pituitary tissue on account no doubt of the small- ness of the dosage of tethelin contained in the amount of the tissue which it was practicable to administer, and cholesterol on account of the secondary deleterious effects of the deposits of this substance which accumulate in the tissues of animals receiving excessive amounts. The absence of any effect, of the administrations upon the life-duration of these various groups of animals rendered them additional "controls" by reference to which the prolongation of life attained by the adminis- tration of tethelin could be gauged. The average duration of life of the tethelin-fed males was found to exceed the average life-duration of the males of all other classes of animals investigated by one hundred and three days, while the life-duration of the tethelin-fed females ex- ceeded that of all other classes by one hundred and eight days. The chance of both of these deviations from normality being "accidental" was computed to be only 1 in 1 1,000. The prolongation of life in mice by the continuous or frequent administration of relatively large doses of tethelin is therefore unmistakable, Furthermore, Senescence is very OLD AGE AND SENESCENCE 517 much delayed in tethelin-fed animals, the loss of weight for a prolonged period being almost imperceptibly gradual, whereas in normal animals it is relatively sudden (Fig. 41, p. 506). We have seen that the tissues of the Nervous System are very rich in lipoids which are either identical with (cholesterol) or related to (phospholipins, etc.), the substances which we know to have an influence upon growth similar to that which we would expect to be exerted by catalyzers of growth. Furthermore their exceptionally high Metabolic Rate encourages the supposition that they produce an abundance of endogenous growth-catalyzers. A predominant develop- ment of nervous tissues should therefore be equivalent in its effects upon metabolism, growth, and life-duration to the continuous adminis- tration of an excess of growth-catalyzers. Now Friedenthal has pointed out that the ratio of brain-weight to body-weight or to the two-thirds power of the body-weight, which he terms the " cephalization-f actor," varies from one species of animal or bird to another in extremely close correspondence with the maximal attainable duration of life. The following are among the figures which he cites in support of this thesis: 1 MAMMALS. Maximal life-duration Cephalization-factor. (according to Hanseman Species. in years. Man. . 2. 67 to 2. 81 80 to 150 Elephant 1.24 to 1.34 90 to 100 Anthropoid apes 0.76 to 0.65 Horse I, ... 0.43 to 0.57 45 Deer v . .. 0.40to0.50 30 Bears 0.36 to 0.50 50 Dogs 0.34 to 0.51 15 to 20 Cats 0.29 to 0.34 20 Oxen 1 Giraffes > 0.30 to 0.40 30 Antelopes J Squirrels 0.16 to 0.20 6 Insectivora 0.06 to 0.18 6 to 10 Mice ..." 0.04 3 BIRDS. Maximal life-duration (according to Hansemann Species. Cephalization-factor. in years. Carrion crow 0.168 100 (?) Parrots 0.147 to 0.177 100 (?) Alpine crow 0.114 50 Buzaard 0.11 Owl 0.113 Finch 0.086 8 Sparrow 0.086 Duck 0.0731 Snipe 0.0585 Quail 0.0495 Heron . 0.0459 15 Pheasant . 0.0343 15 Fowls 0.0249 10 to 20 Ostrich . 0. 0195 1 The life-duration of the mouse computed from the observations cited above has been added to the table. 518 PROCESSES INFERRED FROM INDIRECT OBSERVATION The various estimates of the maximal Duration of Life can only he regarded, excepting in the case of the mouse, as very hazardous approximations, since, even in the case of man, the maximal attainable duration of life has been the subject of far more fables than investi- gations. Probably the mean duration of life would be a better standard of comparison than the maximal duration of life, since the magnitude of the latter estimate may be so greatly affected by a single exceptional observation. On the other hand statistical estimates of the average duration of life are lacking, save for man and mice, and even the estimates for man which are available include accidental deaths and deaths from epidemic infections. However, notwithstanding the approximate character of the estimates, they afford very striking evi- dence of a tendency of Longevity to be associated with a high degree of development of the nervous system. Thus, so far as the effect upon the duration of life is concerned, exceptional development of the nervous system exerts an effect similar to that which is induced by the administration of an excess of a growth-catalyzer. The resemblance between the effects of a high proportion of Nervous Tissues and those induced by administration of a growth-catalyzer extends, however, even to the time-relations of growth, as expressed by the contours of the growth-curve. Thus on comparing the growth- curves for man and mice in Fig. 31 (p. 473), with the growth-curves for cholesterol-fed and tethelin-fed mice in Figs. 40 and 41 (pp. 505 and 506), it is at once apparent that the change in the time relations of the growth of mice which is induced by these catalyzers brings their growth-curve into close approximation to the human curve. The effect of the growth-catalyzers in unusual amount is to apparently prolong the second and abbreviate and accelerate the third growth-cycle, and it is in precisely these characteristics that the human growth-curve, when reduced to the same scale, differs most strikingly from the growth-curve for mice. It is not unlikely, therefore, that the differ- ence in contour of the mouse and human curves of growth is attribu- table to the greater abundance of endogenous catalyzers of growth in the tissues and tissue-fluids of man consequent upon the greater pro- portionate development of his nervous system. REFERENCES GENERAL CHARACTERISTICS OF THE GROWTH-PROCESS: Voit, C.: Zeit. f. Biol., 1866, 2, p. 353. Bowditch: Eighth Annual Report, State Board of Health, Massachusetts, U. S. A., 1877. Roberts: Manual of Anthropometry, London, 1878. Anthropometric Committee, British Assn. Reports, 1879, p. 175; 1883, p. 253. Minot: Jour. Physiol., 1891, 12, p. 97. Porter: Trans. Acad. Sci., St. Louis, 1895, 6, p. 263. Voit, E.: Zeit. f. Biol., 1905, 46, p. 195. Donaldson: Boas Memorial Volume, New York, 1906, p. 5. The Rat, Pub. of the Wistar Institute, Philadelphia, 1915. Loeb, J.: Seventh Internat. Zool. Congress, Boston, 1907. Ostwald: Vortrage und Aufsatze iiber Entwicklungsmech., Leipzig, 1908, Heft 5. OLD AGE AND SENESCENCE GENERAL CHARACTERISTICS OF THE GROWTH-PROCESS: Robertson: Arch. f. Entwioklungsmech., 1908, 25, p. 581; 1908, 26, p. 108; 1913, 37, p. 497. Biol. Centr., 1910, 30, p. 316; 1913, 33, p. 29. Am. Jour. Physiol., 1915, 37, pp. 1 and 74; 1916, 41, pp. 535 and 547. Tables for the Computation of Curves of Autocatalysis, with Special Reference to Curves of Growth, Univ. California Pubs. Physiol., 1915, 4, p. 211. Stratz: Der Korper des Kindes und seine Pflege, Stuttgart, 1909, 3. aufl. . Read: Arch. f. Entwicklungsmech., 1913, 35, p. 708. Martin: Lehrbuch der Anthropologie, Jena, 1914 (consult for literature con- cerning the growth of man). Thompson: Growth and Form, London, 1917. SUBSTRATES OF GROWTH: Hopkins and Willcock: Jour. Physiol., 1906-7, 35, p. 88. Mendel and Mitchell: Am. Jour. Physiol., 1907, 20, p. 81. Mendel and Saiki: Ibid., 1908, 21, p. 64. Stepp: Biochem. Zeit., 1909, 22, p. 452. Zeit. f. Biol., 1912, 57, p. 135; 1912, 59, p. 366; 1913, 62, p. 405. Osborne and Mendel: Science, N. S., 1911, 34, p. 722; 1917, 45, p. 294. Jour. Biol. Chem., 1912, 12, p. 81; 1912-13, 13, p. 233; 1913-14, 16, p. 423; 1914, 18, p. 95; 1917, 31, p. 149. Am. Jour. Physiol., 1916, 40, p. 16. Biochem. Jour., 1916, 10, p. 534. Funk: Jour. Physiol., 1911-12, 43, p. 395; 1912, 44, p. 50. Ergeb. d. Physiol., 1913, 13, p. 125. Jour. Biol. Chem., 1916, 27, p. 1. Hopkins: Jour. Physiol., 1912, 44, p. 425. McCollum and Davis: Jour. Biol. Chem., 1913, 15, p. 167; 1914, 19, pp. 245 and 323; 1915, 20, pp. 415 and 641; 1915, 23, pp. 181 and 231. Jour. Am. Med. Assn., 1917, 68, p. 1379. Hart and McCollum: Ibid., 1914, 19, p. 373. Mendel: Nutrition and Growth, Harvey Lectures, 1914-15, 10, p. 101. McCollum: Supplementary Relationships among our Natural Foodstuffs, Harvey Lecture, 1915-16, 11, p. 151. Funk and Macallum: Jour. Biol. Chem., 1915, 23, p. 413; 1916, 27, p. 51. Macallum: American Medicine, new series, 1916, 11, p. 782. RELATIONSHIP OF THE ENDOCRINE ORGANS TO GROWTH: Gushing: The Pituitary Body and its Disorders, Philadelphia, 1912. Aldrich: Am. Jour. Physiol., 1912, 30, p. 352; 1912-13, 31, p. 94. Schafer: Quar. Jour. Exp. Physiol., 1912, 5, p. 203. Gudernatsch: Am. Jour. Anat., 1913-14, 15, p. 431. Wulzen: Am. Jour. Physiol., 1914, 34, p. 127. Adler: Arch. f. Entwicklungsmech., 1914, 39, p. 21. McCord: Interstate Medical Jour., 1915, 22, p. 354. Pearl: Jour. Biol. Chem., 1916, 24, p. 123. Robertson: Ibid., 1916, 24, p. 385. Smith: Anat. Record, 1916-17, 11, p. 410. University of California Pubs., Physiol., 1918, 5, p. 11. Uhlenhuth: Jour. Gen. Physiol., 1918-19, 1, pp. 23, 33, 305, 315, 473, 525. Robertson and Ray: 1919, 37, pp. 393, 427, 443. OLD AGE AND SENESCENCE: Metchnikoff: The Nature of Man, New York, 1903. The Prolongation of Life, New York, 1910. Minot: The Problem of Age, Growth and Death, New York, 1907. Adami: Principles of Pathology, Philadelphia, 1908, 1, p. 125. Rubner: Kralt und Stoff im Haushalte der Natur, Leipzig, 1909. Friedenthal: Contr. f. Physiol., 1910, 24, p. 321. Woodruff: Arch. f. Protistenkunde, 1910-11, 21, p. 263. Fleischer and Loeb, L.: Proc. Soc. Exp. Biol. and Med., 1911, 8, p. 133. Saundby: Old Age; Its Care and Treatment in Health and Disease, London, 1914. Child: Senescence and Rejuvenescence, Chicago, 1915. Loeb, J.: The Organism as a Whole, New York, 1916. Cramer: Jour. Physiol., 1916, 50, p. 322. CATALYZERS OF GROWTH : King: Biol. Bull., 1907, 13, p. 40. Loeb, L.: Jour. Am. Med. Assn., 1908, 50, p. 1897; 1909, 53, p. 1471. Arch. f. Entwicklungsmech., 1909, 27, p. 89; 1911, 32, pp. 67 and 662. Johnson: Univ. California Pubs., Zoology, 1913, 11, p. 53. 520 PROCESSES INFERRED FROM INDIRECT OBSERVATION CATALYZERS OF GROWTH: Bain: Lancet, 1913, 182, p. 918. Browder: Univ. California Pubs., PhysioL, 1915, 5, p. 1. Robertson and Cutler: Jour. Biol. Chem., 1916, 25, p. 663. Robertson and Ray: Ibid., 1916, 24, p. 347; 1919, 37, pp. 377, 393, 427, 443. Robertson: Ibid., 1916, 24, pp. 363, 385, 397, 409; 1916, 25, pp. 635, 647. Robertson and Delprat: Ibid., 1917, 31, p. 567. Robertson: Endocrinology, 1917, 1, p. 24. R6LE OF CATALYZERS IN CANCER: Wacker: Zeit. f. Physiol. Chem., 1912, 80, p. 383. Burnett: Proc. Soc. Exp. Biol. and Medicine, 1913-14, 11, p. 42. Robertson and Burnett: Jour. Exp. Med., 1913, 17, p. 344; 1915, 21, p. 280; 1916, 23, p. 631. Proc. Soc. Exp. Biol. and Med!, 1913, 10, pp. 140 and 143. Bennett: Jour. Biol. Chem., 1914, 17, p. 13. Sweet, Corson-White and Saxon: Ibid., 1915, 21, p. 309. Robertson and Burnett: Jour. Cancer Research, 1918, 3, p. 75. Luden: Jour. Lab. and Clin. Med., 1916, 1, p. 662; 1917, 3, pp. 93 and 141; 1918-19, 4, p. 849. Jour. Biol. Chem., 1916, 27, p. 273; 1917, 29, p. 463. HEALING OF WOUNDS: Carrel: Jour. Am. Med. Assn., 1910, 55, p. 2148. Robertson: Ibid., 1916, 66, p. 1009. Spain and Loeb, L.: Jour. Exp. Med., 1916, 23, p. 107. Carrel and Hartman: Ibid., 1916, 24, p. 429. Du Nouay: Ibid., 1916, 24, p. 451; 1917, 25, p. 721. Barney: Jour. Lab. and Clin. Med., 1918, 3, p. 480. Clark: Bull. Johns Hopkins Hospital, 1919, 30, p. 117. CHAPTER XXI. PROCESSES INFERRED FROM INDIRECT OBSERVATION: MEMORY AND SLEEP. MEMORY. THE most prominent characteristic of the Nervous System is the facilitation of its functions which their performance brings about. A mental task which is at first difficult becomes easy by frequent repe- tition; an act which may be performed under the guidance of the central nervous system at first only with effort and concentration of the will and attention, becomes by repetition a habit or even a reflex which is performed almost automatically and without any conscious expenditure of effort. Secondary and subsequent to this phenomenon of facilitation is the phenomenon of Fatigue. For example, in the learning of a long passage by rote, as one tries to recall it after the first repetition, recollection is distinctly difficult. With a second repetition recollection is easier, with a third it is easier still and so the progressive facilitation accumu- lates until it becomes possible to repeat a long passage from "Memory," faultlessly and fluently. If, however, the repetitions be still continued or fresh matter added to the lesson a new phenomenon supervenes which is the reverse of that initially experienced. The passage which a little while before was repeated faultlessly cannot now be repeated without mistakes. The attention wanders readily. Recollection becomes increasingly difficult, the consciousness has to be "flogged' ' into activity and finally excessive fatigue compels desistance from the task. The effects of the initial facilitation have not been undone, however, for a return to the task after an adequate interval for recuper- ation reveals the fact that the previous study has implanted memories which disappear from the field of consciousness in many instances only after a lapse of time comparable with the duration of life itself. We meet, therefore, in the exercise of any given intellectual function, with two apparently contradictory facts. Performance facilitates the exercise of the function, and it likewise depresses the exercise of the function. We note, furthermore, that the facilitation and depression become evident at different periods of time, the former in the earlier stages of performance and the latter in its later stages. Many hypotheses have been advanced by philosophers, psychologists and physiologists in the endeavor to imagine a mechanism which could account for the phenomenon of memory. The vast majority of the mechanistic hypotheses, which are the only ones of which we need 522 PROCESSES INFERRED FROM INDIRECT OBSERVATION attempt the consideration, partake of the same general character; they assume that the previous repetition or performance has left some species of more or less permanent modification in the nervous system, and they vary only in the nature of this hypothetical modification. Broadly speaking, the nature of this modification may be conceived in either of two ways which, for convenience sake, we may designate, respectively, the "static modification" and the "dynamic modifi- cation." The static conception, as developed especially by Munk and Ziehen, regards the "trace" or "image," which has been formed in the nervous system in consequence of some act or repetition, as consisting of some structural modification, some physical alteration, an alteration, in other words, in the distribution of cell-matter in space. The objections which may be and have been urged against this view are manifold. A purely physical alteration, namely the redistribution of preformed cell-material in space, would be something of the nature of a strain produced in response to some stress (= stimulus) which might be conceived of as mechanical, electrical, thermal or yet some other type of energy-change capable of inducing modifications of the physical state of matter. Now the remarkable Persistence of Memories proves that the "trace," whatever it may be, is rather permanent and only very slowly fades away. Indeed such investigations as those of Prince or Sidis would appear to indicate that a large proportion of memory-traces may persist in some measure throughout a lifetime. Of course, reinforcement of the trace by occasional "recollection," either conscious or "subconscious" may have occurred from time to time in the interval between the receipt of an impression and its emer- gence from consciousness under abnormal psychological conditions, such as those imposed by Hypnosis, at a much later period of life. Reinforcement of the trace by recollection cannot, however, be the general rule, for otherwise, as Sidis has pointed out, our entire mental life would be occupied in recollecting. The memory trace, or at least some residual fragment of it, is there- fore an extraordinarily persistent modification. The material of which the central nervous system is composed, however, is largely fluid or semifluid, and all our experience teaches as that a fluid cannot retain physical strains for any prolonged period; indeed it is this quality which enables us to recognize a fluid or a jelly and distinguish it from a solid. A modification of the theory of Munk is that which was proposed by Lepine and Duval and has been very widely adopted by a certain school of neurologists and psychologists. This theory is based upon the demonstration by Cajal that the nervous system is divided, like other tissues, into distinct cell-units, or Neurons, which he regarded as being in contact with one another through the medium of their cell- processes or Dendrites, but not physically continuous with one another. It was assumed by Lepine that the formation of a new memory-trace in the nervous system was attributable to the formation of a new den- MEMORY 523 driteHL'ontact, while Amnesia or the phenomenon of forgetting repre- sents the breaking of a contact previously established. To this view there attach most of the difficulties attendant upon Munk's hypothesis and, furthermore, as Meyer has very justly pointed out 1 the invo- cation of such hypothetical structural changes to explain the physical correlates of psychic phenomena must necessarily lead, sooner or later, to the invention of a metaphysical entity to keep the apparatus in order. Meyer expresses this difficulty as follows : " Why does the pro- toplasm stretch toward one neighboring neurone when the organism happens to be in one situation, toward another neurone when the organ- ism is in another situation? General silence with the neurologists. But some psychologists had an answer ready. They brought in their deus ex machina. The Ghost does it. Consciousness, feeling, will, or whatever you call it, turns the bridge in the proper direction as the switchman turns the switch in a railway-yard." The cytological basis of this hypothesis has also been called severely in question since the investigations of Epathy, Bethe and others have demonstrated the existence of fine intercommunicating fibrils which, in many instances at least, establish anatomical continuity between adjacent dendrites. The dynamic conception of the memory-trace, on the other hand, regards it as being formed by a chemical alteration of cell-material along the nervous path which was followed by the stimulus which is subsequently recalled. The superior generality and simplicity of this hypothesis is evident at once. It does not exclude the possible forma- tion of a definite structure as the result of chemical change, on the other hand the persistence of memory traces is at once accounted for since, as we have abundant reason to know, chemical changes within living organisms may be as enduring as life itself. We have seen (Chapter XVIII) that the rate of conduction of impulses in Nerve-fibers is conditioned partly if not wholly by physical changes which underlie the passage of the impulse. We infer this from the low Temperature-coefficient of conduction in peripheral nerve- fibers. In Nerve-cells, on the contrary, the passage of impulses is demons trably accompanied by chemical changes. The temperature- coefficient for the conduction of impulses in the nerve-cells of the respiratory center and the cardiac ganglion, for example, is of the chemical order of magnitude. Furthermore, as Mosso has demon- strated, excitation of the cerebral cortex results in a pronounced disengagement of heat. Repeated attempts to demonstrate a similar evolution of heat in nerve-fibers in consequence of stimulation have failed. The processes which attend the conduction of impulses through nerve-cells, therefore, appear to be of a fundamentally different char- acter from those which accompany the passage of impulses in nerve- fibers. The effect of the chemical change which accompanies the passage of 1 Meyer: Journal of Philos. Psychol. and Scientific Methods, 1912, 9, p. 365. 524 PROCESSES INFERRED FROM INDIRECT OBSERVATION an impulse through the central nervous system is to initially facilitate and ultimately retard the passage of subsequent impulses along the same path. The nature of the initial facilitation has been variously characterized. Thus Maudsley described it as the formation of a trace or thread of a deposit which is followed by the succeeding impulse, while Exner likened it to the "excavation of a channel/' a hypothesis which is generally referred to as the Canalization Hypothesis. In preceding chapters we have had frequent occasion to dwell upon a variety of chemical processes and not a few life-phenomena which display initial facilitation followed by retardation. These are the various processes or phenomena which are governed as to their speed by underlying Autocatalyzed Reactions. It is evident that if the passage of an impulse through the central nervous system were attributable to the occurrence of an autocatalyzed chemical reaction, the deposition of the products of this reaction along the path of the impulse would facilitate the passage of a subsequent impulse, while their accumula- tion in undue amount would constitute an impediment to the further occurrence of the reaction and therefore to the passage of subsequent impulses. The same mechanism thus accounts for both the facilitation and the fatigue which accompany the performance of functions involv- ing the central nervous system. Regarding the nature of the autocatalyst in this reaction we are of course completely in the dark in so far as any direct results of chemical analysis are concerned. We may, however, draw certain more or less probable inferences from our knowledge of the behavior of a par- ticular part of the central nervous system, namely, the Respiratory Center. In this region we have a rhythmic passage of impulses of which the frequency is determined by the alternate facilitation and retarda- tion of conduction which is brought about, as we have seen in a pre- ceding chapter, by the presence of greater or lesser amounts of Lactic Acid, Carbon Dioxide, or other fatty or hydroxy fatty acids in the cir- culating fluids. Evidently, therefore, acids, or at least this particular class of acids, facilitate the passage of impulses through this if not through other regions of the nervous system. Now hyperactivity of the central nervous system results in the accumulation of acid sub- stances in the brain, and we may with some probability infer that the normal activities of the central nervous system are accompanied to a lesser degree by the production of similar substances. THE FATIGUE-PRODUCTS OF NERVE-CENTERS. It has been pointed out by Mosso that the fatigue-products of Nerve-centers and those of Muscle are probably very similar in nature since mental fatigue is accompanied by signs of muscular fatigue and vice versa. Among the products of muscular activity two acids figure very largely, namely Lactic Acid and Carbonic Acid, and, if the products of muscular and of nerve-cell activity are similar, we should expect to FATIGUE-PRODUCTS OF NERVE-CENTERS 525 find that acids are set free in the central nervous system as a result of its activity or fatigue. The actual demonstration of an increase in acidity of the brain-substance as a result of prolonged excitation has proved difficult on account of the slightness of the change of hydrogen ion concentration which is involved, owing to the buffer-action of the tissues and tissue-fluids, and the technical difficulties, almost insuper- able it would appear, which attend the utilization of adequate electro- chemical methods of estimating the hydrogen ion changes in nervous tissues. We can, however, perceive the changed reaction of the brain after excessive stimulation by the employment of a simple indicator, provided, however, that instead of employing the change of color of the indicator as a sign or measure of acidity, we employ the change in its solubility in a solvent which is immiscible in water. If to ten cubic centimeters of a concentrated (two per cent.) and very faintly acid solution of Neutral Red in water we add a single drop of tenth-normal potassium hydroxide the color of the solution does not perceptibly change, but nevertheless a great change is seen in respect to the lipoid-solubility of the neutral red if we shake up the original and the faintly alkaline solutions with Ethyl Acetate, from which any admixture of acetic acid has been previously carefully removed. On shaking up with the faintly acid solution of neutral red the ethyl acetate remains absolutely colorless, while on shaking it up with the faintly alkaline solution the ethyl acetate layer is stained deep yellow. In two ways the indicator is rendered moire sensitive by this method; in the first place a trace of the yellow modification of neutral red, which would be invisible in watery solution owing to the great excess of the red modification, is removed by the ethyl acetate and thereby rendered visible. In the second place, let us suppose that the Coeffi- cient of Distribution: concentration in lipoid layer concentration in aqueous layer is 100 : 1 for the yellow modification of neutral red, and zero for the red modification. Then at any given concentration "b" of hydroxyl ions, if "y" be the concentration of the red modification and "x" that of the yellow modification : x = kf(b)y where "k" is a constant and f (b) is some function of the alkalinity not necessarily known or defined. Now let this solution be shaken up with ethyl acetate, and let the concentration of the yellow modi- fication in the watery layer now be "x," while that of the red modifi- cation is "y," and that of the yellow modification in the ethyl acetate layer is "x?," then we have: xi = kf(b)yi x 2 = lOOxi x? = 100kf(b)yi 526 PROCESSES INFERRED FROM INDIRECT OBSERVATION that is, the concentration of the yellow modification in the lipoidal layer (ethyl acetate) is 100 times its concentration in the watery layer and, provided f (b) were a linear function, it would be the same con- centration as that which would be produced in the watery layer by 100 times the concentration of hydroxyl ions. In other words the sensi- tiveness of the indicator is multiplied by the distribution-coefficient of the lipoid-soluble modification between the two immiscible solvents. In addition to this there is, as has been stated, an apparent or " physio- logical" increase in the sensitiveness of the indicator due to the physical separation of the two colors. Two frogs may be taken and a powerful stimulus applied to the skin of one of them by means of an induced current for a prolonged period (half an hour) while the other is left undisturbed. The brains of both animals are then rapidly removed, divided longitudinally and the two parts of each placed in a two per cent, neutral aqueous solution of neutral red for from four to five minutes. The two brains are then removed from the neutral red solution at exactly the same moment and dropped into neutral ethyl acetate. Within five or ten minutes there is seen to be a distinct difference between the colors of the cut surfaces of the two brains. The cut surface of the brain which has been stimulated remains deep red, but the indicator diffuses out of the unstimulated brain, and the depth of color diminishes until it is only pink. The differences in color increase for some time, and in some instances after the lapse of an hour the unstimulated brain may be almost colorless, owing to extraction of the dye by the ethyl acetate, while the stimulated brain retains a reddish pink hue. Evidently the stimulated brain behaves like a faintly acid aqueous layer, the unstimulated brain like a faintly alkaline aqueous layer. The development of acid as a fatigue-product of nerve-centers may thus be clearly inferred. It might be imagined that in this experiment the increased acidity of the brain may be apparent and not real, being due to acids carried to the brain by the blood from the tetanically contracting muscles of the stimulated frog. It has been shown by Gobau, however, that pre- cisely the same result is obtained if the frog employed for stimulation is previously curarized, in which case the muscles are immobile. Acids are therefore produced in the brain in consequence of its activity and in the respiratory center, if we may take this area as repre- sentative of the whole, certain specific acids accelerate the passage of impulses through it. We have thus experimental verification of the view that central nervous phenomena are self-catalyzed. The cata- lyzer which is responsible for the formation of Memory-traces, however, is not probably any substance so simple as lactic or carbonic acids, which as we have seen, are stimulants of the respiratory center. These substances are so soluble in water that they would very rapidly be washed out of the nervous tissues and the persistence of memory- traces would be inexplicable. It is more likely that we have here to APPLICATION OF THE FORMULA OF AUTOCATALYSIS 527 deal with a colloidal fatty acid which is deposited along the path of an impulse and remains to accelerate or, if it is in excess, to retard a subsequent impulse. THE APPLICATION OF THE FORMULA OF AUTOCATALYSIS TO CENTRAL NERVOUS PHENOMENA. The time-relations of any Voluntary Movement are primarily governed by events which occur in the central nervous system. This may readily be inferred from the fact that it requires, not a single impulse or stim- ulus to produce any coordinated movement, but a stream of impulses FIG. 46. Photograph of a drawing-board specially constructed to record the time- relations displayed in the execution of a simple volition (the drawing of a straight line). which must be maintained throughout the duration of the act which is performed. A single stimulus, when applied to voluntary or striated muscle, only produces a single rapid twitch; a prolonged tetanic or semitetanic movement such as that involved in the performance of any muscular exertion is only possible to evoke by a rapid succession of stimuli. Moreover the performance of a coordinated muscular act such as that of bending the arm, involves a simultaneous discharge of stimulatory impulses to the flexor, and inhibitory impulses to the opposing extensor muscles of the limb. The time-relations of a simple voluntary movement, such as that implied in drawing a straight line with a pencil upon a board, may be accurately investigated by a method which was originally proposed by 528 PROCESSES INFERRED FROM INDIRECT OBSERVATION Loeb and Koranyi. A drawing-board is made up of alternate strips of metal and wood and is carefully polished so that the junction of the strips is as nearly as possible indistinguishable to the touch. The metallic strips are connected together in a circuit which includes a signal- magnet and a metallic pencil. When a line is drawn upon the board with the metallic pencil the moment at which the pencil touches or leaves a metallic strip is indicated upon a cylinder of smoked paper by the signal-magnet (Fig. 46) . In this way the length of line traversed at any instant in the entire process can very readily be determined. It is convenient to provide a ruler, firmly affixed to the board, to guide the movements of the pencil and to start the line from a check or crotch in the ruler, the position of which on the board with reference to the nearest succeeding metallic strip has been accurately determined. When the relationship between the time and the extent of movement in drawing a straight line is investigated in this way it is found that the Autocatalytic Formula: log = ka(t - ti) a x applies with remarkable accuracy, "a" being the total length of the line, "x" the length of line drawn at time "t" after the motion of the pencil first began, and "t," the time taken to reach the middle of the line. The following is an illustrative result: Subject R.M.M.rf Formula: log -^ = k (t - 38.75) ol . x . x t k inches. 1/100 sees. 1 10.00 0.051 2 15.50 0.050 3 17.50 0.046 4 21.00 0.047 5 ............ 22.50 0.044 6 , ... 25.00 0.045 7 ....... 26.00 0.042 8 . , . . . ... . . . . 27.50 0.041 9 29.00 0.040 10 30.75 0.040 11 32.00 0.038 12 33.50 0.038 13 35.00 0.038 14 , . i 36.50 0.037 15 38.00 0.037 16 39.50 0.037 17 40.75 0.042 18 42.00 0.043 19 43.50 0.042 20 . ... . . . . . . ... . 45.00 0.041 ............. 47.00 . 0.039 22 ..... . . . ... 48.25 0.041 23 v * . . . . . . . 50.00 0.041 24 . -:.'.'.'. . . 52.00 0.044 25 V '. ."' . . ... . . 53.75 0.041 26 :.. 55.50 0.043 27 57.50 0.044 28 59.50 0.049 29 61.75 0.050 30 66.00 0.054 APPLICATION OF THE FORMULA OF AUTOCATALYSIS 529 The values in the third column are, as would be required by the formula of autocatalysis, almost constant. The performance of this particular type of central nervous activity is therefore autocatalyzed. Turning now to the much more complex phenomenon of Memory we are in possession of quantitative data which have been most elabor- ately compiled by the psychologist Ebbinghaus. The method which he employed was to read and reread a series of meaningless syllables at a definite rate, 0.40 seconds being expended in the perusal of each syllable. The data recorded are the numbers of repetitions which were found to be necessary to attain the perfect memorization of the given number of syllables in the series. Hence the time in seconds which was employed in learning each series was 0.4 X n X r where "n" and "r" were the number of syllables in the series and the number of repetitions respectively. Excepting in the case of the first observation (that is, the number of syllables learnt in a single repetition) the syllables were read in conjunction with a sufficient quantity of other material to make the total length of each period of reading approximately the same. The following were the results obtained: Number of syllables. Number of repetitions. Time in seconds. 7 1.0 2.8 12 16.6 79.7 16 30.0 192.0 24 44.0 422.4 36 55.0 792.0 If we apply to these results the formula of autocatalysis, calling "a" the maximal number of syllables which Ebbinghaus could have memorized by any number of repetitions, "x" the number actually learnt or the extent of the trace or deposit formed in time "t," and "t/' the time consumed in learning half the maximal number, we find that the following equation most nearly expresses the results: 0.001468 t - 0.526 In the following table the experimental values of "x" and those calculated from the formula are compared : Time in seconds x (observed) x (calculated). 2.8 .... ....... 7 10.1 79.7 ....... ..... 12 12.2 192.0 ......... . . 16 15.8 422.4 ........... 24 24.2 792.0 ........... 36 35.4 The only deviation of significant magnitude is that between the observed and calculated numbers of syllables which may be learnt in a single repetition. This, however, may most probably be attributed to the conditions under which this number was determined, differing as they did, by the non-inclusion of other reading matter, from the conditions which pertained in the remaining observations. 34 530 PROCESSES INFERRED FROM INDIRECT OBSERVATION The view that the formation of the Memory-trace is due to an auto- catalyzed chemical reaction, therefore, not only enables us to interpret some of the most striking qualitative phenomena of intellectual proc- esses, but also to predict their quantitative alteration with successive repetition. The quantitative data obtained by Ebbinghaus are among the most readily interpretable and at the same time accurate measure- ments of this kind which we possess, but a variety of measurements which have been made on the rate of learning by telegraph-operators, typists, and so forth, all yield " curves of learning" which very strikingly resemble the curve which represents the progress of an autocatalyzed chemical reaction, and in some cases, it appears, two or more of such curves may be superimposed to yield "cycles of learning" just as we have cycles of growth in a growing organism. SLEEP. The various theories of sleep which have been proposed are no less numerous than those which have been propounded to account for the phenomenon of memory. A Vasomotor Theory of Sleep has been advanced by Howell, who considers that it is attributable to cerebral anemia, due to a diminished blood-supply to the brain, following the general fall of arterial pressure which accompanies sleep. While this may very possibly be a contributing factor to the phenomenon of sleep, yet, on the other hand, it is at least equally conceivable that the vaso- motor-phenomena which accompany sleep are merely secondary mani- festations of the processes which induce sleep, and that the actual onset of sleep is due primarily to other factors. The close connection of sleep with Fatigue on the one hand, and with the absence or monot- ony of Sensory Stimulation on the other, indicates very clearly that a condition of the nervous tissues consequent upon prolonged activity is a potent factor predisposing the central nervous system to the relatively suspended activity of sleep. The accumulation of Fatigue Products in the brain, when it has exceeded the amount which causes maximal facilitation of the passage of nervous impulses, begins to retard the passage of impulses, and this retardation increases with the degree of accumulation. With continued wakefulness, as many observers have pointed out, the Threshold of Sensory Stimulation rises. A stronger stimulus than usual is required to traverse the clogged and overloaded channels, and consequently the environment, by exclusion of the countless slight fluctuating impres- sions which lend variety to our surroundings, becomes more and more monotonous, fewer and fewer "channels" of the brain are traversed by impulses, larger and larger areas become quiescent through lack of traversing stimuli, until finally sleep supervenes, and the whole of the brain except those portions, chiefly in the medulla, which are vital to the maintenance of the circulation and respiration, and some SLEEP 531 detached fragment which may be occupied in weaving dreams, has subsided into quiescence. It is the variety of our environment and the intensity of rapidly succeeding sensory impulses which keep us awake, by forming new "channels" which intersect with other channel-systems, i. e., arouse "associations" and keep up a continuous activity over the whole area of consciousness. If the Field of Consciousness is limited, either by fatigue or by the limitation of incoming sensory impressions, one group after another of channel-systems or interconnected memory- traces sink into quiescence until only the least fatigued or the most intensely stimulated channels are awake. When the stimulation is nowhere sufficient to rise above the threshold of consciousness, we have sleep, but where the stimulation is intense, and yet excessively circumscribed, we have the condition of Hypnosis. The extraordinary vividness of the impressions which are formed under hypnosis is due to the isolation of these impressions and to the fact that for the moment the brain is, for all effective purposes, limited to and circumscribed by the areas which are directly stimulated. 1 Inhibitive and conflicting impressions are temporarily in abeyance. The customary method by which we recollect past events is the Association of a present event with an incident which recalls the past. In other words a stimulus of the present moment happens to traverse a previously formed system of trace-deposits. If, however, only a small portion of the brain be active the chance of a subsequent impulse traversing it must obviously be less than when the area of stimulation is larger. The cutting off of sensory impressions in sleep and the diminution of the extent and variety of "canalization" or trace-for- mation throughout the upper portion of the central nervous system which accompanies sleep is therefore conducive to Amnesia or lack of ability to recollect the intellectual events which occur under these circumstances. This fact is well illustrated by phenomena which fre- quently attend the onset of sleep. A certain sequence of ideas arises in the consciousness we think of it, as we say, dreamily then suddenly this train of ideas vanishes and another takes its place, and we find that we cannot recollect the first. This amnesia is occasionally so surprising in itself that the wonder of it excites us to the extent of awakening. So the cessation of canalization in one trace-system leads, by the blocking off of impulses, to its cessation in an adjacent system, and amnesia spreads over a wider and wider area, until finally sleep supervenes. The fabric of intercommunicating trace-systems which constitutes the waking consciousness shows larger and larger rents of amnesia, the fragments of the fabric are less and less bound together, until at last the entire fabric seems to be blotted out, or one 1 The impressions received during hypnosis are usually separated from the waking impressions by a gap of amnesia, but during the actual period of hypnosis the extra- ordinary vividness of the impressions received is testified by the almost automatic response of the body to commands or suggestions which are received in this condition. 532 PROCESSES INFERRED FROM INDIRECT OBSERVATION shred may remain, as in a dream, to be faintly recalled or completely forgotten in awakening, according to whether or not our customary waking perceptions (traces) traverse the point of union of the dream- shred with the whole fabric of the reawakened consciousness. That the onset of sleep is in reality due to the accumulation of Fatigue-products which are washed out during the period of quiescence by the circulatory fluids, has been very strikingly demonstrated by Pieron. This observer has shown that if the blood-serum or Cerebro- spinal Fluid of a dog which has been kept awake for an abnormal period be injected directly into the fourth ventricle of the brain of a normal dog, even if this latter animal has recently slept, it falls at once into a profound slumber. The effect is much greater if cerebro- spinal fluid or the fluid from the ventricles of the brain is employed than if blood-serum be used. Frequently with blood-serum nothing more than a moderate somnolence is elicited, whereas when cerebrospinal fluid is employed the slumber which is induced may be so profound that the animal will remain asleep in any attitude in which he may be placed. Pieron has made many interesting observations upon the chemical nature of this sleep-inducing substance, or Hypnotoxin as he designates it. He finds that it is destroyed by heating to 65 C. and by oxidation, is precipitable or coagulable by alcohol and is non- diffusible. It is evidently, therefore, a colloidal substance of some complexity, and chemically unstable. THE FADING OF MEMORY-TRACES. It is a matter of common experience, and a fact which has been experimentally verified, that a person who has been deprived of sleep beyond the normal period of wakefulness does not require the full sum of the periods of sleep which he has lost in order completely to recover from his desire to sleep. We must therefore conclude that not only do Fatigue-products disappear from the brain during sleep but, furthermore, that they disappear the more rapidly the greater their concentration. We have seen that the initial effect of the fatigue-products of the cen- tral nervous tissues is to cause facilitation of the passage of nervous impulses and the formation of Memory-traces. The phenomenon of forgetting must therefore be essentially of the same nature as the phe- nomenon of refreshment by sleep, i. e., it must consist in (or depend upon) the disappearance of the products of their functional activity from certain nerve-tracts. Ebbinghaus has carried out a number of excessively painstaking investigations upon the rate at which meaningless syllables which have once been learned by heart are forgotten. Ebbinghaus was his own subject. Series, each consisting of thirteen meaningless syllables, were read and reread in such a manner that each syllable was presented to the senses for a period of 0.41 seconds at each repetition. When it was found just possible to completely recall the series correctly, the total time (= ti) consumed in memorizing the series was noted. FADING OF MEMORY-TRACES 533 After the lapse of certain definite periods of time the series were relearned, and the time (= t] -- t) necessary to relearn them was also noted. Then the difference (= t) represented the time saved by the previous repetitions, or in other words the time which would be consumed in learning that proportion of syllables which was remem- bered. The percentage - X 100 was employed by Ebbinghaus (and ti has been employed by his successors in this field of investigation) as the most convenient measure of the extent of forgetting. It is, of course, not actually equivalent to the amount of memorized material which has been forgotten, for the time required to memorize syllables is, as we have seen, not proportional to their number. Nevertheless the outline of the relationship between the time which has elapsed since the material was learned and the amount of material forgotten is sufficiently clearly revealed by the successive values of - - X 100 ti noted by Ebbinghaus to show that this phenomenon, like that of refreshment by sleep, occurs most rapidly in the beginning, when the mass of deposit undergoing destruction or dispersal is greatest. The following were the results obtained by Ebbinghaus the time being reckoned from the end of the first period of learning to the end of the second . Time in hours. - X 100 ti 0.33 41.8 1.00 . . 55.8 8.80 . . . . . . .... 64.2 24.00 66.3 48.00 72.2 144.00 74.6 744.00 78.9 The negative acceleration of this process is extraordinarily high, for although over 55 per cent, of the time saved by the first period of learning is lost in one hour, yet during the succeeding twenty-three hours only 9 per cent, more is lost. In other words the Velocity of Forgetting decreases very rapidly with the passage of time; it never, under normal conditions, undergoes any increase in rapidity with time. The process of forgetting is therefore essentially different in mechanism from the process of memory-formation. It is very improbable that the fading of a memory-trace can be due to chemical changes in the substance forming the trace, for no chemical reactions are known which diminish so greatly in rapidity with time, continue to proceed, and yet do not attain completion for such pro- longed periods as the memory-traces persist. A reaction which was 55 per cent, completed in one hour would either have ceased before twenty-four hours, or else would be much more than 66 per cent, com- pleted. A chemical reaction, to display such extraordinary falling-off 534 PROCESSES INFERRED FROM INDIRECT OBSERVATION in velocity with time, would have to be polymolecular, i. e., involve a large number of simultaneously reacting molecules, and polymolecular reactions do not actually occur, or rather they take place in successive monomolecular, bimolecular or trimolecular stages. Tim e in 6-46 hou r units FIG. 47. Curves illustrating the analogies between the fading of a memory trace, the extraction of protamine from spermatozoa by acid and the dissolution of dried casein by dilute alkali. If, however, we compare the curve of forgetting with the curve which expresses the rate of issuance of a colloid (or possibly of a crystalloid) from a colloidal into a fluid menstruum, we cannot fail to recognize FADING OF MEMORY-TRACES 535 at once their essential similarity. In the accompanying figure (Fig. 47) curve 1 represents the rate of issuance of potassium caseinate from suspended Casein particles into dilute potassium hydroxide solu- tion; 2 represents the rate of extraction of Protamine (salmin) from dried salmon-spermatozoa by dilute hydrochloric acid; and 3 represents the Curve of Forgetting, as illustrated by the results of Ebbinghaus cited above. Comparing these curves it is evident that by a suitable modification of parameters any one of them might be employed in place of the others to illustrate the processes which they severally depict, and that each of them represents the time-relations of a process in which the negative acceleration is so marked as to forbid its repre- sentation by any known chemical reaction-formula, or by the similar formulae which represent the diffusion of crystalloids in fluid media. It has been found that the issuance of a protein (and therefore, probably of other colloids) from a colloidal menstruum is governed primarily by Capillary Forces so that the time-relations of the washing-out process are similar to those exhibited in the rise of a fluid in a capil- lary tube or of a liquid in a column of sand or a strip of filter-paper. We may infer that the fading of a memory-trace is attributable to some similar phenomenon and may not improbably be due to the wash- ing out of a colloidal substance, which forms the memory-trace, by the circulating fluids. This would explain at once the rapidity of the initial stages of forgetting and the extraordinary persistence of the last traces of the memory-deposit, for complete extraction of a colloid from a colloidal menstruum by an external liquid is a matter, not of hours, but, as may be computed by exterpolation from actual measure- ments, may actually require the lapse of periods of time which are vastly in excess of the total duration of the life of man. REFERENCES. MEMORY: Maudsley: Body and Mind, London, 1873. The Pathology of Mind, London, 1879. Munk: Ueber die Funktionen der Gehirnrinde, Berlin, 1881. Exner: Pfliigei's Arch., 1882, 28, p. 487. Entwurf zu einer physiologischen Erklarung der psychischen Erscheinungen, Wien, 1894. Ebbinghaus: Ueber das Gedachtniss, Leipzig, 1885. Loeb and Koranyi: Pfliiger's Arch., 1890, 46, p. 101. Mosso: Arch. f. Anat. u. Physiol., Physiol. Abt., 1890, p. 129. Phil. Trans. Roy. Soc., London, 1892. 183, p. 299. Lepine: Revue de Medicine, 1894, p. 727. Duval: Compt. rend, de la Soc. Biol., 1895, p. 85. Smith: Psychological Review, 1896, 3, p. 21. Bryan and Harter: Ibid., 1897, 4, p. 27; 1899, 6, p. 345. Loeb: Comparative Physiology of the Brain and Comparative Psychology, New York, 190C. James: Principles of Psychology, London, 1901. Robertson: Arch ; Internat. de Physiol., 1908, 6, p. 388. Biochem. Zeit. Festband. f. H. J. Hamburger, 1908, p. 287. Folia Neurobiologica, 1912, 6, p. 553; 1913. 7, p. 309. Gobau: Ann. et Bull, de la Soc. de Med. de Gand., 1910, 76, No. 4. Pieron: L'Annee Psychologique, 1913, 19, p. 91. Principles of Psychology, London, 1901. 536 PROCESSES INFERRED FROM INDIRECT OBSERVATION HYPNOSIS: Bernheim: Suggestive Therapeutics, trans, by Herter, New York, 1888. Moll: Hypnotism, London, 1890. Sidis: The Psychology of Suggestion, New York, 1911. Prince: The Unconscious, New York, 1915. Ochorwicz: Hypnotisme, Richet's Dictionnaire de Physiologie, Paris. SLEEP: Sidis: An Experimental Study of Sleep, Boston, 1909. Manaceine: Sleep, its Physiology, Pathology, Hygiene and Psychology, London, 1912. Pier on: Le Pro bl erne Physiologique du Sommeil, Paris, 1913. FORGETTING: Ebbinghaus: Vide supra. Patrick and Gilbert: Psychological Review, 1896, 3, p. 469. Swift: Psychological Bulletin, 1906, 3, p. 185; 1910, 7, p. 17. Bean: Columbia Contributions to Philosophy and Psychology, 1912, 20, No. 3. Robertson: Folia Neurobiologica, 1914, 8, p. 485. RATE OF EXTRACTION OF COLLOIDS FROM COLLOIDAL MENSTRUA: Cameron and Bell: U. S. Dept. Agr., Bureau of Soils, Bull. No. 30. Jour. Physical Chem., 1906, 10, p. 658. Ostwald: Zeit. f. Chem. u, Ind. der Kolloide, 2d Supplement, 1908, 2, p. xx. Robertson: Jour. Phys. Chem., 19^0, 14, p. 377. Jour. Biol. Chem., 1913 14 p. 237. Pfluger's Arch., 1913, 152, p. 524. Robertson and Miyake: Jour. Biol. Chem., 1916, 25, p. 351; 1916, 26, p. 129. PART V. THE PRODUCTS OF TISSUE-ACTIVITY CHAPTER XXII. THE WASTE-PRODUCTS. THE CARBONACEOUS WASTE-PRODUCTS. The chief carbonaceous waste-product is, of course, Carbon Dioxide. Only a trifling proportion of the excretion cf carbon dioxide takes place through the urine, feces and sweat, the lungs playing the pre- ponderating part in accomplishing the elimination of this product. The total production of carbon dioxide in twenty-four hours varies with the quality and quantity of food ingested, with the quantity of muscular work performed, and with the rate or loss of heat from the body, but in an adult male doing moderate work it may be estimated in round numbers at four hundred liters at ordinary temperatures and atmospheric pressure. The carbon-dioxide output is derived from the oxidation of the carbon in the metabolized foodstuffs. It arises, therefore, in conse- quence of the absorption of oxygen by the tissues. Carbon dioxide is, however, not the only oxidation-product of cellular activities, and hence the carbon dioxide which is given off by an animal is rarely the molecular equivalent of the oxygen which is absorbed in the same period. The ratio: - , is termed the Respiratory Quotient O 2 absorbed and it varies in a very characteristic manner with the nature of the ingested foodstuffs. Thus the carbohydrates contain a -greater pro- portion of oxygen than any of the other foodstuffs, the oxygen being, in fact, molecularly equivalent to the hydrogen which they contain. The hydrogen in a carbohydrate may, therefore, be regarded as having been completely oxidized beforehand, and the carbohydrates behave, so far as the absorption of oxygen and evolution of carbon dioxide are concerned, as if they consisted of pure carbon and underwent the reaction : c + O 2 = co 2 53$ WASTE-PRODUCTS hence the respiratory quotient for the oxidation of pure carbohydrates is equal to unity. This probably represents the maximal value of the respiratory coefficient which may be obtained with normal animal tis- sues. Figures in excess of this which have occasionally been observed have been attributed by some observers to the formation in the tissues of fat from carbohdrates with the liberation of carbon dioxide: + O 2 = Ci6H 32 O 2 + 8CO 2 + 8H 2 O Glucose. Palmitic acid. Respiratory quotients in excess of unity have been observed in hiber- nating animals immediately prior to their winter-sleep, and in animals and birds fed with an enormous excess of carbohydrates. The respiratory quotient for the oxidation of Fats is necessarily much lower than it is for carbohydrates, since the fats do not contain more than about one-sixth of the oxygen which is required to convert the hydrogen which they contain into water. An important proportion of the absorbed oxygen is therefore excreted in the form of water, and the carbon dioxide which is discharged from the body falls very much short of the molecular equivalent of the oxygen absorbed, the respira- tory quotient for the ordinary dietary fats being 0.71. The Proteins contain about half the oxygen needed to oxidize their hydrogen and the respiratory quotient is intermediate between the value for carbohydrate and fats, namely 0.81. 'The respiratory quotient for Alcohol is lower even than for fats, namely 0.67. From these considerations it is evident that the value of the respira- tory coefficient must be capable of yielding important information as to the particular class of foodstuffs which is being utilized for the performance of a given function. Thus for man, under ordinary con- ditions of work and nourishment, the respiratory quotient lies between 0.8 and 0.9, but when hard Muscular Work is being performed it rises and may even approach the ideal value of unity for the oxidation of carbohydrates. Part of this rise, especially during the initial stages of a work-experiment, or in experiments occupying only a short period, may possibly be ascribed to the "washing out" of carbon dioxide accumulations from the tissues by the more rapid respiratory and cardiac movements. It must be recollected, however, that the rapidity of the respiratory movements in exercise is conditioned by the enhance- ment of the carbon-dioxide content of the blood, so that but a slight proportion of the increased carbon-dioxide output during exercise can justifiably be attributed to the increased ventilation of the body, and, furthermore, the effect of muscular work upon the respiratory quotient endures for a long period, until in fact, the carbohydrate-reserves have been so far depleted that we may surmise that other foodstuffs are now being utilized for the production of muscular energy. The rise of the respiratory quotient during the performance of muscular work therefore affords us confirmatory evidence of the view that muscular CARBONACEOUS WASTE-PRODUCTS 539 energy is derived, in the first place, from the oxidation of carbo- hydrates. On the other hand, in Starvation, the respiratory quotient falls to a value intermediate between that characteristic for the oxidation of fats and the value for proteins, for in starvation the carbohydrate reserves are quickly depleted, and thereafter the energy which is dis- sipated by the body is derived from the oxidation of the fat reserves and the tissue proteins. Extraordinarily low values of the respiratory quotient have occa- sionally been obtained with Hibernating Animals during their winter- sleep. Thus Pembrey obtained figures as low as 0.25 with hibernat- ing dormice. For hibernating bats Hari obtained higher figures, but even these values were generally less than the normal value for the oxidation of pure fats. The origin of these low values has been the subject of numerous surmises. It appears to be incontestable that they represent incomplete oxidations, which do not proceed so far as to result in the formation of carbon dioxide. A question much more difficult to decide, however, is whether the excess of oxygen intake over carbon-dioxide output in the winter-sleep is stored in the animal's tissues, or excreted in the form of compounds other than carbon dioxide. It was at first supposed that the oxygen excess was stored in the tissues in the form of partially oxidized foodstuffs, as, for example, carbohydrates derived from fats. It has been pointed out, however, that the total accumulation of oxygen throughout the duration of the winter-sleep would necessitate the production of a quantity of carbo- hydrate far in excess of the total carbohydrate-content of the animals under any conditions. It has been ascertained that the urine of hibernating animals contains notable quantities of products of incom- plete oxidation such as Lactic Acid, and it is probable that a con- siderable proportion of the excess of absorbed oxygen is excreted in the urine in these forms. Not only does the ratio of carbon-dioxide evolved, to oxygen ab- sorbed, rise during the performance of muscular exercise, but the total carbon-dioxide output increases in direct proportion to the work performed. This has been shown in a very striking manner by the experiments of Johansson who first measured his carbon-dioxide out- put per hour at rest and then during the performance of the Muscular Work involved in repeatedly lifting a weight. He found that his carbon- dioxide output rose to the value CO 2 = Np + q where " q" was the output at rest, "N" the number of times the weight was lifted and "p" the increase in output induced by lifting the weight once. The effect of the Temperature of the environment upon the carbon dioxide output is opposite in cold-blooded and warm-blooded animals. 540 WASTE-PRODUCTS In cold-blooded animals, in which the temperature of the tissues approx- imates to that of the environment, the rate of oxidations is increased, as might be expected, by a rise in external temperature, and the carbon-dioxide output is even more than proportionately increased, since the respiratory quotient generally undergoes a slight rise with temperature also. This is illustrated by the following experiments of C. J. Martin on the carbon dioxide output of the Australian Lizard Cyclodus gigas. COz output per kilogram Temperature of Temperature of and hour, the air. the animal. mg. 5 . . 5.5 13 9 9.2 42 15 15.2 53 20.5 20.4 55 25. .. 24.5 64 30. .... . . . . V . . . 29.3 78 35. -.-; v . . . 34.8 97 39. 38.5 292 The effect of rising temperature upon the carbon-dioxide output of warm-blooded animals is, within certain limits, the reverse of this. The Body-temperature of the warm-blooded animals varies but slightly with the temperature of the environment and this uniformity of tem- perature is secured by a number of cooperating factors, among which the radiation of heat from the surface of the body, the loss of heat by the latent heat of evaporation of perspiration, and the adjustment of the production of heat by the oxidations of the body to the need for heat to maintain the normal temperature of the tissues. The increase of metabolism which low temperatures induce in the warm-blooded animals is probably brought about, in part at least, by the stimulation of the skin by cold air inducing reflex movements, such as shivering or reflex alterations of muscular tone which necessitate an enhanced combustion of carbohydrates with the performance of a minimum of external work. The regulation of the temperature of the body between the normal "comfortable" temperature-limits of the environment is mainly brought about by the modification of the purely physical factors of radiation and evaporation which govern the rate of loss of heat from the body. Below the external temperature of 20 C. (68 R), however, the "chemical regulation" of the bodily temperature becomes an exceedingly important factor, the rate of metabolism rising continu- ously, and^considerably with falling temperature. Above 30 C.-35 C. (86 F.-95 F.) the effect of temperature upon the oxidations of the body vanes with the humidity of the air. The greater part of the heat-loss at these high temperatures is accomplished through the evaporation of perspiration, and if the humidity of the atmosphere be so great as to interfere with this method of heat-dissipation the regulatory mechan- isms of the body become inadequate, the bodily temperature rises and with it the rate of oxidation and the total output of heat, just as it NITROGENOUS WASTE-PRODUCTS 541 would in cold-blooded animals. It is to this that the exhausting effects of the Tropical Climate are to be referred. A temperature of 86 F. in an atmosphere saturated with moisture is almost unbearable, and physical work is, for Europeans at least, an impossibility, while a temperature of 110 F. in a perfectly dry atmosphere can be endured, and even a considerable amount of physical work performed, without any exceptional discomfort, by persons in normal health whose tem- perature-regulating mechanisms are in good order. Among the remaining carbonaceous waste-products under normal physiological conditions may be enumerated Methane which is derived from bacterial fermentations in the intestine but is exhaled mainly through the lungs. The quantity of methane produced by carnivora and animals which subsist upon a mixed diet, such as ourselves, is normally a very small proportion of the total carbon output, but in herbivora it may become very appreciable. Oxalic Acid is regularly found in normal urine in very small amounts, the normal excretion being about 0.02 grams in twenty-four hours. Its origin is unknown. As it is a frequent product of bacterial fermentations it may have an alimentary origin, and, again, the administration of sodium oxalate leads to the appearance of the unchanged oxalic acid in the urine, and a number of foodstuffs, particularly fruits and vegetables, contain oxalates which would therefore appear in the urine. On the other hand, the output of oxalic acid continues on a pure protein diet and, on a normal diet, is stated to be enhanced by the administration of con- siderable quantities of gelatin, so that we may conjecture that the urinary oxalic acid is in part produced by the metabolism of the tissues. The output of oxalic acid is also stated to be increased in diabetes. Lactic Acid is only found in the urine in partial asphyxia, or after the most extreme muscular exertion; its appearance in the urine indicates imperfect oxidation of carbohydrates or else extraordinarily excessive production by the muscular tissues. THE NITROGENOUS WASTE-PRODUCTS. Of the various nitrogenous waste-products, Urea: /NH 2 co is quantitatively the most important. The daily output of this sub- stance varies with the quantity of protein which is ingested, but for the adult man subsisting upon a mixed diet the daily excretion is about thirty grams, for a woman somewhat less. This corresponds to from 84 to 90 per cent, of the total nitrogenous output. The quantity of urea which is excreted varies directly with the quantity of protein ingested. We have seen in preceding chapters that animal tissues do not store up proteins and that their storage-capacity 542 WASTE-PRODUCTS for amino-acids is limited. The excess of amino-acids absorbed from the intestine is converted into urea by a series of steps which we are about to discuss, and this is excreted promptly in the urine. On the other hand the excretion of urea upon a diet low in proteins, but abundant in fats and carbohydrates, may actually be less than in star- vation, because the fats and carbohydrates spare the tissue-protein from destruction for the production of the energy which is dissipated by the body. The question of the region of the body in which urea originates has been the subject of a great many investigations. Since it is so promi- nent a constituent of urine, the kidneys naturally fall first under sus- picion of being the organs in which the manufacture of this material takes place. This possibility has been the subject of experimental inquiry by a number of investigators. If the kidneys produced urea to the extent of an important proportion of the total output, then excision of the kidneys should lead to the disappearance of urea from the body, or at any rate should not lead to its accumulation. If, however, the kidneys simply eliminate urea which is produced primarily by other organs, then excision of the kidneys should lead to the accumulation of urea in the organs and tissue fluids. This- is what actually occurs, and the accumulation of urea under these circumstances and in conditions involving inefficient excretion by the kidneys, as in Nephritis, has been repeatedly established. We must therefore look elsewhere than to the kidneys for the main source of the urea which they excrete. Front a variety of different experimental results we can definitely affirm that the Liver plays a very important role in the production of urea; whether it is the exclus- ive source of this substance or not cannot be regarded as definitely established, but a very large proportion of the total output originates in this organ. Thus if blood be perfused through the various organs in such a manner that the same blood passes without renewal through the tissues over and over again, no accumulation of urea in the blood is noted in the case of the kidneys or of muscular tissues, but a very pronounced accumulation occurs in the blood which is perfused through the liver. The portal vein, which carries the blood containing absorbed food- stuffs from the alimentary wall to the liver, runs parallel with, and very close to, the inferior vena cava. By making an incision in the adjoining sides of these veins and sewing the edges together, an oper- ation which is known as Eck's Fistula, the portal circulation is short- circuited and the blood from the intestine, with its load of food- products, no longer passes through the tissues of the liver. The liver is, however, still nourished by the circulation from the hepatic artery. Animals upon which this operation has been performed will survive for prolonged periods, and it was found by Pawlow and Nencki that in such animals the urea excretion is greatly diminished while the ammonia excretion is very considerably increased; in other words that ammonia NITROGENOUS WASTE-PRODUCTS 543 to a certain extent takes the place of urea in the urine of such animals. Confirmatory evidence is supplied by the effects of degenerative changes of the liver upon the urea output. In cirrhosis of the liver and in the liver-degeneration which is induced by Phosphorus -poisoning there is a decided diminution of the urea output, and a concurrent increase in the ammonia output. It is impossible to settle this question by extirpation of the liver in mammals, since they do not survive the operation for a sufficient period to permit observation of the excretory products. In birds however, this severe operation may be performed without immediately fatal results. The birds do not, it is true, survive the operation for more than about twenty-four hours, but the time during which they live is sufficient to enable us to ascertain the effect of the removal of the liver upon the excretory products. Unfortunately urea is not the normal end-product of protein catabolism in birds; its place being taken by Uric Acid, which forms from one-half to three-fourths of the total nitrogenous output. However, the uric acid which is excreted by birds is undoubtedly the physiological equivalent of urea. In fact when urea is administered to birds it is excreted in the form of uric acid, so that were the tissues of birds to form urea it would neverthe- less be excreted in this form. The effects of extirpation of the liver in geese were investigated by Minkowski, with the following results: Per cent, of total nitrogen in the form of: Uric acid. Ammonia. Before extirpation ........ 60 to 70 10 to 18 After extirpation 3 to 6 45 to 60 These results are decisive, and the origin of at least ninety per cent, of the uric-acid output in birds must be in the tissues of the liver. Taking all of these different experiments together, therefore, and recol- lecting that the uric-acid excretion of birds is the physiological equiva- lent of the urea output of mammals, we are justified in inferring that the liver is a predominant, if not the sole source of the urea output of mammals. Nevertheless some urea output continues in animals which have an Eck fistula, even when the hepatic artery is also ligated, so that blood is cut off altogether from the liver, and the output of urea is definitely increased under these circumstances by the subcutaneous administration of amino-acids. We can hardly doubt therefore that other tissues besides the liver possess the power of manufacturing urea, although the size and functional activity of the liver enable it to play a predominant role in this, as in other chemical phenomena in which it plays a part. The question which next arises is that of the chemical origin or pre- cursor of urea. A direct origin from Arginine is immediately suggested by mere inspection of the structural formula of this amino-acid : /NH 2 NH = C< \NH.CH 2 .CH 2 .CH 2 CH(NH 2 ) COOH 544 WASTE^PRODUCTS and since the discovery by Kossel and Dakin of the existence of an enzyme, Arginase in aqueous extracts of the liver, spleen, thymus and intestinal mucosa which directly splits arginine with the production of urea, and Ornithine : /NH 2 HN = C< + H 2 X NH.CH 2 .CH 2 .CH 2 .CH(NH 2 ) .COOH Arginine. -/NHj CO + CH 2 .(NH 2 )CH2.CH2.CH(NH 2 ).COOH Urea. Ornithine. there can be no doubt that a proportion of the urea output originates in this manner. It can only be a small proportion, however, since urea forms over eighty per cent, of the total nitrogen output and only a very small percentage of the nitrogen intake is in the form of arginine radicals. The origin of the greater part of the urea output is undoubtedly to be traced to Ammonia formed by deaminization from the various amino- acids. We have seen that the decrease of urea output which accom- panies interference with the liver-functions also results in a correspond- ing increase of the ammonia output in the urine, and this fact in itself would point to ammonia as a precursor of urea. It can, however, be directly shown that when ammonia in the form of Ammonium Carbonate is supplied to the liver, it is transformed therein into urea. Thus Nencki and Pawlow have shown that the percentage of ammonia con- tained in the blood from the portal vein is considerably higher than it is in the blood from the hepatic vein, showing that the ammonia is retained by the liver as the portal blood passes through it. Further- more, when ammonium carbonate is administered to animals it appears in the urine as urea, and, finally, von Schroeder perfused the isolated liver of the dog with ammonium carbonate and obtained, not only the retention of ammonia observed by Nencki and Pawlow, but also an actual replacement of the perfused ammonium carbonate in part by urea. Ammonium Formate was similarly transformed. The conversion of ammonium salts into urea by the tissues of the liver has therefore been confirmed in a variety of ways. Urea is the diamide of carbonic acid and may be derived from carbonic acid by the successive introduction of amino-groups, an inter- mediate stage of the process being the formation of Carbamic Acid: /O.NH 4 /NH 2 c=o - c=o > c=o \).NH 4 \NH 2 H 2 O H 2 O Ammonium carbonate. Ammonium carbamate. Urea. NITROGENOUS WASTE-PRODUCTS 545 Now it has been shown by Macleod and Haskins that there is an equilibrium in aqueous solutions between ammonium carbonate and Ammonium Carbamate, so that if the ammonium carbamate is removed by transformation into urea a continuous renewal of the ammonium carbamate is to be expected, and consequently a quantitative conversion of the ammonium carbonate into urea. The formation of ammonium carbamate as an intermediate product in the synthesis of urea in the body is shown by the fact that if alkalies be administered to animals in considerable quantity carbamates appear in abundance in the urine. A direct conversion of ammonium carbamate into urea has been accomplished by Drechsel by simply passing an alternating current through its solution, i. e., by alternate oxidation and reduction which is, of course, equivalent to dehydration. We may infer, summing up the results of these various investigations, that ammonia, derived from amino-acids by the process of deaminization, is converted by union with carbon dioxide into ammonium carbonate, which spontaneously undergoes partial transformation into ammonium carbamate. The latter substance is converted by alternate oxidation and reduction in the liver into urea which is subsequently expelled from the body by the kidneys. In Acidosis, whether induced by disordered metabolism or by the ingestion of acids in excess, this process is impeded and the ammonia is utilized in part to neutralize the excess of acids in the blood and tissues. The output of Ammonia in the urine, therefore, rises in acidosis and is, in fact, a most valuable means of detecting and esti- mating the severity of that condition. Next to urea, but as a rule far inferior to it in amount, the most abundant nitrogenous constituent of the urine is Creatinine: /NH CO HN = C< X N(CH 3 ).CH 2 this substance may be regarded as an anhydride of Creatine, or methyl guanidine acetic acid: X NH 2 HN = c< X N(CH 3 ).CH 2 COOH which, it will be remembered, is an abundant constituent of muscular tissues. The daily output of creatinine in man is from 1.0 to 1.7 grams or from four to six per cent, of the total nitrogenous excretion. Our views regarding the probable origin of creatinine have undergone very important modifications in recent years, thanks to the fundamental investigations of Folin, Van Hoogenhuyze and Verploegh, and Mel- lanby. It was formerly assumed without any doubt that the source of the creatinine in the urine was the creatine in the muscular tissues. This must now be considered to have become uncertain, and in any 35 546 WASTE-PRODUCTS case we have come to attach a very fundamental significance to the creatinine excretion in the urine. It was first pointed out by Folin that with varying nitrogenous intakes the behavior of the creatinine output is fundamentally different from that of the output of urea. The latter rises and falls almost in direct proportionality to the quantity of protein in the food. The creatinine output, on the contrary, remains almost unaltered whether the protein content of the diet be high or low. The creatinine output is not, therefore, derived from the diet. Thus, for example, Folin compared the urea and creatinine excretipn on a high protein diet and a low protein diet, with the following very striking results: High protein diet. Low protein diet. Volume of urine 1170. c.c. 385. c.c. Total nitrogen . ... . 16. 80 grams 3. 6 grams Urea-nitrogen . . . . . . . 14.70 " 2.2 Creatinine-nitrogen .... 0.58 " 0.6 The urea output, it will be seen, fell on the low protein diet to one- sixth of that obtained on the high protein diet. The creatinine output, on the contrary, remained almost unaltered. The statement that the creatinine which is excreted in the urine is not derived directly from the foodstuffs must be qualified to this extent, that if creatinine be contained preformed in the diet, the greater part of it is excreted in the urine unaltered within twenty-four hours. On the other hand, if creatine be administered with the food it does not appear in the urine either in the form of creatine or creatinine. In fact it usually appears to be excreted by some other channel or else retained by the body, for Folin in many instances administered crea- tine without causing any increase even in the total nitrogen of the urine. It has been suggested by Mellanby that bacteria in the intestine decompose the creatine and retain it in their tissues. However this may be, these observations render it certain that the creatine which is contained preformed in a meat-diet is not the source of the creatinine in the urine. Since the output of creatinine is so extraordinarily independent of fluctuations in the diet, Folin regards it as originating in the Endog- enous Metabolism of the tissues themselves, while a great part of the urea arises from the destruction by deaminization of amino-acids which have never become part of the living protoplasm of the body, and therefore represents a product of Exogenous Metabolism. The exogenous metabolism rises and falls with the intake of foodstuffs, but the endogenous metabolism persists practically unchanged under a variety of nutritional conditions. It represents the "wear and tear" or irreversible spontaneous decomposition of the tissues. It is questionable, however, whether the creatinine output represents the endogenous metabolism of the whole body or whether it does not, on the contrary, arise from the endogenous metabolism of the muscular NITROGENOUS WASTE-PRODUCTS 547 tissues only. The daily output of creatinine, although so constant in a given individual, varies in different individuals with the weight, and more especially with the degree of muscular development. Obese persons, notwithstanding their high body-weight, have a low creatinine output, wliile comparatively lean persons, who by virtue of muscular development have a like weight, exhibit a high creatinine output. It is true that muscular work on a normal diet does not increase the creatinine output, but then we have seen that on a normal mixed diet the muscles do not derive their energy from the metabolism of their own substance (protein) but from the oxidation of carbohydrates. When, however, muscular work is performed during starvation, the creatinine output is definitely increased. In other words the actual destruction of muscular tissue results in an increase of creatinine excretion. It appears very probable that the normal products of the disinte- gration of tissue-protein are similar to or identical with the substrates out of which tissue-protein is synthesized, namely, the amino-acids, for we have seen that the process of tissue-synthesis is a balanced reaction which is retarded by its products, and this can only be true if the products of the synthesis break down, in the first place, into the substances which form the substrates of the forward reaction. The amino-acids which are thus set free are cast into the general stock of circulatory and storage amino-acids, undergo their share of exogenous metabolism or deaminization, and participate with the ingested amino- acids arising from the foodstuffs in determining the Nutrient-level of the tissue-fluids. If the nutrient-level falls, as in starvation, the amino- acids of tissue origin form a large proportion of the whole mass of cir- culating amino-acids, and their deaminization results in a continual drainage which, in turn, results in a steady loss of tissue-substance. There must, in fact, be an endogenous or tissue-source of urea, for otherwise urea excretion would ultimately fall to zero in starvation, which it never does. In fact, even in starvation the urea output still exceeds very decidedly the creatinine output. On the other hand, if the tissues must use their own substance for the performance of external work, at any rate in muscular tissues, the breakdown of the protein or of amino-acids resulting therefrom takes another course, with the production of creatinine. The effect of this must be to initiate a process analogous to repair or Regeneration by the resynthesis of the lost tissue-proteins from amino-acids. Creatine is not a normal constituent of the urine of adult men and, as has been stated above, the ingestion of creatine leads to no increase in the creatinine output, nor does it lead to the appearance of any. creatine in the urine. In the urine of women, on the contrary, creatine is found during menstruation and after delivery, and the ingestion of creatine leads to the appearance of a small proportion of the creatine in the urine. In the urine of children creatine is a regular constituent. According to Krause it disappears from the urine of boys at about five 548 WASTE-PRODUCTS or six years of age, but persists in the urine of girls until puberty. The ingestion of creatine in children is also followed by an increase in the creatine output in the urine. The adult has therefore acquired a power of destroying or utilizing creatine which is imperfect in women and only slightly developed in young children. Apart from the question of the nature of the tissues in which creatine and creatinine originate we have to consider the problem of the chemi- cal precursors or parent-substances from which they originate. A very obvious possibility is that they may arise from Arginine. /NH 2 HN = C \NH.CH2.CH 2 .CH 2 .CHNH 2 .COOH by breaking the hydrocarbon-chain and methylation of one of the nitro- gens in the guanidine nucleus. It has been stated that creatine may arise from proteins in the autolytic decomposition of tissues in the absence of bacteria but no other evidence of its formation from arginine has yet been adduced. Creatinine is a reducing agent and decolorizes cupric hydroxide in alkaline solutions, but does not precipitate cuprous oxide as the reduc- ing sugars do. It is precipitated by Picric Acid, but if treated with picric acid in alkaline solutions it yields a red coloration which turns yellow upon the addition of acids (Jaffe's Reaction). If an alkaline solution is treated with Sodium Nitroprusside the mixture turns ruby red (Weyl's Reaction) and then yellow. If this yellow solution is treated with excess of acetic acid and boiled, it becomes first green and then blue (Salkowskfs Reaction). JafiVs reaction is utilized by Folin for the colorimetric estimation of creatinine in urine. Creatine is esti- mated by converting it into creatinine by boiling with dilute acid and then reestimating the creatinine. Uric Acid is an exceedingly important constituent of the urine, since it represents, in man, the end-product of the purine metabolism. The average output per day on a mixed diet is 0.7 grams, and the ratio of uric acid to urea varies between 1 : 50 and 1 : 70. Uric acid is derived from the Purine Bases by oxidation; it is 2, 6, 8, trioxypurine : HN CO I I i i OC C NH HN NH/ It may be prepared synthetically from urea and glycocoll. On heating in sealed tubes with hydrochloric acid, glycocoll, carbon dioxide and ammonia are produced. It is capable of acting as a weak acid and forms two series of salts, the Monourates, containing one, and NITROGENOUS WASTE-PRODUCTS 549 the Diurates, containing two molecules of base. The so-called quadri- urates are non-existent. Uric acid yields a variety of characteristic color reactions, among which the Murexide Test, already described in connection with the purine bases, must be included. Uric acid is a reducing agent and reduces an alkaline cupric hydroxide solution; the quantity of uric acid which is present in urine is, however, insufficient to produce an appreciable precipitation of cuprous oxide. If a drop of uric acid dis- solved in sodium carbonate be placed upon a filter-paper moistened with silver nitrate solution, reduction occurs with the production of a yellow or brown spot (Schiff's Reaction). If a weak alkaline solution of uric acid in water is treated with a soluble zinc salt a white precipitate is produced which gradually turns blue if exposed to light and air, or immediately, if treated with sodium persulphate (Ganassini's Reaction). With a certain mixture of phosphoric and phosphotungstic acids uric acid yields a blue coloration (Folin and Macallum's Reaction), the origin of which is unknown. The elimination of uric acid is definitely increased by a diet which contains excess of purines or of Nucleic Acids. This is due to the fact that the adenine and guanine, split off from the nucleic acids, are transformed in the tissues into Hypcxanthine and Xanthine, by the deaminizing enzymes adenase and guanase. The hypoxanthine is sub- sequently converted into xanthine and the xanthine into uric acid by a specific oxidizing enxyme which is found in a variety of animal tissues, and is designated Xanthine-oxidase : HN CO HN CO HN CO ! I I I II HC C NH\ OC C NH\ OC C NH, J| || \ CH + O -> | || VJH + O -* | || >CO N _C_ N + HN C N * HN C NH/ Hypoxanthine. Xanthine. Uric acid. Nevertheless, the elimination of uric acid continues on a purine or nuclein-free diet. In a series of experiments on himself and others, Folin was able to reduce the daily elimination to 0.3 grams on a diet of cream and starch, but this minimum could not be reduced. Evi- dently, therefore, there is, as in the case of amino-acids and other foodstuffs, an Endogenous Metabolism of purines as contrasted with an Exogenous Metabolism. That the endogenous metabolism represents the actual breaking down of tissues is shown by the fact that if destruc- tion of tissue is remarkably augmented, as in pneumonia, leukemia, or in severe burns, the uric acid excretion rises decisively. There is no evidence that mammalian tissues can synthesize uric acid from any other source than purines. It is true that the elimination of uric acid, and of purine bases also, is increased by an increase in the dietary intake, but this is true whether the increase be nitrogenous or non-nitrogenous, and it follows very rapidly upon the intake of food. Time 10 to 11 ...... Urea, grains. . ."1.07 11 to 12 12 to 1 . . 1.13 . 1 . 07 1 to 2 (meai at 1.30) 2to 3 . 0.64 . 1.12 3to 4 4to 5 . . . . ' 5 to 6 .... . . 1.16 . . 0.84 . . 1.16 6 to 7 . ... . . 1.20 7 to . 8 '. 8 to 9 9 to 10 10 to 11 . . 1.37 . . 1.47 . 1.33 1.33 550 WASTE-PRODUCTS Thus Hopkins and Hope, after fasting for six hours, consumed a meal of bread and potatoes, pratically purine-free, with the following results: Uric acid, milligrams. 26 27 24 21 22 38 40 56 39 30 33 24 23 Thus a slight rise in the urea output occurred about six hours after the ingestion of the food, and continued for some time, but a sharp rise in the uric acid output occurred within two hours, and the excre- tion fell tc nearly the normal value again before the urea excretion began to rise. It is not known where this uric acid originates, but it would appear to be manifestly connected with the activities of the alimentary canal,. and to be endogenous in origin. It is for this reason that the uric acid and purine output is greater during the day than it is at night. In birds and .reptiles the relationships are quite different. These possess the power of synthesizing uric acid, most probably from Ammonia and Lactic Acid, since, if the liver be extirpated in birds, the place of the uric acid in the excreta is taken by ammonia, and large amounts of lactic acid are excreted concurrently. An increase of uric acid elimination in birds follows the administration of lactic acid and other hydroxy-acids and dibasic acids of the aliphatic series. This power is, however, lacking in the mammalia. In the majority of mammals, uric acid is not the end-product of the purine metabolism, but undergoes in part or almost wholly, trans- formation into Allantoin which is excreted in the urine: HN co H 2 N oc + H 2 + O = HN .This transformation, which is known as Uricolysis, is brought about by an oxidizing enzyme, Uncase, which occurs in tissue-extracts prepared from the liver, kidney and other organs. It trantforms uric acid almost quantitatively into allantoin. It is probable, however, that the destruc- tion of uric acid does not stop at this stage but proceeds further and, ultimately, to the formation of urea and other products. Thus Ascoli NITROGENOUS WASTE-PRODUCTS 551 and Izar have shown that if an extract of liver which has completely destroyed a given sample of uric acid in the presence of oxygen be excluded from oxygen, the uric acid is gradually reformed. This is what one would expect if we had here to deal with a reversible oxidation. The curious feature of their results is, however, that the addition of allantoin had no effect upon the production of uric acid, appearing to indicate that the production of allantoin was not an intermediate step in the resynthesis. The power of uricolysis is absent from the tissues of man and the chimpanzee a fact which would have gladdened the heart of Huxley, could he but have known it. All other mammals, so far as we know, contain uricase in their tissues. The following results, cited after Hunter and Givens, show the relative proportions of uric acid and allantoin in the urine of various mammals. The "Uritolytic Index" is the proportion, expressed as a percentage of uric acid, which has been converted by the animal into allantoin. Orders and species. Marsupialia: Opossum . Rodentia: Rabbit Guinea-pig Rat . . Ungulata: Sheep . Goat . Cow . Horse . Pig. . Carnivora : Raccoon Badger Dog . Coyote Primates : Monkey . Chimpanzee Man , Total purine nitrogen, gms. . 0.04 0.2 to 0.6 1.0 8.0 1.6 0.3 . 0.25 O.ltoO.3 . 0.15 . 0.045 ... . 0.2 Percentage of purine-allantoin- nitrogen. Allantoin. Uric acid. 76.0 19.0 91.0 93.7 64.0 81.0 92.1 88.0 92.3 92.6 96.9 97.1 95.6 66.0 2.0 6.0 3.7 16.0 7.0 7.3 12.0 1.8 5.4 1.9 1.9 2.6 8.0 90.0 Bases. 6.0 3.0 2.7 20.0 12.0 0.7 0.5 5.8 2.0 1.2 1.3 1.8 26.0 8.0 Uricolytic index. 79 95 94 96 80 92 93 88 98 95 98 98 97 89 2 Allantoin has been isolated by Hunter from the blood of the ox, pig, horse and sheep, but could not be detected in the blood of man. It is not by any means certain, however, notwithstanding the inability to convert uric acid into allantoin, that the tissues of man cannot destroy uric acid in some other manner. Thus, Taylor and Rose fed a human subject for three days on a diet very low in purines, namely milk, eggs, starch, and sugar. For three days following, a part of the protein, namely three grams per day out of a total of ten was given in the form of "sweetbread" nitrogen (sheep's pancreas). For four days succeeding this twice as much "sweetbread" nitrogen was given, namely six out of ten grams, and this was succeeded by a 552 WASTE-PRODUCTS period of four additional days on a purine-free diet. The following were the results obtained: 1st period, 4th period, purine-free diet. 2d period. 3d period. purine-free diet. Total urinary N . . . 8.9 8.7 9.1 8.80 UreaN + NH 3 . . . 7.3 7.1 7.1 7.05 CreatinineN . . . 0.58 0.55 0.56 0.47 Purine N (total) . . 0.11 0.17 0.26 0.10 Uric acid 0.09 0.14 0.24 0.07 Undetermined N . . 0.91 0.88 1.18 1.18 The intake of purine nitrogen in the second period was 0.17 and in the third 0.34 grams per day, so that the increased output only accounted for one-half of the intake. The purine was not simply stored, to be excreted later, for as soon as the purine-rich diet ceased the excretion fell to the figure previously obtained on a purine-free diet. The only alternatives that remain are either that part of the purine was never absorbed from the intestine or else that the tissues of the subject destroyed the purines in some manner which did not result in the formation of uric acid or allantoin. We may recall the observa- tions of Ascoli and Izar, cited above, which tend also to the conclusion that there are means of destroying uric acid in the tissues which do not involve the production of allantoin as an intermediate stage. In persons afflicted with Gout deposits of uric acid form in various tissues and particularly in the joints. The origin of these deposits has been the subject of much investigation. There is a definite increase in the uric-acid content of the blood in such persons, although the uric- acid output in the urine is not above the normal. Evidently, there- fore, the kidneys are functioning abnormally and in such a way as to constitute a barrier to the excretion of uric acid. The limiting con- centration in the blood at which transmittal through the renal epi- thelium begins is raised, and hence the uric acid, dammed back in the blood, accumulates therein. This alone, however, is not a sufficient cause of gout, for uricemia occurs also in nephritis, and in lead poison- ing, without the production of gouty deposits. It has been suggested that the solubility of uric acid in the blood is diminished in gouty persons, but no positive evidence of this has been advanced. The origin of the tendency of uric-acid deposits to form in the joints when they do occur at all is, however, rendered clear by the fact upon which emphasis is laid by Taylor, that Cartilage, possibly owing to its high content of sodium salts, diminishes the solubility of sodium urate in water, so that deposits are precipitated upon it from saturated solu- tions. The solubilities of the monourates of potassium, sodium and ammo- nium at 37 C. in water have been determined by Gudzent as follows: Salt of uric acid. Solubility in grams per liter. Potassium t t 2.7002 Sodium 1.5043 Ammonium 0.7413 NITROGENOUS WASTE-PRODUCTS 553 The solubility of sodium urate in blood is, however, no less than three times its solubility in water (Taylor). This is not due to the formation of diurates, since at the reaction of the blood diurates cannot exist. The nature of the factor which so greatly increases the solubility of uric acid is unknown. It was formerly considered possible to remove uric acid from the body by administering Alkalies, the assumption being that the greater alkalinity of the blood resulted in the formation of the more soluble diurates. We now know that the alkalinity of the blood is only increased to an almost imperceptible extent by this means and that the maximum alkalinity attainable would not suffice to form diurates, or indeed to influence perceptibly the solubility of uric acid. Nevertheless, the administration of certain alkalies may be assumed to facilitate the solution of uric acid by the formation of a certain proportion of the more soluble potassium salt, or of the Lithium Urate which is the most soluble salt of uric acid. The most remarkable effect upon the elimination of uric acid is, however, that of phenylquinoline-carbonic acid or Atophan: CH c COOH \ CH C CH 6 v CH N The administration of this substance and of other quinoline-carbonic acid derivatives has been shown by Nicolaier to increase the amount of uric acid excreted by the kidneys to an extraordinary extent, even to twice or three times the normal amount. No other physiological effects are noted and no other constituent of the urine is altered in amount. The increased elimination occurs on a purine-free diet and has been shown by Folin and Lyman to be accompanied by a fall in the uric acid content of the blood. In other words the hyperexcretion of uric acid is due to the increased permeability of the kidneys for this substance, just as the glycosuria following phloridzin administration is due to increased permeability of the kidneys for glucose. The hyperexcretion does not persist if the administration be continued, the daily output sinking within a few days to only slightly above the normal level, probably because the available supply of urates in the blood and tissue-fluids has become exhausted. There is, however, a continuous slight hyperexcretion throughout a prolonged period of administration, and when nuclear tissues are administered in the diet a greater proportion of uric acid is excreted in consequence than is usually the case. The formation of uric acid from the nucleic acids is thus facilitated by atophan, but this effect is probably only a secondary 554 WASTE-PRODUCTS one, depending upon the reduction of the concentration of the urates in the tissue-fluids, and the tendency of the tissue-enzymes to spon- taneously reestablish the normal equilibrium between the blood and the tissues. The only amino-acid which normally occurs in urine is Glycocoll, or amino-acetic acid, which, in very small amounts, appears to be a constant constituent. If, however, an excess of leucine or alanine be introduced into the circulation they will appear in the urine. It would appear that, normally, deaminization and utilization are too rapid to permit of the accumulation of amino- acids in the blood in sufficient amount to cause elimination by the kidneys. If, however, the rate of deaminization be slowed, as, for instance, in degenerative changes of the liver induced by chloroform-necrosis or phosphorus-poisoning, then a variety of amino-acids may appear in the urine. It is also stated by Loewy that the amino-acid content of the urine is increased at high altitudes. When the urea, creatinine, uric acid and glycocoll of the urine are added together, there is always a considerable remainder of nitrog- enous excretion. Part of this arises from the sulphur-containing and conjugated excreta which are about to be described, part is stated by Abderhalden and Pregl to be present in the form of Polypeptides which yield glycocoll, leucine, alanine, glutamic acid and phenylalanine on hydrolysis. When all the nitrogen in hitherto defined substances is summed up, however, there is still a small remainder which, although it arises from substances excreted in small amount, may nevertheless be of physiological importance. It is derived in part from exogenous and in part from endogenous metabolism. CONJUGATED EXCRETA. A variety of substances occur in the urine which arise from the union of a genuine excretory product with another molecule which serves as a vehicle to accomplish its elimination. Such excreta are, for example, the Conjugated Glucuronic Acids which are normally pres- ent in the urine in small amounts and are greatly augmented by the ingestion of certain poisons, of which a partial list has been given in a previous chapter (Chapter III). The function of the glucuronic acid moiety of the molecule appears to be in the main to render harmless the associated substance which is usually of a toxic character. Only definite classes of toxic substances are eliminated in this manner, however. The Glucuronates which normally occur in the urine are in the main the phenyl, indoxyl and skatoxyl glucuronates, the latter two in very small amounts, The phenol, indoxyl and skatoxyl radicals are derived, it is believed, mainly from putrefactive decomposition of aromatic amino-acids, particularly tyrosine and tryptophane, by the intestinal bacteria. These substances are in themselves very toxic, but their CONJUGATED EXCRETA 555 conjugates with glucuronic acid are harmless. Upon boiling with dilute acids or occasionally even on allowing urine to stand, they decompose, setting free glucuronic acid and the associated radical of the conjugate. The origin of the glucuronic acid in urine is unknown. The most natural assumption is to suppose that the toxic substances which are eliminated in this way combine in the body with glucose, and that the oxidation of glucose is by this so hindered, that it only proceeds as far as the conversion of the primary alcohol-group into a carboxyl- group. Certainly the phenyl-glucuronic acid is a compound of the glucoside type, i. e., the phenyl radical is attached to the glucu- ronic acid by the aldehyde-group. On the other hand if camphor be administered in large amounts to phloridzinized dogs, although the excretion of glucuronates is very greatly increased thereby, the excre- tion of glucose is either not diminished at all or only slightly diminished, a fact which would appear to indicate some other source than glucose for the glucuronic acid. A very important excretory conjugate is the conjugated sulphuric acid, indoxyl-sulphuric acid or Indican: C.O.SOzOH /\ -'- C 6 H 4 CH NH which yields Indigo when treated with oxidizing agents. This substance arises by conjugation of indoxyl with sulphuric acid and is the form in which the greater part of the indoxyl output is present in the urine. The indoxyl output varies with the extent of putrefactive processes in the intestine. Any measure of Intestinal Stasis, such as that induced by tying off a loop of small intestine, results in an increase of the indican output. The subcutaneous injection of indol leads to an increased output of indican, while the administration of an excess of Tryptophane in this way does not. Evidently the tissues do not decom- pose tryptophane in such a way as to liberate indole, while the intes- tinal bacteria, like the majority of putrefactive bacteria, generate a large proportion of indole from tryptophane, which, after absorption is oxidized to indoxyl and then excreted in the form indicated above. It must be remembered that the indican output, although generally running parallel with the degree of intestinal stasis or putrefaction, is not a reliable measure of intestinal putrefaction when taken by itself, for the output depends, not solely upon putrefaction, but also upon the proportion of tryptophane which is contained in the proteins of the diet. Thus, if a large part of the protein intake be supplied by Gelatin, which contains no tryptophane, the indican output becomes very small although putrefactive processes may not be diminished in 556 WASTE-PRODUCTS the slightest degree. Then, again, even upon a standard diet, the out- put of indican may be expected to vary greatly with the type of infect- ing organisms in the intestine. Thus Herter has shown that Bacillus coli communis produces indole but only traces of skatole, which is the methyl derivative of indole, while certain anaerobic putrefactive bacteria produce skatole, in preference to indole, from tryptophane. Skatol does not appear to be normally excreted in the urine, at least in the form of a conjugated sulphuric acid. Phenol-sulphuric Acid and Cresol-sulphuric Acid are constant con- stituents of urine, and, as in the case of indican, the output is obviously derived from the products of intestinal putrefaction. It is probable that these substances, of which the total excretion may amount to fifty milligrams per day, originate from the putrefactive decomposi- tion of Tyrosine and Phenylalanine. In general it may be said that while aliphatic alcohols, terpenes and many phenols are excreted in the urine in conjugation with glucuronic acid, the greater part of the phenols and polyphenols are excreted in conjugation with sulphuric acid. Yet a third vehicle of excretion is that afforded by conjugation with Glycocoll, or ammo-acetic acid. Thus Benzole Acid, appears in the urine after administration in the form 9f the conjugated Hippuric Acid : C 6 H 6 COOH + CftNHzCOOH = C6H 5 COHNCH 2 COOH + H 2 O Benzole acid. Glycocoll. Hippuric acid. Hippuric acid is a very abundant constituent of the urine in Herbivora, comparatively scanty in the urine of Carnivora, and inter- mediate in amount in the urine of partially herbivorous animals like ourselves. The daily excretion in man, subsisting upon a normal mixed diet, is about 0.7 grams, but after eating quantities of vegeta- bles or fruits it may rise as high as 2 grams. The synthesis of hippuric acid from benzoic acid and glycocoll is accomplished within the tissues of the kidneys themselves. This, in fact, was the first synthetic process which was definitely shown to take place in animal tissues (by Schmiedeberg and Bunge) and also the first to be performed by admixture of the components of the reaction with macerated tissue. It is not improbable, however, that some measure of hippuric acid synthesis may also occur in other organs. When large amounts of benzoic acid are administered to animals the elimination of glycocoll is far in excess of the glycocoll which could be obtained by simple hydrolysis of the protein. " Thus McCollum and Hoagland brought a pig into a condition of minimal nitrogen metab- olism by administering a diet of starch containing 75 calories per kilogram body-weight. To this diet was then added varying amounts of benzoic acid, and finally hydrochloric acid and benzoic acid were given together. The total nitrogenous output and its partition among the various nitrogenous fractions in the urine were determined ifr the different periods of the experiment with the following results: CONJUGATED EXCRETA 557 Period. No. of days. Food. Total N. Urea N. NH 3 N. Creatinine N. Hippuric acid + other N. I 12 Starch, 75 cal. per kilo + 2.56 1.43 0.21 0.488 0.424 alkali salts II 4 Same + 4 g. benzoic acid 2.63 1.29 0.21 0.456 0.681 III 7 Same + 1 g. benzoic acid 2.23 0.58 0.22 0.484 0.948 IV 5 Same + 16g. benzoic acid 2.86 0.55 0.38 0.437 1.492 V 5 Same + 16 g. benzoic acid 4.03 0.54 1.44 0.424 1.632 + 10 g. of 25 per cent. HC1 It will be seen that despite the great increase of hippuric acid excre- tion induced by these large dosages of benzoic acid the total daily nitrogen elimination was unaffected. Evidently body-protein was not attacked to provide the glycocoll needful for the synthesis of the hip- puric acid. The glycocoll was evidently derived at the expense of the urea-fraction, and the endogenous catabolism, in so far as it is repre- sented by the creatinine output, remains unaffected. On the other hand the acidosis induced by hydrochloric acid resulted in a large increase of the total nitrogen output, the chief part of the increase being Ammonia which performs the protective function of neutralizing a part of the excess of acid. The urea and creatinine output were alike unaffected by the administration of the acid. The glycocoll moiety of hippuric acid must therefore be traced to the same origin as urea, and this, it will be remembered, is the amino- acids of the tissue-fluids. No less than thirty-five per cent, of the nitrogen of the food may be excreted as hippuric acid, and no protein contains this percentage of glycocoll. It is evident that glycocoll may be synthesized from other amino-acids. It might be imagined that the benzoic acid unites with other amino-acids which thereafter under- go partial oxidation until only the residue of glycocoll is left. Injec- tion of such compounds synthetically prepared, however, leads to no increase in the hippuric acid output. It seems probable, therefore, that glycocoll may form a normal disintegration-product of many amino-acids, that under ordinary circumstances it is finally deaminized, but that when toxic substances that will pair with it, namely aromatic acids, are present in the tissue-fluids, deaminization is prevented by the conjugation. The power of the tissues to synthesize glycocoll is of very great importance, since it not only enables the body to protect itself against such poisons as benzoic acid, but also enables suckling animals to synthesize their tissue-proteins from a protein which is totally lacking in glycocoll, namely the casein of milk. When the administration of benzoic acid is pushed beyond the limit of the glycocoll available from the proteins of the diet the protec- tive mechanism breaks down and free benzoic acid appears in the urine. Under no circumstances, it appears, are tissue-proteins attacked 558 WASTE-PRODUCTS for this purpose nor are the proteins of the blood broken down to fur- nish glycocoll, for the ratio of albumins (containing no glycocoll) to globulins in the blood-serum remains unaltered by benzoic-acid administration. In the metabolism of birds, Ornithine, or diami no valeric acid plays CH 2 (NH 2 )CH 2 .CH2.CH(NH2)COOH the part which is taken by glycocoll in the metabolism of mammals, or, at all events, to the extent of being the substance utilized to detoxicate and eliminate benzoic acid. The conjugated acid which appears in the urine of birds when benzoic acid is administered to them is Ornithuric Acid, which splits into benzoic acid and ornithine when it is hydrolyzed. AROMATIC OXYACIDS. The putrefaction of proteins in the intestine results in the formation of Paraoxyphenylacetic Acid and Paraoxyphenylpropionic Acid as inter- mediate stages in the decomposition of tyrosine, and they pass in small amounts unchanged into the urine. It has been observed, from the middle ages, that human urine in certain very rare instances may regularly darken on exposure to air and ultimately turn black. The individuals exhibiting this peculiarity, which is designated Alcaptonuria, are very rare, and yet the condition constitutes a definite peculiarity of metabolism which has often been described, and has been very carefully investigated. The darkening is due to the spontaneous oxidation of dioxyphenyl acetic acid or Homogentisic Acid: CH /\ . ; . :i:;i HC COH HOC CH CH CH 2 I COOH which is a constituent of the urine of these persons. The individuals who display this peculiarity do not appear to suffer any inconvenience from it, and cases only reach the physician through the alarm created by the extraordinary appearance of the urine after standing, or by failure to secure an insurance-policy, for dioxyphenyl acetic acid is a reducing-substance and may be reported by a physician who is unfa- miliar with the typical indications of the disease, as glycosuria. The WASTE-PRODUCTS OF THE SULPHUR METABOLISM 559 reduction of cupric hydroxide solution by the urine of an alcaptonuric individual is, however, accompanied by darkening or even blackening of the fluid, so that no confusion of diagnosis should be possible even on superficial observation. The homogentisic acid in alcaptonuria arises from the tyrosine and phenylalanine radicals in the proteins of the food. If the diet contains little tyrosine or phenylalanine the output sinks, if much it rises. The administration of tyrosine or phenylalanine by mouth, or of glycyltyrosine hypodermically, leads to quantitative excretion of the aromatic nucleus in the form of homogentisic acid. The elimination continues, although it is reduced, in starvation, and this, together with the fact that it may be enhanced by subcutaneous or intravenous administration of the parent acids, shows that the homogentisic acid is not derived from intestinal cleavage or putrefaction. Evidently the alcaptonuric is unable to complete the oxidation of the aromatic nuclei of Tyrosine and Phenylalanine, just as the Diabetic is unable to complete the oxidation of /3-oxybutyric acid. Curiously enough, however, as Garrod and Neubauer have shown, tryptophane is normally utilized by persons who display alcaptonuria. According to Garrod there is but one degree of alcaptonuria and that is complete. Either the excretion of homogentisic acid amounts to several grams a day or it is absent from the urine, and usually the condition is present from earliest childhood. It is evidently the exog- enous metabolism only of tyrosine and phenylalanine which is affected for no defect of development or loss of weight in the adult occurs such as we would expect to happen, were tissue-protein destroyed to produce the homogentisic acid. It is the circulating amino-acids, which normally undergo complete combustion after deaminization, which are the source of this substance. It is probable that homogentisic acid represents a normal inter- mediate product in the oxidation of the oxyphenyl-oxypropionic acid which results from the deaminization of tyrosine. The curious fea- ture of the transformation, however, resides in the fact that whereas tyrosine has only one hydroxyl-group in the benzene nucleus and that in the para position, homogentisic acid has two, one in the ortho and the other in the meta position. It is found, however, that this is the only class of dioxyphenols which is oxidized by normal persons, other dioxyphenols being excreted in the form of conjugates in the urine. The alcaptonuric therefore differs from the normal person in that his inability to oxidize diphenols extends to the single class which normal individuals can oxidize, namely those in which the hydroxyl-groups occupy the ortho and meta positions relatively to the side-group. WASTE-PRODUCTS OF THE SULPHUR METABOLISM. The waste-products of the sulphur metabolism are of three types, namely Inorganic Sulphates, Ethereal or Conjugated Sulphates and the 560 WASTE-PRODUCTS Neutral Sulphur compounds in which the sulphur is not present as a sulphuric acid radical. These three fractions have been found by Folin to vary in a char- acteristic manner with the abundance of proteins in the diet. On high and low protein diets respectively the following daily output of the various sulphur-containing excreta was observed: Protein-rich diet. Protein-poor diet. Volume of urine . 1170 c.c. 385 c.c. Total nitrogen . . 16.80gm. 3.60gm. Total sulphur (SO) 3.64 " 0.76 " Inorganic SO 3 . . 3.27 " (90. per cent.) 0.46 " (60.5 per cent.) Ethereal SO 3 . . 0.19 " ( 5 . 2 per cent.) 0.10 " (13.2 per cent.) Neutral SO 3 . . 0.18 " ( 4 . 8 per cent.) 0.20 " (26.3 per cent.) It will be observed that a reduction of the total sulphur output to one-fifth, reduced the output of inorganic sulphates to one-seventh, and of ethereal sulphates to one-half, while the output of neutral sulphur remains unaltered. Folin draws an analogy between the neutral sulphur output and the creatinine output among the nitroge- nous excreta, and regards the neutral sulphur as originating from the degeneration of tissue-protein, the Endogenous Metabolism, while the inorganic sulphates represent the extent of Exogenous Metabolism or the destruction of circulating amino-acids which have not become constituents of living tissue. The ethereal sulphates, representing conjugated phenols, indican and so forth, have usually been regarded as indicative of the extent of Intestinal Putrefaction. The relatively slight degree to which they are reduced by a reduction of protein intake to one-half is adduced by Folin as an indication that they may possibly arise from the endog- enous metabolism of tissues. It is to be noted, however, as Hopkins has pointed out, that we have no right to assume that Intestinal Putre- factions are reduced proportionately to the reduction of the protein intake. On the contrary, the proportion of the protein intake which reaches the lower intestine without absorption is as a rule very small, unless it chances to be a form of protein which is indigestible, such as raw egg-albumen, or which contains a large glycocoll-fraction, such as gelatin. A large proportion of the putrefaction in the lower intestine must be attributed to the protein contained in the mucous secretions of the intestine itself. Thus Whipple has shown that toxic proteoses of bacterial origin may be absorbed from an isolated loop of intestine, from which the contents have previously been removed. Hence reduction of the protein intake only reduces one, and not necessarily the larger source of intestinal putrefaction, and the reduction of ethereal sulphates to one-half, by a reduction of protein intake to one- fifth, is probably the utmost that could be expected. We may, there- fore, ascribe to the ethereal sulphates, as to the inorganic sulphates, a primarily exogenous origin. Another channel of sulphur excretion is the Bile, wherein sulphur WASTE-PRODUCTS OF THE SULPHUR METABOLISM 561 is contained in the form of Taurine, which, combined with cholic acid, forms the taurocholic acid fraction of the mixed bile-acids. Taurine is amino-ethyl sulphuric acid, and its relationship to the sulphur- containing amino-acid of the tissue-proteins, Cystine, is shown in the following formulae : CH 2 S S CH 2 CH 2 (SO 2 OH) CHNH 2 CHNH 2 CH 2 NH 2 I I COOH COOH Cystine. Taurine. The taurine thus excreted is mainly reabsorbed and either reexcreted as taurocholic acid or else transformed into products which are elim- inated in the urine. It will be observed that the relationship of taurine to cystine is a very simple one, decarboxylation and oxidation of the sulphur serving to convert the cystine into taurine. This being the case it is of very great interest to note that the excretory products to which these compounds give rise are very diverse, for as Salkowski originally showed, and his results have been confirmed and amplified by Schmidt, von Adelung and Watson, the administration of taurine in large doses to man by mouth, or subcutaneous or intravenous injection, leads to a large increase in the Neutral Sulphur output, over eighty per cent, of the taurine being excreted within twenty-four hours in a "neutral" form which Salkowski has identified as Tauro- carbamic Acid. Now the administration of cystine in moderate dosage, or of polypeptides containing cystine, leads to an increase in the inor- ganic sulphates only, and a very large dosage is required to elicit an increase of neutral sulphur. The fact that the administration of cystine, whether by mouth or intravenously, results in an increased output of inorganic sulphates suggests that a portion of the endogenous sulphur metabolism may be represented in the inorganic sulphates, for, as we have previously argued in connection with a possible endogenous origin of urea, if the circulating amino-acids stand in equilibrium with the tissue-amino- acids, as the results of Van Slyke indicate, and these latter in equi- librium with the tissue-proteins, then the disintegration-products of tissue-proteins must be the amino-acids themselves, for otherwise protein synthesis would go on indefinitely and unchecked. But the amino-acids, including cystine of course, when once released from the tissues must be thrown into the common supply and undergo their share of exogenous metabolism. Indeed it may be questioned whether the neutral sulphur output really represents the metabolism of cystine in the tissues of the body considered collectively, or whether it does not possibly represent the destruction of a special fraction of the cystine which is converted by the liver into taurine, and a series of products obtained from the sulphur-containing compounds of the nervous system, cartilage, etc., in which sulphur is present in radicals other 36 562 WASTE-PRODUCTS than cystine. Among the constituents of the neutral sulphur fraction may be enumerated Sulphocyanides which are found in traces in the urine and also in the Saliva, Chondroitin-sulphuric Acid, and a number of poorly-defined nitrogenous acids which have been designated the Oxyproteic Acids. In rare instances Cystine is found to occur in the urine in notable quantities, as much as 0.5 to 1.5 grams being excreted in one day. This condition, known as Cystinuria, is a much more serious abnor- mality than alcaptonuria, which it resembles in being due to a defect of metabolism, because the large excretion of this sparingly soluble amino-acid often leads to the formation of deposits or calculi in the bladder. According to Garrod, cystinuria is a rarer disease than alcaptonuria, but it reaches the physician more frequently because of the serious natiire of the symptoms which arise. The failure to oxidize cystine, which is characteristic of the cystinuric patient, fre- quently extends to other amino-acids, and amines, such as Cadaverine and Putrescine, derived from the decarboxylation of Lysine and Orni- thine may also appear in the urine, and occasionally, leucine and tyrosine. In such cases cystinuria is evidently an expression of a general defect of the deaminizing-mechanism. An experimental cystinuria may be induced in animals by the administration of halogen-benzenes, such as monochlorbenzene or monobrombenzene. The halogen-benzene is paired with cystine and excreted in this form as Mercapturic Acid, in combination with glu- curonic acid. The excretion of cystine in these cases is accompanied by a diminution of the output of inorganic sulphates. The presence of cystine in the urine may be suspected if hexagonal crystals are deposited which are soluble in ammonia and insoluble in acetic acid. If a few crystals are dried, placed on a slide and covered with a cover-glass underneath which is introduced a drop of strong hydrochloric acid, as each crystal is touched by the acid a cluster of fine prisms is seen to spring from it, consisting of cystine hydrochloride (Wollaston's Test). In passing it may be stated that the Phosphorus of the diet is wholly or almost wholly excreted in the form of phosphates in the urine and the feces. URINARY PIGMENTS. A variety of urinary pigments have been described by different investigators, but only three pigments have been definitely character- ised. These are Urochrome, a pigment to which the yellow color of urine is mainly due, Urobilin which is voided in the form of a colorless chromogen, Urobilinogen, which is converted into urobilin by exposure to air under the influence of light, and Uroerythrin, which is frequently but not invariably present. On saturating urine with ammonium sulphate, urochrome remains in solution while urobilin is precipitated. When a solution of urobilin PROPERTIES AND COMPOSITION OF URINE 563 is dissolved in ammonia and a little zinc chloride solution is added the mixture turns red with a green fluorescence; urochrome, on the contrary, does not yield fluorescent solutions. Both of these pigments are closely related to the bile-pigments and, therefore, to hemoglobin. They yield the pyrrole reactions and strongly resemble substances which are obtainable from Bilirubin by reduction. Urobilin, or its parent-substance urobilinogen is a con- stant constituent of the feces, but before the identity of the two pig- ments was realized the urobilin in the feces received a separate name, Stercobilin. The quantity of these pigments in the urine is distinctly increased in all fevers, also in hemorrhage and in conditions involving the destruction of red blood-corpuscles, and in diseases of the liver. Uroerythrin is the pigment which frequently gives a red color to urinary sediments, particularly to sediments of uric acid, which, owing to its presence, may appear like grains of cayenne pepper. It does not yield fluorescent solutions and is rapidly decolorized by light. The normal color of solutions is pink, but strong sulphuric acid changes this to carmine, and alkalies to green. Uroerythrin is believed not to be related to bilirubin but to be derived from Skatole. The quantity is increased by muscular activity, profuse perspiration, alcohol, immod- erate eating, fevers and diseases of the liver. The presence of urobilinogen in the feces and the probable deriva- tion of uroerythrin from skatole render an alimentary origin of these pigments very probable. It is likely that urochome and urobilin arise by bacterial decomposition of the bile-pigments in the lower intestine. In confirmation of this view it is found that strong Intestinal Putre- faction leads to an increase of the urobilin output while exclusion of bile from the intestine reduces the output to zero. If the exclusion of bile from the intestine be due to mechanical occlusion of the bile-ducts, then bile-pigments, but not urobilin, appear in the circulation and in the urine. THE PROPERTIES AND COMPOSITION OF URINE. The volume of the urine which is voided daily necessarily varies very greatly with the quantity of water which is drunk, the quantity of water contained in the food, the amount of fluid lost from the body by perspiration and a variety of other factors such as the presence or absence of Diuretics such as Caffein or Theobromin in the diet, or hyper- activity of the posterior lobe of the pituitary body which may lead to a chronic hyper secret ion of a dilute urine containing no sugar; a con- dition known as Diabetes Insipidus. The Specific Gravity of the urine necessarily varies with its volume, usually fluctuating between 1.008 and 1.030. The reaction is usually acid, but immediately after a meal an alkaline reaction, the "alkaline tide" may frequently be observed, and on a purely vegetable diet the urine is not infrequently alkaline. The sulphur and phosphorus 564 WASTE-PRODUCTS in the proteins of a meat-diet are oxidized wholly or in part to the highly dissociated sulphuric and phosphoric acids which decrease the alkali-reserve of the blood and tissues and are excreted as acid salts in the urine, while the alkaline salts in vegetables are oxidized to carbon- ates or bicarbonates and excreted as such. According to Fitz and Van Slyke the titratable acidity of the urine (employing phenolphthalein as an indicator) runs remarkably parallel, in conditions of Acidosis, with the decrease of the alkali-reserve. In order to observe this parallelism, however, we must add to the titrat- able acidity the amount of Ammonia in the urine which has been furnished by the tissues as a means of neutralizing a portion of the excess of acid. This can be estimated by the method of Sorensen, the Formol Titration, which depends upon the fact that formaldehyde in faintly alkaline solutions unites with ammonia to form hexamthylene- tetramine, which has a neutral reaction : 4NH 4 C1 + 6 HCHO + 4 NaOH = N4(CH 2 ) 6 + 10 H 2 O + 4 NaCl The urine is first rendered very faintly alkaline to phenolphthalein, then neutral formaldehyde is added and the quantity of alkali which must be added to render the urine alkaline again is determined by titration. This is equivalent to the ammonia which has been converted into hexamethylene-tetramine. 1 The relationship observed by Fitz and Van Slyke is expressed by them in the following formula, which is an adaptation of the formula of Ambard for the excretion of urea and chlorides: Bicarbonates in the plasma = 80 where D.is the titratable acidity plus the ammonia output, W the weight of the individual and C the concentration of acids in the urine, or -, where V is the volume of urine. The figure 80 represents the maximum yield of carbon dioxide in volumes per cent, which may be obtained by treating blood-serum with sulphuric acid. Reduction of the alkali-reserve below this point results in the urinary excretion of an excess of acid radicals which is expressed by the factor: This relationship is purely empirical and the agreement between the calculated and observed values of' the alkali-reserve cannot be relied 1 The NH groups of amino-acids will react with formaldehyde in the same way as ammonia. The concentration of amino-acids in the urine is so small, however, that, as a rule it may be neglected. NORMAL COMPOSITION OF URINE 565 upon to within ten per cent. It nevertheless is of value as serving to show that titratable acidity of the urine, if added to the ammonia, or protective basic output, is a real indication of the presence or absence of acidosis. We have seen that the diurnal output of most of the nitrogenous excreta is profoundly influenced by the diet. No normal composition of the urine can therefore be formulated which is not subject to wide fluctuations which are nevertheless within the limits of diversity which may be exhibited by a single normal individual under varying dietary conditions. The following may, however, serve to illustrate the com- position to which the urine of a normal individual subsisting upon a moderate and mixed diet would more or less closely approximate : % NORMAL COMPOSITION OF URINE. (Illustrative Analysis.) The following represents a normal twenty-four-hour sample of urine of volume 1500 c.c. and specific gravity 1.010-1.015: Constituent. Weight in Approximate grams. percentage. Water . 1440.0 96.0 Solids 60.0 4.0 Urea 35.0 2.33 Uric acid 0.75 0.05 Hippuricacid 0.7 C.05 Oxalic acid 0.015 0.001 Aromatic oxy-acids 0.06 0.004 Creatinine 1.0 0.07 Thiocyanic acid (as KSCN) .... 0.15 0.01 Indican 0.01 0.001 Ammonia 0.65 0.04 Sodium chloride 16.5 1.10 Phosphoric acid (P 2 O 6 ) 2.5 0.15 Total sulphuric acid 2.5 0.15 Silicic acid 0.45 0.03 Potassium (K 2 O) 2.5 0.15 Sodium (Na 2 O) 5.0 0.30 Calcium (CaO) 0.25 0.015 Magnesium (MgO) 0.30 0.02 Iron 0.005 0.0004 REFERENCES. THE CARBONACEOUS WASTE-PRODUCTS: Pembrey: Jour. Physiol., 1901, 27, p. 66; 1903, 29, p. 195. Johansson: Skand. Arch.%. Physiol., 1901, 11, p. 273. Hdri: Pfliiger's Arch., 1909, 130, p. 112. Warburg: Ergeb d. Physiol., 1914, 14, p. 253. Krogh: The Respiratory Exchange in Animals and Man, London, 1916. MacLeod: Physiology and Biochemistry in Modern Medicine, St. Louis, 1918. Lusk: The Science of Nutrition, Philadelphia, 1919. THE NITROGEINOUS WASTE-PRODUCTS: Abel and Muirhead: Arch. f. exp. Path. u. Pharm., 1893, 32, p. 467. Hopkins and Hope: Jour. Physiol., 1898-99, 23, p. 271. Kossel and Dakin: Zeit. f. physiol. Chem., 1904, 41, p. 321; 1904, 42, p. 181. Macleod and Haskins: Jour. Biol. Chem., 1905-6, 1, p. 319. 566 WASTE-PRODUCTS THE NITROGENOUS WASTE-PRODUCTS: Van Hoogenhuyze and Verploegh: Zeit. physiol. Chem., 1905, 46, p. 415; 1908, 57, p. 161; 1909, 59, p. 101. Folin: Am. Jour. Physiol., 1905, 13, p. 66; Jour. Am. Med. Assn., 1914, 63, p. 823. Mellanby: Jour. Physiol., 1907-8, 36, p. 447. Ascoli and Izar: Zeit. f. physiol. Chem., 1908-9 58, p. 529; 1909, 62, p. 347. Mendel: Ergeb. d. Physio! , 1911, 11, p. 418. Taylor: Digestion and Metabolism, Philadelphia, 1912. Dakin: Oxidations and Reductions in the Animal Body, London, 1912. Taylor and Rose: Jour. Biol. Chem., 1913, 14, p. 419. Hunter and Givens: Ibid., 1914, 18, p. 403. Hunter, Ibid., 1916-17, 28, p 369. Denis and Minot: Ibid., 1917, 31, p. 561. CONJUGATED EXCRETA: Hopkins. Guy's Hospital Gazette, 1907, 21, p. 424. McCollum and Hoagland. Jour. Biol. Chem., 1913-14, 16, p. 321. Jolles: Zeit. physiol Chem., 1915, 94, p. 79. Sherwin: Jour. Biol. Chem., 1917, 31, p. 307. Dubin: Ibid., 1917, 31, p. 255. OXYACIDS AND SULPHUR DERIVATIVES: Folin: Vide supra. Garrod: Inborn Errors of Metabolism, London, 1909. Schmidt, von Adelung and Watson: Jour. Biol. Chem., 1918, 33, p. 501. PIGMENTS: Garrod: Jour. Physiol., 1894-95, 17, pp. 349 and 439; 1897, 21, p. 190. Garrod and Hopkins: Ibid., 1896, 20, p. 112. ACIDITY OF URINE: Fitz and Van Slyke: Jour. Biol. Chem., 1917, 32, p. 495. PART VI. THE ENERGY-BALANCE OF THE ORGANISM, CHAPTER XXIII. THE ANIMAL BODY AS A MACHINE. THE APPLICABILITY OF THE LAW OF THE CONSERVATION OF ENERGY TO LIVING ORGANISMS. To all of our not very remote forebears and to the majority of those of our contemporaries who vote, legislate and govern in this our present day, Life was, or is, a thing apart from the Universe, independent of cosmic laws, controlling rather than expressing the forces of nature. The inversion of this primitive idea which was ultimately to result in the attainment of our present conception of life, as the outcome of forces which it does not of itself create, originated in the investigations of that greatest of French chemists, Lavoisier. The clue to the true nature of the processes of combustion had previously been provided by the discovery by Priestley that air con- tains a substance which is essential to combustion and is consumed thereby. It was Lavoisier, however, who showed that this gas is absorbed by and becomes combined with the burning substance, and the amplification of this discovery led to the enunciation of the law of the Conservation of Matter. The corresponding law in the domain of energy-transformation was not formulated until 1845, over fifty years later. Nevertheless it is to Lavoisier also that we must accredit the investigations which first established the applicability of the law of the Conservation of Energy to animals. It has frequently happened in the history of scientific investigation, that a truth which was not generally apprehended or clearly enunciated at the time has never- theless been tacitly assumed in advance of their period by investi- gators possessing exceptional powers of insight and discovery. It is a mistake to suppose that successful scientific discovery is the outcome of purely logical processes of thought in the mind of the investigator. The great discoverer appears to be distinguished from equally diligent but less successful investigators quite as much in his possession of a 568 THE ANIMAL BODY AS A MACHINE species of intuitive sympathy with the order of nature, as in his purely intellectual endowments as these are ordinarily understood. There can be no question at all that both Lavoisier and Faraday, without ever having formulated it in so many words, and certainly without adequate proof of its validity, nevertheless assumed the truth of the law of the conservation of energy and were guided in their investiga- tions by this assumption. Lavoisier had shown in 1790 that the oxygen absorbed and trans- formed into other substances by a man or animal is increased by the performance of Muscular Work and by exposure to a low temperature. Work and the production of Bodily Heat were thus correlated with the occurrence of chemical reactions which were known to liberate energy, i. e., combustions. The next step was to institute a direct comparison between the heat of combustion of a carbonaceous material and the heat-evolution of an animal, a comparison which has since then been repeated many times, and with ever-increasing exactitude. The material chosen by Lavoisier as a standard for comparison was pure carbon. He measured the amount of heat evolved in the conversion of the carbon into carbon dioxide, and he then measured the amount of heat and carbon dioxide given off by a guinea-pig in a period of ten hours. The heat-evolution was estimated from the latent heat of ice which was melted by the heat of the burning carbon in the one experiment and by the heat of the animal's body in the other. It was found that the guinea-pig communicated 31.8 calories to the ice, while 25.4 calories were yielded by burning enough carbon to furnish the amount of carbon dioxide exhaled by the animal in the same period. The figures are not equal and we now know why. Apart from experi- mental errors arising from the unavoidably imperfect technic of the estimation, the animal burnt, not only carbon during the period of its incarceration in the ice-chamber, but also hydrogen. Were Carbohydrates, in which the hydrogen is fully neutralized by oxygen already present in the molecule, the sole source of energy, then the comparison instituted by Lavoisier would have been adequate, but the Fats and Proteins contain an excess of hydrogen, of which the heat of combustion must be added to that of the carbon in order to establish the chemical origin of animal heat and work. Nevertheless the figures obtained by Lavoisier were sufficiently comparable to afford decided encouragement to the view which he himself expressed : " La vie est une fonction chimique." In 1793 Lavoisier was condemned to death and executed by the apostles of Liberty, Equality and Fraternity. His crime appears to have consisted in his being a man of superior intellect and education who had dared to express his opinion that the French Academy of Sciences should be preserved and not suppressed, as the National Convention desired. His appeal for liberty to live and serve was thus answered by the president of the tribunal which condemned him: LAW OF THE CONSERVATION OF ENERGY 569 "La Republique n'a pas besoin de savants" which was true, until 1870, let us say, or 1914. It is to a Roman politician that we owe the very popular and oft-quoted doctrine that "The Voice of the people is the Voice of God." On this occasion the spokesman of the people assured one of the greatest discoverers that humanity has produced, that a republic had no need of him or of his kind. To a Swedish physicist, Oersted, we owe a different doctrine, which he expressed in these words: "The Laws of Nature are the Thoughts of God." If we should estimate the value of these two doctrines by their fruits, then doubtless we would prefer the doctrine of the physicist who produced telegraphy to that of the demagogue who planned a brutal and senseless murder. Contemporary events will doubtless, in time to come, furnish us with an abundance of additional means of estimat- ing the relative value of these theories. The work which had been thus initiated by Lavoisier, was carried on by his pupil Liebig, who, however, mainly devoted his attention and his life's work to the firm establishment of the Law of the Conser- vation of Matter in its application to living organisms. The methods of organic analysis which he devised, and the investigations which he undertook, laid the foundations of analytical biochemistry as we know it today. The energy-transformations of life were destined to become the preoccupation of Liebig's pupil, Voit, and of a series of investi- gators who owed to Voit their inspiration. Thus, to the second and third generations of investigators succeeding Lavoisier, fell the task of achieving the fruition of his labors. In order to render possible an accurate comparison of the kind which was attempted by Lavoisier it was first of all necessary to ascertain Heats of Combustion of the various foodstuffs. The actual fuels burnt by the animal machine are carbohydrates, fats and proteins, and it is evidently with the heat of combustion of these substances, and not merely that of carbon, tjiat we should compare the heat-evolution of an animal. The Calorific Values in heat-units per gram for the different repre- sentatives of the three main classes of foodstuffs do not vary greatly among themselves. The molecules of the Fats and Proteins are so large that the differences of composition or structure which they display affect the total heat of combustion but slightly, while the Carbohydrates uniformly contain the proportion of oxygen which is requisite to burn their hydrogen and hence the combustion-value for each carbohydrate is very nearly proportional to the carbon which it contains and this in turn is proportional to the weight of the molecule. The following are the calorific values of various foodstuffs, as esti- mated by complete combustion in a calorimeter, the heat-output being expressed in terms of the large calorie, or quantity of heat re- quired to raise the temperature of one kilogram of water from C. to 1 C. 570 THE ANIMAL BODY AS A MACHINE Cals. Proteins: Casein . . . ......... .... r . . 5.86 Egg-albumin 5.74 Serum-albumin 5.92 Average 5.84 Fats: Tissue-fat . 9 . 48 Butter-fat - 9.23 Olive oil 9.33 Average 9.35 Carbohydrates : Glucose 3.74 Cane-sugar 3.96 Milk-sugar . 3.95 Maltose 3.95 Starch 4.18 Average 3.96 The figures usually employed for the fats and carbohydrates as they actually occur in a mixed diet are those which were originally estimated by Rubner, namely: One gram of fat = 9.3 calories One gram of carbohydrate = 4.1 calories the high value for carbohydrates being employed on account of the predominance of starch among the carbohydrates of an ordinary mixed diet. The heat-value of carbohydrates and fats for the body must be the same as that indicated by the combustion-calorimeter, since the products of combustion are in both cases identical, namely, carbon dioxide and water. The case is far different for the Proteins, however, because these are not completely burnt, the nitrogen being excreted in the form of urea, creatinine and so forth, which are substances still capable of yielding heat when they are completely oxidized. Further- more, the proteins as they actually occur in the diet are not com- pletely digested and assimilated, a proportion of indigestible or diffi- cultly assimilable material being evacuated in the feces. The true heat-value of protein to the animal body is therefore not indicated by the combustion-calorimeter. The determination of the actual calorific value of protein in the animal body was first carried out by Rubner. His procedure was as follows: The calorific value of dried muscle-tissue was determined in the combustion-calorimeter, and the heat- values of the urine and feces upon an exclusive meat-diet were also determined. Subtract- ing the heat-value of the excreta from that of the food, and also a small correction representing the heat of solution of the urea in the urine, it was found that an average of about 4.1 calories per gram was actually available to. the animal from the protein in its diet. The LAW Of THE CONSERVATION OF ENERGY 571 actual calorific value of a protein to an animal is therefore the same as that of a carbohydrate, both being far inferior in heat-value to the fats. The necessary data for the accurate evaluation of the comparison which Lavoisier attempted were by now assembled and the com- parison, when actually carried out by Rubner in 1894, established beyond any doubt the validity of the principle of the Conservation of Energy in the phenomena of life. The experiments were carried out upon a dog, because there existed at that time no calorimeter, of sufficient size to contain a man, which would accurately measure the heat evolved during a period of twenty-four hours. The heat actually imparted by the dog to the calorimeter in twenty-four-hour periods was measured and this was compared with the heat-value of its food computed from the nitrogen in the urine (1 gram Nitrogen = 6.25 grams protein = 25.63 calories) and from the output of water and carbon dioxide. The following are the details of his comparisons, the "food" in starvation consisting, of course, of the proteins and fats of the animal's own tissues: Number of Heat calculated Heat directly Difference in Food. days. from metabolism. determined. percentage. -1.42 2 Fat . . \ . 5 1510.1 1498.3 -0.97 2488.0 Meat and fat / ' ' ' 6 2249 ' 8 2276.91 \ . . . 7 4780.8 4769.3 / iiyr A I V Xf^rtJJ.O ^~t\J.V I 0.42 iviGo/t \ n-ic\n c\ Ant*f\ *\ t , _i_n j.^? When one considers the complexity of these estimations, the multi- tude of factors which participate in determining their outcome, and the elaborate character of the apparatus employed, the coincidence of the calculated and actual output is so exact as to leave no room for doubt that the law of the conservation of energy applies no less to animals than to other machines. The energy which the animal dis- sipates is derived from the combustion of foodstuffs, just as the energy dissipated by a locomotive is derived from the oxidation of its fuel. In the living, as in the inanimate machine, the potential energy of the fuel is released by oxidation and reappears in the form of heat and work. An even more exact balance between income and output was how- ever sought for and found by the American investigator, Atwater. The extraordinary degree of accuracy which was attained in his investigations was rendered possible by the invention of the Atwater- Rosa Calorimeter, which was of sufficient capacity to hold a man and yet so technically perfect that when a measured amount of heat was generated within the calorimeter by an electric current, the quantity of heat liberated could be measured to within 0.01 per cent. (Figs. 48 and 49). The amount of protein burnt by the subject was estimated 572 THE ANIMAL BODY AS A MACHINE from the nitrogen in the urine and in the feces. The carbon which would be derived from this quantity of protein was deducted from the total carbon output and the difference yielded the total non-protein carbon, or carbon derived from carbohydrates and fat. The carbo- hydrates in the food were measured and the corresponding quantity of carbon deducted from the total non-protein carbon. The difference represented carbon derived from the fat. In this way the quantities of each of the three classes of foodstuffs consumed were estimated and FIG. 48. Schematic diagram of the Atwater-Rosa-Benedict respiration-calorimeter. 02, oxygen introduced as consumed by subject; 3, H2SQ4 to catch moisture given off by soda-lime; 2, soda-lime to remove COz', 1, H2SO4 to remove moisture given off by subject; Bl, blower to keep air in circulation; V, vacuum jacket; C, tank for weighing water which has passed through calorimeter each hour; W, thermometer for measuring tem- perature of wall; Ai, thermometer for measuring temperature of the air; R, rectal ther- mometer for measuring temperature of subject. (After Lusk.) the energy which their combustion could yield was computed in the manner indicated above and compared with the actual heat-evolution of the subject. The results of forty days' experimentation with three different subjects yielded the following averages: Calculated daily output . . . . . . ,/,., . ' . . 2717 calories Observed daily output 2723 " Difference, 0.2 per cent. A further refinement of technic consisted in the simultaneous esti- mation of the carbon-dioxide output and the oxygen intake, from LAW OF THE CONSERVATION OF ENERGY 573 which the Respiratory Quotient could be calculated. Deducting the protein carbon from the total carbon output, and the oxygen required to oxidize the protein from the total oxygen intake, the ratio of the non-protein carbon dioxide to the residual oxygen intake, or the non- protein respiratory quotient; afforded a measure of the proportion of fat to carbohydrate actually consumed by the subject of the experi- ment. Thus a non-protein respiratory quotient of 0.707 indicates the FIG. 49. General view of the respiration-calorimeter laboratory at Middletown, Connecticut. The calorimeter-chamber is seen, with window open upon the right. The principle of its construction is that of an ordinary refrigerator, namely, a chamber surrounded by a series of confined air-spaces. The inner chamber is of copper. This is succeeded by a wall of zinc and two walls of wood, each pair of walls being separated by about three inches of air-space. Gain or loss of heat through the metallic walls of the chamber is prevented by keeping the zinc wall at the sa.me temperature as the copper. Any difference of temperature between these two walls is indicated by a thermocouple and a galvanometer. Heat is supplied to the air-space surrounding the zinc wall by passing an electrical current through coils of resistance-wire. Cooling is accomplished by currents of water. The heat generated in the chamber is removed partly in the form of the latent heat of vaporization of the water exhaled from the lungs and partly by means of cold-water absorbers. The quantity of heat evolved is computed from the amount of water passing through the heat-absorbers and its rise in temperature during its passage. (After Benedict and Milner.) combustion of pure fat, a quotient of 1 .00 indicates the combustion of pure carbohydrate (cf . Chapter XXII) and intermediate values repre- sent the combustion of a mixture of these foodstuffs, the composition of which can be estimated by a simple calculation. It now remained, in order to complete the demonstration of the validity of the law of the conservation of energy in the animate world, to investigate the source of the energy which is expended by an animal in the performance of external work. In the experiments hitherto 574 THE ANIMAL BODY AS A MACHINE enumerated the subjects were at rest, and although their respiratory and cardiac muscles were contracting and the skeletal muscles main- tained in tone or even contracting, yet, the whole of the organism being enclosed within a heat-insulated system, the effect of all these move- ments ultimately appeared and was estimated in the form of heat. The case is different when, as in many of Atwater's experiments, the subject was made to perform external work, by operating a stationary bicycle which was so arranged that the rotation of the wheels raised a weight. The energy output was not in this case expressed entirely in the form of heat, but in part in the form of Mechanical Work. We can express this work in terms of heat-units, however, just as we can express heat in terms of electrical units or electrical units in terms of mechanical work again. Since no energy is ever lost and all forms of energy are equivalent to one another, the heat-value consumed in performing mechanical work can be directly calculated from the known mechanical equivalent of heat. The following are the results which Atwater obtained in the investigation of this problem: Calories. Income per Output per twenty-four twenty-four Difference. Days. hours. hours. per cent. Rest experiments: 7 experiments with E.G. . 25 2268 2259 -0.4 1 experiment with A.W.S. 3 2304 2279 -1.1 3 experiments with J.F.S. 9 2118 2136 +0.8 1 experiment with J.C.W. 4 2357 2397 +1.7 Average ." . , 41 2246 2246 0.0 Work experiments : 2 experiments with E.G. . 8 3865 3829 -0.9 4 experiments with J.F.S. 12 3539 3540 0.0 14 experiments with J.C.W. 46 5120 5120 0.0 Average ... 66 4682 4676 -0.1 To within one part in a thousand the output of heat plus work was equal to the calorific value of the foodstuffs consumed . We can hardly doubt that this minute discrepancy was of purely technical origin and that these experiments represent the culmination of the proof which Lavoisier had sought a hundred years previously, that the energies of life are derived simply and solely from the chemical energy of the foodstuffs. The fundamental importance of these investigations cannot be over- rated, for they reveal to us in the clearest possible manner the fact that life is the outcome of a complex of forces which it does not create. We are enabled by them to confidently state that if there is such an entity as "Vital Force" created and generated out of nothing by living organ- isms, then the inconspicuousness of its effects is commensurate with the inconspicuousness of its origin. They must be confined to somewhat Jess than a one thousandth part of the total activity of the organism. ISODYNAMIC VALUES OF THE FOODSTUFFS 575 We must be careful, however, in formulating any such fundamental conclusion not to go too far. We must beware of overstepping to the slightest extent the sure ground of fact which our evidence affords, and we must therefore candidly admit that while the evidence accumulated by the remarkable series of investigations which we have briefly and inadequately outlined, clearly justifies the conclusion that no source of energy is contributed to or resides in the organism that is not com- prised in the chemical energy of its foodstuffs, and the heat of its environment, yet we cannot definitely reject the possibility that forces of evanescent magnitude which are not comprised in either of the above categories may influence, in the manner of a catalyzer, the rate of discharge of energy from the organism. We cannot disprove this, but then, on the other hand, if one should choose to assume the existence of such forces the burden of proof clearly rests upon the originator of the hypothesis. In the interpretation of life-phenomena, so far as we have as yet been enabled to subject them to measurement, such an assumption has proved to be altogether unnecessary, and hence our present state of knowledge affords for it no foundation whatever. No sure ground is possible in scientific discovery unless we proceed from the known to the unknown. The assumption that hitherto unknown forces are involved in life cannot assist but only retard its interpreta- tion until and unless every previously known possibility has been exhausted in a vain endeavor to reconcile the facts. But the existence of unknown possibilities manifestly cannot be contradicted upon a priori grounds, and a dogmatic insistence upon the sufficiency of the known has only too frequently, in the history of science, served but to pave the way for a subsequent recantation. THE ISODYNAMIC VALUES OF THE FOODSTUFFS. Since the products of the combustion of the Fats and Carbohydrates in the diet are the same, namely carbon dioxide and water, it was sug- gested at an early period in the investigation of metabolism that these components of the dietary might be mutually interchangeable in equicalorific quantities. This possibility was experimentally realized by Rubner, who fpund that 100 grams of fat in the diet could be replaced by 232 grams of starch or 234 grams of cane-sugar, the equi- calorific values estimated from the heat of combustion being 229 grams of starch and 235 grams of cane-sugar. The same conclusion was ulti- mately reached by Atwater in a series of experiments in which the subjects were made to perform external work, so that part of the energy of the foodstuffs had to be expended for this purpose. The procedure of the experiments was designed to test the efficacy of the fats as sub- stitutes for carbohydrates in a variety of ways. Thus the diet was insufficient to maintain bodily equilibrium, so that there was a loss of weight throughout the duration of the experiments due to the consump- tion of the subject's tissues. The loss of body-substance on the diet 576 THE ANIMAL BODY AS A MACHINE containing carbohydrates could thus be compared with that experienced on the diet containing fats, and the relative value of these constituents of the dietary as tissue-sparers could thus be estimated. The external Mechanical Work performed in both sets of experiments was as nearly as possible the same, and equivalence of total energy-consumption on the two diets would therefore indicate equal availability of fats and of carbohydrates for the performance of mechanical work. The following table summarizes .the results of these experiments: Experiment number. Time, days. Heat derivable from food, calories. Heat equivalent of external work, calories. Total energy- output, calories. Calories equivalent to gain ( +) or loss ( -) of tissue. 40 J.C.W. carbohydrate-diet . 4 4180 518 5251 -1071 41 J.C.W. fat-diet .... 4 4150 522 5304 -1154 44 J.C.W. carbohydrate-diet . 4 4602 571 5125 -523 43 J.C.W. fat-diet .... 4 4496 548 5155 -659 47 J.C.W. carbohydrate-diet . 4 4366 562 5173 -807 46 J.C.W. fat-diet .... 4 4 % 473 551 5193 -715 53 J.C.W. carbohydrate-diet . 3 5132 587 5104 +28 52 J.C.W. fat-diet .... 3 5120 607 5309 -189 Average of four experiments with carbohydrate-diet .... 15 4532 558 5167 -635 Average of four experiments with fat-diet 15 4524 554 5236 712 The substantial equivalence of the fats and carbohydrates as sources of heat and work and sparers of tissue in these experiments is evident. There is some indication that the loss of tissue on the fat-diet is greater than it is on a carbohydrate-diet, and this is especially evident in the experiment in which the total calorific value of the diet was relatively high. The reason for this probably lies in the fact that, as Zeller has recently shown, if the preponderance of fat over carbohydrates in the diet be too great, even when the total calorific value of the diet is kept constant, acetone bodies appear in the urine and an Acidosis arises necessitating the production of Ammonia by the tissues tc neutralize the excess of acid radicals in the blood. The output of nitrogen is consequently increased and loss of body-substance accelerated. This effect only appears in normal individuals, however, when less than ten per cent, of the total calories are given in the form of carbohydrate. Up to this limit, therefore, the carbohydrates in the diet may be replaced by fat without influencing very appreciably the total heat-output or wastage of tissue-materials. In Diabetes, of course, the limit of toler- ance for fats is much lower than this. In the replacement of the fats by carbohydrates we are limited in another direction. So far as the mere question of heat-equivalence is concerned the complete replacement of the fats in the dietary by carbohydrates is doubtless entirely feasible, more especially since the conversion of carbohydrates into body-fat is a regular concomitant of ISODYNAMIC VALUES OF THE FOODSTUFFS 577 insufficient utilization of the carbohydrates of the diet for the produc- tion of heat and work. We have seen (Chapter XX), however, that certain essential Substrates of Growth, or raw materials for the synthesis of protoplasm are contained in the animal fats and, so far as we are yet aware, in no other abundant constituents of the diet. The total replacement of fats by carbohydrates, therefore, is likely to result in unbalanced tissue-waste through the lack of non-synthesizable atom- complexes which do not necessarily contribute any appreciable share to the energy-output. The total replacement of animal fats by Vegetable Oils is for a like reason impracticable. The proportion of animal fat which is requisite for maintenance is, however, very small, and pro- vided this small residuum is retained, the fats of the dietary may be replaced by carbohydrates in equicalorific proportions without affect- ing the balance of energy-input and -output. In the case of the Proteins a number of complications arise which limit in a variety of directions the application of the principle of isodynamic values. In the first place the proteins are the medium through which the body acquires its nitrogen. Their complete replace- ment by fats or carbohydrates is therefore obviously impossible. Then, again, different types of protein are not even isodynamic with each other, for those which lack or are deficient in certain amino-acids, such as Gelatin, Zein or Gliadin will not replace the protein in a normal mixed diet however great an excess of the incomplete protein may .be employed (Chapter XX). No nitrogen balance is possible unless the missing amino-acids are supplied, and upon a diet containing an abundance of nitrogen the output will continuously exceed the intake. If, however, the missing amino-acids are added to these proteins, as, for example, tyrosine, cystine and tryptophane to gelatin, then the attainment of nitrogenous equilibrium becomes possible because all of the constituent parts of tissue-protein are then present in the diet. Although gelatin cannot replace other proteins in the diet, yet it is possible to attain nitrogenous equilibrium on a smaller amount of normal dietary protein if gelatin be also present. If the total heat- requirement of the normally fed animal be supplied solely in the form of carbohydrates and fats a certain daily loss of nitrogen will occur which is due to the consumption of tissue-proteins. If 7.5 per cent, of the heat- value be now supplied in the form of gelatin the excess of loss over intake is diminished by 23 per cent. If, however, 60 per cent, of the heat-value of the food is supplied by gelatin the saving of tissue-protein is only 35 per cent., and if the whole of the heat-value be supplied in gelatin only 37.5 per cent, of the tissue-wastage is spared. The principle of isodynamic values is therefore manifestly inapplicable to the quantitative relationship between gelatin and the other dietary constituents unless a sufficiency of other protein be at the same time supplied to furnish the full requirement of tyrosine, cystine, and tryptophane. A further limitation upon the application of the principle of isody- 37 578 THE ANIMAL BODY AS A MACHINE namic values to the protein constituents of the dietary, arises from the fact that an increase of protein in diet actually stimulates the total metabolism, so that more food is burnt and more heat evolved on a diet high in protein than upon a diet which contains less protein. This phenomenon, which Rubner terms the Specific Dynamic Action of pro- teins, is very well displayed by the effect of administering protein to a starving animal. One might suppose that if a starving animal is losing a certain amount of tissue-protein daily, the administration of this amount of protein daily would suffice to balance the nitrogenous input and output. This is not the case, however, for on increasing the nitrogenous input an increase of nitrogenous output also occurs and the balance remains negative. A further increase of nitrogenous input calls forth a still greater metabolism of protein until, on an exclusively protein diet, a balance between intake and output is attained with an output of nitrogen no less than three and one-half times that which is observed in the starving animal. In man the quantity of protein thus required to obtain nitrogenous equilibrium is greater than he can conveniently consume, and even when nitrogenous equilibrium has been attained the carbon balance remains negative, since not only the nitrogenous metabolism, but the metabolism of fats and carbohydrates is stimulated by protein. The effect of protein is therefore to greatly increase the heat-evolution of the body, and the replacement of fat or carbohydrate by protein in a diet which is just sufficient to maintain equilibrium results in rendering the diet inadequate to replenish the tissue-loss. The proteins cannot, therefore, replace fats or carbo- hydrates in isodynamic proportions. The origin of the specific dynamic action of the proteins has been sought by Lusk, who investigated the effects of individual Amino- acids upon the heat-output in starving dogs. He found that while glycocoll and alanine greatly increase the production of heat, and leucine and tyrosine slightly, glutamic acid is devoid of action. A mixture of 5.5 grams each of glycocoll, alanine, glutamic acid and tyrosine produced as much increase of heat-output as 100 grams of meat. THE PROTEIN REQUIREMENT IN THE DIETARY. From the preceding considerations it must be evident that the proteins are the most wasteful constituents of the dietary, since they increase the consumption of other constituents as well as that of pro- tein itself. The proteins are also the most expensive foodstuffs from a commercial point of view and this is particularly true of the proteins of animal origin, for while there is little wastage of energy or materials in the growth of the vegetable constituents of the diet, a very large wastage occurs in the synthesis of animal proteins for human consump- tion. An ox or sheep may, for our immediate purpose, be regarded as an ambulatory factory of protein. In order to supply this factory with raw materials, vegetable proteins, carbohydrates and fats must first PROTEIN REQUIREMENT IN THE DIETARY 579 be grown at the expense of the constituents of the soil and the pre- occupation of space that might be otherwise utilized. Not only must an amount of vegetable food be provided equivalent in heat-value to the animal foodstuffs which we desire to synthesize, but an enormous excess, to supply the radiation of heat and mechanical work performed by the animal throughout the period of its growth. The Animal Proteins therefore represent a consumption and expenditure of food- materials totally disproportionate to their calorific value. The vegetable proteins, on the other hand, are also expensive because the proportion of protein in the majority of vegetable tissues, with few exceptions, is extremely small. As a measure of national economy, therefore, if we view the matter solely from a financial standpoint, a restriction of the protein-con- sumption to the minimum consistent with health and efficiency would seem to be highly desirable. Now the consumption of protein food- stuffs and particularly of animal proteins varies very greatly among different peoples. The following pre-war statistics are furnished by Ostertag : Meat consumed per day per capita, in grams. Australia . .... >\ * ..'.. . 306 United States of Ameri ca 149 Great Britain ..... , . . . . . ... . . 130 France . . -.. .*% ..... . . 92 Belgium and Holland . ... . . . :. . . . 86 Austria-Hungary 79 Russia ....."... ",' . . . . . .- . ..' -. 59 Spain 61 Italy. . , ,V -. ...A ...;. -. ...; ":.-., :.. ....... 29 Japan 25 It will be observed that the consumption of meat in the English- speaking countries far exceeds that which prevails elsewhere. Either the English-speaking countries and particularly Australia are waste- fully dissipating their food-values, or else a large proportion of the population of Europe is chronically suffering from suboptimal con- sumption of protein. The standard requirement of protein, partially derived from meat and in part from vegetables and cereals, was computed by Voit to be 118 grams for the average man not engaged in heavy labor, and 90 grams for a woman. This estimate was based upon a statistical comparison of the actual consumption by presumably normal persons subsisting upon a mixed diet. The necessity for this intake of protein has of recent years, however, been sharply challenged by Chittenden and others of the American school of physiologists and biological chemists. The statistical method of estimating protein-requirements is based upon the assumption of the exercise of free choice by the individual and the underlying supposition is made that prevailing diets represent a species of " survival of the fittest/' It is obvious, however, 580 THE ANIMAL BODY AS A MACHINE that if this criterion were to be applied in Japan it would yield far different estimates from those which would result from its application in England. As Taylor has observed, the customary dietary of dif- ferent races has in no small degree been fashioned by their ethnological development. "In some lands races were compelled to adopt cultiva- tion of the soil, in other places, fishing, in some areas the chase remained, long into relative civilization, one of the chief methods of securing food. The variations in ethnological development brought about by enforced cultivation of the soil, as contrasted with the state of affairs in a tribe of hunters, are well illustrated in different tribes of our American Indians. Depending upon the method of sustaining the life of the tribe, the standard diet of the tribe varied. Only under modern conditions of transportation have the instincts and tastes of man had opportunity for full choice in diet. Compulsion to some extent and in some degree there has always been." Chittenden was able not only to maintain nitrogenous and calorific equilibrium for prolonged periods on a much lower protein intake than that recommended by Voit, but he was able to keep athletes in a condition fitting them for extreme exertion. According to Taylor the Nitrogenous Metabolism of a man of 70 kilos may be summarized as follows, the nitrogenous output being expressed in terms of grams of protein : Grams per day. Nitrogen output on protein-free diet with carbohydrates . . . 10 to 15 Nitrogen output in starvation, lowest level 15 to 20 Nitrogenous and caloric equilibrium, with ample ingestion of carbo- hydrate 30 Nitrogenous and caloric equilibrium, largely with fat 40 Normal protein input, safety margin of 100 per cent 70 Nitrogenous and caloric equilibrium on a pure protein diet . . . 750 Nitrogenous and calorific equilibrium can therefore be attained on a diet rich in carbohydrates with a daily intake of only one-third of the amount of protein recommended by Voit. It cannot be positively affirmed that this low protein intake would also suffice to permit normal growth in children or adolescents. It has been argued that as a great part of even this small protein intake is simply deaminized and burnt in the Exogenous Metabolism there must be plenty to spare for tissue-synthesis. It has never been demonstrated, however, that the exogenous metabolism is reducible below a certain level. In fact the deaminization of amino-acids with production of urea continues even in starvation. There is apparently, in so far as protein is con- cerned, no level of the nutrient-reservoir at which a large overflow does not occur. If the overflow and inflow are nearly balanced and the over- flow (i. e., exogenous metabolism) is irreducible upon a diet of given composition, then it is clear that the outflow of nutrients to the tissues may be just sufficient to maintain repair and yet quite inadequate to synthesize additional tissue, despite the fact that the intake is far above PROTEIN REQUIREMENT IN THE DIETARY 581 that which would, in the absence of the overflow, be necessary for this purpose. In order to establish the adequacy of a maintenance-income of protein for growth it would be necessary to show that the rate of exogenous metabolism, which appears to be governed, at least in part, by the Thyroid, is reduced when tissue-accretion occurs. The experi- mental indications are quite the reverse and tend to show that tissue- accretion is not a cause, but may be a consequence of lowered exogenous metabolism. It is clear, however, that adults may maintain themselves in nitrog- enous and calorific equilibrium upon a much lower protein intake than is customary in many countries, and the question therefore arises whether a restriction of the protein intake, particularly in the English- speaking countries, may not be nationally and economically desirable. We should be cautious in deciding this question upon an insufficiency of evidence. A multitude of factors enter into the question besides the merely financial factor. In the first place it may be stated that no harmful effect of a high protein diet in normal persons has ever been demonstrated. No particular disease is noticeably more common among people accustomed to a high protein intake than among those accustomed to a low protein intake. On the contrary diseases traceable to lowered resistance of the peripheral tissues, such as Trachoma, are decidedly more abundant among people whose diet is deficient in protein, although it must be admitted that the dietary of these peoples is probably deficient in other respects beside that of protein-content. A high protein intake does not throw a "load upon the kidneys" which is deleterious in normal persons, and in any case the "load" is very easily lightened by a copious intake of water. On the other hand, taking Australia as an extreme instance of a community which is accustomed to a high protein intake, we find from the pre-war statistics of the Commonwealth Government that the Death-rate was extraordinarily low, nearly one-half that which prevailed in Italy and Austria, lower in fact than in any other country excepting New Zealand, which is also a community of high protein consumption. The Cancer death-rate was intermediate between that of Italy and that of France, two communities each consuming far less meat per capita than the Australian. 1 The birth-weight of Australian infants of British parentage exceeds that of British infants born in England by over ten ounces. 2 No trace of deleterious influence of the high proportion of meat in the dietary is thus perceptible. On the other hand the diver- sity of climatic and social and economic conditions forbids us from drawing the opposite conclusion that the high protein intake is posi- tively beneficial. It may be pointed out, however, that an unusually low, and also an exceedingly high rate of Exogenous Metabolism are alike deleterious to 1 Official Year-book of the Commonwealth of Australia, 1914. 2 T. Brailsford Robertson: University of California Publications, Physiology, 1915, 4, p. 207. Amer. Jour, of Physiol., 1915, 37, p. 1. 582 THE ANIMAL BODY AS A MACHINE the general welfare and efficiency. Physicians seek in some instances to correct the former condition by the administration of thyroid extract or of other preparations which are believed to stimulate metab- olism. It is quite possible, however, that the effects which are desired might also, in those instances in which no manifest disease of the thyroid is present, be elicited by an adequate increase in the protein intake of the patient. This possibility is merely mentioned in order to illustrate the probable nature of the effects and utility of a protein intake in excess of our minimum needs. We must recollect that it is not the energy-output which suffices merely to maintain life, to gain the means of living for another day, which is of genuine value in the eyes of civilized mankind. The products of human effort which we prize are wholly the outcome of the small surplus of energy which we col- lectively generate over and above the minimum which will support life and propagate the species. This small surplus, which is minute in comparison with the aggregate expenditure, is the origin of all that we cherish, and, even in purely economical terms, the cost of its production is negligible in comparison with its value. In the absence of any evidence of deleterious influence, a reasonable excess of protein -intake, such as that usual in the United Kingdom or America, should not be discouraged in advance of a clear demonstration that it plays no part in the generation of efforts which, in the aggregate, may outweigh the costliness of the practice. It must be admitted, however, that even upon this basis it is difficult to defend the extraordinarily excessive meat-consumption which has hitherto been customary in Australia. THE NORMAL DIET. The normal dietary of a variety of different classes and occupations of society in the United States has been investigated by Atwater both from the standpoint of composition and that of Calorific Value. The following table summarizes some of his results. It must be recollected, however, that the quantity of food actually digested, assimilated and utilized, was in each instance a little less than the quantity which was ingested. Composition of the diet. Calories per day. 3560 3605 3530 3880 3705 3405 8850 6905 5740 4462 2910 3465 Protein, Fat, Carbohydrate, Occupation. grams. grams. grams. Farmers' families . .',..,- 101 128 476 Mechanics' families . 113 153 420 Professional families . .. ' V 110 136 442 Five college-student clubs ' . 127 181 402 Sixteen men's student-clubs . 105 147 465 Four women's student-clubs 101 139 414 Stonemason, hard work . 180 365 1150 Blacksmith, hard work . 200 304 365 Footballer . . . . . i^. 181 292 557 Sandow ~ 244 151 502 Teacher's families, Indiana . 111 110 349 Official's families, Pennsyl- vania 98 155 396 NORMAL DIET 583 It will be observed that the habitual performance of hard physical labor is correlated with a high calorific intake. The increase of intake affects, as a rule, all three classes of foodstuffs. The increase of the Protein intake is surprising in view of the fact that proteins are not a normal source of muscular energy. This apparent contradiction, which has been observed in all countries, has been explained in several diverse ways. Advocates of a high plane of protein nutrition have advanced the tendency to increased consumption of protein by those who perform hard physical labor, as evidence that the increased speed of metabolism induced by protein facilitates the functional activity of the tissues, including muscular tissues. Advocates of the low plane of protein nutrition, on the contrary, have urged that the high protein intake of these persons is essentially accidental, arising simply from the fact that they ingest larger quantities of all foodstuffs and, maintaining the normal admixture of the three types of food material, incidentally consume more protein. This, however, was certainly not true in the case of the blacksmith and the professional athlete, Sandow, whose dietaries were investigated by Atwater. A more reasonable suggestion than either of the above is probably that which has been put forward by Voit, that as persons accustomed to hard labor are usually more muscu- lar than sedentary individuals, the total protein intake required to support the greater quantity of Protoplasmic Tissues, maintaining their wear and tear, and at the same time the exogenous metabolism, is greater than it is in persons, even of like weight, in whom a considerable part of the weight is made up of adipose tissues, for example. The figures obtained by Atwater are certainly suggestive from this point of view, for the total mechanical work performed by Sandow in a brief daily exhibition and a period of practice or exercise was evidently not nearly equal to that performed by a manual laborer in an eight- or ten- hour day. But by exercises and a mode of life carefully directed to that end, Sandow had brought about in himself an extraordinary degree of muscular development, far exceeding that of the ordinary laborer, in harmony with Voit's suggestion we find that his intake of protein was nearly two and a half times the normal, while his intake of fat was normal and his intake of carbohydrates only slightly above the the average. It has already been pointed out that the vegetable foodstuffs are, as a rule, distinguished by their relatively low content of protein. This arises from the fact that carbohydrates assume a structural role in plants \vhile in animals their place as structural materials is taken by proteins. It is from this fact that one of the several objections to the practice of Vegetarianism arises. A purely vegetable diet is, if nitrogenous equilibrium is maintained, an exceedingly voluminous one. The indigestible residues of cellulose are large, the feces very bulky, and the fecal masses occlude a proportion of otherwise digestible and assimilable materials which are voided with them. The wastage in a vegetarian diet is for this reason alone a considerable item. A much 584 THE ANIMAL BODY AS A MACHINE more serious source of waste, however, is the incomplete utilizability of the small proportion of protein which the vegetable diet does con- tain. We have seen from the researches of London (Chapter XI) that the intestinal epithelium exerts a preliminary selective action upon the amino-acids which are submitted to it for absorption, rejecting a proportion of those which are present in unwonted excess. Now the proportions of the various Amino-acids in the proteins of vegetable origin differ very decidedly from those which obtain in proteins of animal origin and therefore, on a purely vegetable diet, the arnino-acids presented for absorption are in abnormal proportion to one another. A portion of the amino-acids derived from' vegetable proteins by diges- tion are therefore rejected and voided in the feces. The following table shows the percentage of the nitrogen in various types of food- stuffs which is actually assimilated : Percentage of nitrogen Type of food. actually assimilated. Flesh 98 Fish . . . . . . - <" . . . . 97 Eggs . ' , " . . . -. 95 Milk . ..... . , . 94 to 95 Peas, Beans . . . . ,. . . . 85 Corn 83 Wheat-flour 81 Rice ,......, ' ; 80 Potatoes .,.,... . 78 The following shows the relative proportion of wastage on a purely vegetable diet, an average mixed diet and a high meat-diet (Atwater and Langworth) : Nitrogen in grams per day Percentage Type of diet. , > ^ ^ of nitrogen In food. In urine. In feces. wasted. Vegetable diet ...... 13.8 13.9 3.9 28 Mixed, average meat .... 19.4 15.6 2.4 13 Mixed, large amount of meat .33.1 24.5 2.9 9 Even the amino-acids which fail to undergo assimilation, however, do not represent all the wastage which occurs on a purely vegetable diet, for the process of selection and rejection which initiates in the intestine continues in the tissues, and the rejected excess of unutilizable radicals simply enters the exogenous metabolism and, while it is avail- able for the production of heat, is useless for the maintenance of the integrity and repair or synthesis of tissues. This fact is very well illustrated by the experiments of K. Thomas, who, subsisting upon a diet of starch and sugar, estimated the minimal daily loss of tissue- protein and then added to his diet food materials of various types in order to determine the relative power of the proteins which they con- tained to save the body from loss of tissue-protein, or, as he terms it, the Biological Values of the various proteins. The following were some of his results: NORMAL DIET 585 BIOLOGICAL VALUES OF VARIOUS PROTEINS, ESTIMATED IN TERMS OF THE PERCENTAGE OF BODY-PROTEIN WHICH THEIR INGESTION WILL SPARE FROM Loss. Cow : s milk 100 Casein 70 Fish Rice Cauliflower . Crab-meat . Potatoes . . ... 95 , . . . 88 . . . . 84 . . . . 79 . . . . 79 Nutrose . .-. . . Spinach . .'.... Peas . . . : . . . Wheat-flour .... Cornmeal . 69 . 64 . . 56 . . 40 . . 30 It is evident, therefore, that the nutritive value of peas, for example notwithstanding their remarkably high protein content in comparison with other vegetables is much less than we might infer from their composition, and approximately double the normal protein intake required on a diet in which peas and beans are the only important source of protein. Recollecting that peas and beans are the only generally available vegetable articles of diet in which proteins are at all abundant, the difficulty of securing nitrogenous and calorific equilibrium upon an exclusively vegetable diet must be apparent. The herbivorous animals can accomplish it by eating an enormous bulk of food, for which their intestines are specially adapted by their length and capacity. A pro- portionately bulky diet would insure grave digestive disorders in the average human being to whom it was habitual. Even more serious difficulties than this, however, confront the would- be vegetarian. We have seen (Chapter XX) that certain constituents of the diet which are associated solely with Animal Fats are absolutely essential both for maintenance and for growth. These are lacking in a diet composed of customary articles which are solely of vegetable origin. The fat-soluble essentials for growth and maintenance do not occur in the fatty tissues of plants, in seeds and fruits, but in the forage- parts. They are acquired from these by the herbivorous animals and stored by them in their body-fat. To obtain a sufficiency of these substances from vegetables in our diet, we would be compelled to con- sume an excessive quantity of vegetable material of very low nutritive value, containing a very large proportion of indigestible residue. It may therefore be stated, and experience seems to have fully justified this deduction, that continued maintenance of weight and health, and, above all, growth, are impossible of attainment by human beings who confine themselves strictly to a vegetable diet. Happily there are few people who are so fanatical in their vegetarian- ism as to attempt to subsist solely upon vegetables, fruits and cereals, and the so-called vegetarian usually partakes fairly freely of Milk and Eggs. On a mixed diet which contains a good proportion of these articles there is no difficulty in securing a thoroughly satisfactory nitrogenous and calorific equilibrium, and experience has demon- strated that a dietary of this character may maintain a high standard of bodily health and vigor. It is not improbable that occasional indi- 586 THE ANIMAL BODY AS A MACHINE viduals would positively benefit by adopting a dietary of this type. Others, again, might not improbably find that while it fully sufficed for the maintenance of weight and health and the satisfaction of the appetite, yet better digestion and improved well-being would be attained on a dietary containing some proportion of meat. To the majority, appetite, taste and habit apart, it would probably be indif- ferent which alternative was adopted. Without positively encouraging such dietetic experiments, especially where children are concerned, the physician will probably, unless there are certain indications to the con- trary, do well to allow a vegetarian of this type to indulge his whim. The absolute vegetarian, however, who declines even to partake of milk or eggs, must be solemnly warned of the danger he is incurring and the almost inevitably unhappy outcome of his fanaticism, while his children should be shielded, if possible, from the outrage of the perpetration of his delusion, and irreparable detriment of their bodily welfare. On the other hand an exclusive flesh-diet, which has been advocated no less warmly than vegetarianism in certain ill-informed quarters, is only a shade less undesirable than an exclusively vegetable diet. The wastage again becomes very large on account of the stimulation of metabolism resulting from the high plane of protein intake, and an abnormally large consumption of food becomes necessary to maintain nitrogenous and calorific equilibrium. The insufficiency of the carbo- hydrate intake provides little of the proper nutriment for the muscular tissues, the power of continued exertion is impaired, and the tendency to certain types of auto-intoxication is probably enhanced. The diet is so completely digestible that the fecal bulk is too small to maintain the proper tension and tonus of the lower intestine, and the resultant stasis favors Intestinal Putrefaction. The abundant variety of mineral constituents contained in the vegetable items of the customary dietary is replaced by the relatively limited variety and quantity of mineral constituents in flesh. The high protein intake implies a high sulphur intake, and therefore the formation of large quantities of sulphuric acid, which reduce the alkali-reserve and impose a tendency toward Acidosis. On the whole, it must be evident from the above discussion that the only safe prescription for continued employment by persons of all ages is that which the good housekeeper instinctively recommends, namely an abundant and varied diet. The requirements of the body are so numerous and so varied in their character and in the sources from which they must be derived that in our present state of knowledge a dragnet policy of sweeping into the body a large variety of dietary articles, is the only one which will ultimately ensure a sufficient intake of every possible requisite. All precise dietary prescriptions, however well supported by selected individual experiences, are premature where the majority of humanity are concerned, and a diet of half -raw meat, recommended on the ground that, being muscle, it must contribute to our strength, should be viewed with no less suspicion than a diet of CALORIFIC REQUIREMENT AND THE SURFACE-LAW 587 nuts, advocated because some of our arboreal ancestors were perforce accustomed to partake of these indigestible delicacies rather freely. The physician, of course, will find it imperative from time to time to impose quite severe restrictions upon the dietary of certain types of patient, of diabetics, for example, or of persons afflicted with nephritis, or with certain types of indigestion, and often he will achieve very great success by this simple means. His very success, however, constitutes in certain cases a positive danger to other people, through the possible conversion of his patient into a dietary propagandist seeking to pro- mulgate a "system" arising out of the measures which were found effective in bringing about the recovery of his own health. A brief but clear and simple statement by the physician of the precise object of the dietary imposed, and its limited applicability, might, not infre- quently, suffice to stifle a dietetic fad at its birth. THE CALORIFIC REQUIREMENT AND THE " SURF ACE-LAW." The average Starvation-metabolism of a vigorous man engaged in light work and weighing 70 kilos is about 2240 calories or 32 calories per kilo. To maintain calorific equilibrium this heat- value must be contained in the food, and a certain excess to compensate for the stimulation of metabolism or Specific Dynamic Action of foodstuffs. On a normal mixed diet this amounts to from 11.1 to 14.4 per cent, of the starvation-minimum (Rubner). This would indicate calorific equilibrium on an intake of from 2488 to 2562 or, in round numbers, 2500 calories or 36 calories per kilo of body-weight. The total metabolism varies very greatly in different species of animals, the metabolism per kilo being much higher in small animals than in large. This may be inferred from the relative consumption of Oxygen per hour and kilo body-weight by different species. The following results are cited after Rubner: Grams of oxygen Weight, consumed per kilo Species. kilos. per hour. Calf 115. 0.481 Sheep 66. 0.490 Turkey , . . 6.2 0.702 Dog 5.6 0.902 Goose 4.6 0.677 Rabbit 3.43 0.735 Hen 1.51 0.846 Duck 1.22 1.382 Finch 0.025 13.000 Sparrow 0.022 9.595 The greater metabolism of the smaller animals arises, according to Rubner and Richet, from the greater area of external surface in pro- portion to their volume which they present. If the linear dimensions of a solid are increased in the proportion of 1 : 2 the surface is increased in the proportion of 1:4, but the volume in the proportion of 1:8, so that the ratio of surface to volume falls to one-half. The surface of 588 THE ANIMAL BODY AS A MACHINE a regular solid varies as the two-thirds exponent of the volume or as W ! , if we measure volume in terms of weight. Now Rubner has observed that the metabolism per unit of Body-surface is much more uniform in different species than the metabolism per unit of body- weight. E. Voit has determined the heat-production in resting animals of various sizes per kilo and also per square-meter of surface with the following results : Calories produced. Per sq. M. Species. Weight in kilos. Per kilo. surface. Horse 441. 11.3 948 Pig 128. 19.1 1078 Man 64.3 32.1 1042 Dog 15.2 51.5 1039 Rabbit 2.3 75.1 776 Rabbit (without ears) 2.3 75.1 917 Goose 3.5 66.7 969 Fowl 2.0 71.0 943 Mouse 0.018 212.0 1188 The metabolism per kilo in these different species displays the greatest diversity, ranging from 11 calories per kilo in the horse to 212 calories per kilo in the mouse. The metabolism per square-meter of body-surface is very nearly the same in all of the different species investigated, ranging, with the exception of the rabbit, from 900 to 1200 calories per square-meter. Metabolism bears therefore a far closer relationship to surface than it does to weight and the relation- ship extends to different individuals of the same species and explains in part the high metabolism of infants. This relationship, which was discovered by Rubner in 1883 and emphasized by Richet in 1885, was at first interpreted to mean that the main factor governing metabolism was the rate of Radiation of Heat from the surface of the body. Doubt was thrown upon this interpretation, however, by the discovery that the production of heat by warm-blooded animals of different sizes continues to be propor- tional to the body-surface even when the temperature of the sur- roundings is uniform or nearly uniform with that of the body, so that the heat-loss through radiation is a negligible proportion of the total energy-production. On referring to the preceding table it will be noted that in a rabbit deprived of its ears, although the radiating sur- face is much diminished, yet the production of heat remains unaltered. Although the metabolism per unit of surface varies very much less than the metabolism per unit of weight, yet the proportionality of metabolism to surface-area is not nearly so exact as many observers have in the past decades considered it to be. Thus Benedict, in a critical examination of the ratio of Basal Metabolism to surface in eighty- nine men, sixty-eight women and a large number of infants found very marked deviations from the rate of strict proportionality. As Benedict has stated : " It is obvious that any basis of comparison which involves variations of 40 per cent, with men, of 43 per cent, with women, and CALORIFIC REQUIREMENT AND THE SURFACE-LAW 589 80 per cent, with normal infants, cannot be considered as a physiological law." Benedict draws attention to the great importance of specific Stimulators of Metabolism, which may be contained in the diet or in the products of the activity of certain tissues. Thus after prolonged severe Muscular Exertion the metabolism is stimulated for a long period following the cessation of exercise and the consumption of foodstuffs for the production of mechanical work. Yet the ratio of bodily sur- face to volume has undergone no change in consequence of the exercise, nor has the temperature of the body risen. Whatever may be the mechanism which brings it about it is clear that products of muscular exercise (and the same is true of acidosis) induce a stimulated combus- tion of foodstuffs, and therefore, in the absence of ingested food, an increased destruction of tissue. 50 40 30 20 10 / \ <^-N \ _S> -i^ ^== ' - _ === _ i^K= YRS. |0 20 30 40 50 60 70 80 CAL. PER SQUARE METER PER HOUR. FIG. 50. Chart, prepared by Du Bois, showing the basal metabolism as measured in calories produced per square meter of body-surface per hour from birth until the age of eighty-five years in human males. Between maturity and the eighty-fifth year there is a gradual fall in the intensity of metabolism of 13 per cent. (After Lusk.) Benedict infers that the total metabolism, or metabolism at rest with- out food, is determined by two main factors; the first the mass of Protoplasmic Tissues (parenchyma) and the second the variable concen- tration of specific Stimulators of Metabolism in the tissues. It was, in fact, assumed by Voit that the total metabolism is actually propor- tional to the mass of cellular as distinguished from Sclerous Tissues in the body and this view is supported by the steady decrease in metabolism which is characteristic of the period between maturity and old age in man (Fig. 50). The increase in basal metabolism per unit of weight or surface which occurs to a very striking degree during the first year of post-natal growth is, however, only to be interpreted by also taking into consideration the second factor suggested by Benedict, namely the variable concentration of stimulators of metabolism which determines the Metabolic Rate of the tissues. Ihe rise in metabolism which occurs in early growth and just before puberty, therefore, indi- cates an accumulation of stimulators of metabolism which are not improbably the Endogenous Catalyzers of growth. 590 THE ANIMAL BODY AS A MACHINE We must still admit that the ratio of basal metabolism to surface, although variable, is much less variable than the ratio of metabolism to weight, length, temperature, or any other dimension or characteristic of the individual. The possibility has not been sufficiently considered, however, that many details of structural proportion in the body may be correlated with superficial area rather than with weight, and that the observed relationship of metabolism to surface may be thus only an indirect one, representing a relationship of metabolism to a group of structural elements which vary as the two-thirds exponent of the body-weight or volume. Thus Dreyer has shown that the blood- volume and the sectional areas of the aorta and the trachea of animals of different size are proportional to W*, that is, to the surface. Frieden- thal has pointed out that the sum of the non-protoplasmic materials (reserve-materials, skeletal constituents and fibrous tissues) in the animal body increases more rapidly with total size than the proto- plasmic tissues. This is, in fact, inevitable, for the need of binding and supporting tissues increases in proportion to the strains to which the body is subject and these increase not only in proportion to the mass but to the mass X linear dimensions of the body. A small mass of protoplasm requires no binding tissues to support it, but a large mass of cells would collapse of their own weight without binding, cementing and supporting tissues, and the greater the distance of any mass of protoplasm from the center of gravity of the whole, the greater in that proportion will be its tendency to break away. Friedenthal concludes, in fact, that the protoplasmic or Parenchymatous Tissues only increase in proportion to the two-thirds exponent of the total weight, i.e., in proportion to the surface. Since these are the tissues of highest metabolic rate, their mass, together with the proportion of Endogenous Catalyzers which they contain, might be expected to play a leading part in determining the rate of basal metabolism. THE NUTRITION OF CHILDREN. During the early period of post-natal -development the sole normal source of food among the mammalia is Milk. The milk of different species of animals, however, is very far from being of constant composi- tion, and we may infer that the optimal admixture of foodstuffs for sucklings varies greatly with the species. The following table repre- sents the composition of milk of several species, determined by Abder- halden. One hundred parts by weight of milk contain : Species. Casein. Albumin. Total protein. Fat. Sugar. Dog 4.8 2.6 7.4 11.6 3.2 Pig 3.8 1.5 5.2 9.5 3.3 Sheep 4.1 0.8 4.9 9.3 5.1 Goat 2.9 0.8 3.7 4.3 3.6 Guinea-pig ...4.8 0.6 5.4 7.0 2.0 Cow 2.9 0.5 3.4 3.7 5.0 Horse 1.3 0.8 2.1 1.1 5.9 Ass 0.8 1.1 1.9 1.4 6.2 Human . . . . 0.8 1.2 2.0 3.7 6.4 NUTRITION OF CHILDREN 591 Human milk contains more Albumin and much less Casein than cow's milk. This may be only one among many reasons, not readily deter- minable by analysis, why Artificially-fed Infants rarely thrive as well as breast-fed infants. This fact, which has so often been demonstrated and in such a diversity of ways, may be illustrated by the following tabular comparison of the growth of South Australian male infants which were in every respect normal, but which in the one group were fed for at least the first few weeks at the breast, while in the other group modified cow's milk was the source of nutriment: Average weight in ounces of South Australian male infants. Age in months. 1 Breast-fed. 155 Bottle-fed. 117 2 3 187 . 206 141 169 4 . 224 193 5 . 254 226 6 . 270 242 7 ... 287 267 9 . 311 280 The nutritional requirements of children are much greater in pro- portion to their weight than those of adults. The heat-production of infants at various ages is thus summarized by Murlin: Heat production of infants recently fed and sleeping. Calories per square- Calories per kilo meter of surface Age. and hour. and hour. Birth 1.87 25 2 to 4 months 2.38 35 6 to 12 months 2.45 42 Underfed and atrophic infants produced more, and overweight infants less than the heat-output of normal infants. It must be remembered, however, that these figures are subject to considerable modification by a variety of factors, among which Exercise, for example crying, the type and quantity of Clothing worn and the Temperature of the surrounding atmosphere are the most important. The Heat-production per kilo body- weight in an infant during the first year is about 80 calories, while that of an adult does not exceed 36 calories per kilo. The heat-production of the Newborn Infant is much less than at later months, in many cases not exceeding 48 calories per kilo. The heat-production per square-meter of surface also rises during the first year. The allowance of 100 calories per kilo which is adopted by many physicians upon the basis of the older estimations of Heubner is undoubtedly excessive for the average infant. Even taking 80 calories per kilo as a basis, however, the food required by an infant of 10 kilos at one year of age is one-third of that required by an adult weighing seven times as much. This high food-requirement arises from three sources: Firstly the high average Metabolic Rate and the high proportion of Parenchymatous 592 THE ANIMAL BODY AS A MACHINE Tissues in young animals, secondly the larger proportion of surface to volume involving a greater Radiation of Heat than in the adult, and thirdly the energy absorbed in the building up and retention of new tissue. In older children we must add to these the incessant Muscular Activity which characterizes a healthy child. Taylor states that a resting boy of ten years should have a metabolism of about 40 calories per kilo per day, but when engaged in play the diet of the child may have to be as high as 100 calories per kilo per day to maintain calorific equilibrium. "The diet of a child must, therefore, cover the basal metabolism, the natural increment of growth, and the enormous output for physical exercise. It is the inability to judge these fractions correctly that is responsible for so much underfeeding of children. There are furthermore the additional deprivations so often inflicted on children by the application of fad-notions of diet. The relative caloric input of a normal child leading an outdoor life is to be compared to that of a man at heaviest physical work. Protein in excess is not needed, that is clear; but total calories are needed, in the form of sugar and fat." The craving of healthy children for sugar is therefore the expression of a normal and healthy need arising from the high con- sumption of glucose by the Muscular Tissues. It should be satisfied by a discreet allowance of sugar and an abundant allowance of poly- saccharides. THE ENERGY-EQUIVALENT OF GROWTH. The storing-up of tissue-substance which is possessed of a definite calorific value, necessarily results in the retention by the growing animal of a proportion of the energy- value of its food, and, furthermore, a considerable proportion of the , heat- value of the diet, varying with age and the rate of growth, is additionally consumed and dissipated in performing the work of storage. This is doubtless attributable to the fact that at all stages of growth, as at all stages of any chemical trans- formation, the forward and reverse reactions are proceeding side by side. In growth the products of the reverse reaction (tissue-degrada- tion) participate in a side-reaction (Exogenous Metabolism) and are thus partially consumed and their energy- value dissipated in the form of heat, mechanical work, and the energy-values of the excreta. Hence we find that in a given species of animal, the slower the accretion of tissue the greater the energy consumed per kilo of tissue built up, since the reverse reaction in such a case is proceeding for a longer time. The following are illustrative results obtained by Aron: GROWTH OF DOGS. Calories consumed Animal Calories consumed Increase in weight per gram of number. in fifty days. in fifty days. tissue-increase. B 19,950 1570 12.7 C 13,925 1000 13.9 . VIII 9,500 780 16.4 XII 10,750 838 15.6 ENERGY-EQUIVALENT OF GROWTHS 593 From these results it is clear that an animal gaining 1000 grams in fifty days needs fewer calories for this gain than one gaining 1000. grams in one hundred days, the reason being, as indicated above, that in the former instance the animal needs to be "maintained" for only one-half-as long as the latter. The following are comparable observa- tions made by Aron upon Filipino children: From week. 21 26 31 From week. 4 9 To Number MARIA INOCENCIA. Increase in grams. Calories. week. of days. 26 31 35 To week. 9 13 35 31 28 From 3500 3650 4225 to Per day. 3600 3 4225 17 4811 21 MIGUELO PRIEGA. Increase in grams. Per day. 17 24 Number of days. From to 35 3550 4175 28 4175 4850 Per day. Per kilo. 350 to 375 100 to 105 450 115 to 120 500 125 Calories. Per day. Per kilo. 350 to 400 100 450 to 475 105 Hence, during the entire period of the investigation, Maria Inocencia increased in weight at the rate of 14 grams per day and consumed an average of 450 calories per day and 115 calories per kilo. Miguelo Priega, on the other hand, increased in weight at the rate of 21 grams per day and consumed about the same number of calories per day and a considerably smaller number per kilo. According to Rubner, the energy consumed per kilo in doubling the Birth-weight of animals is always very nearly the same, excepting in the case of man, namely about 4000 calories. The following data are presented by Rubner in support of this thesis : Species. Horse . Cow . Sheep . Hog . Dog . Cat . Rabbit Energy-consumption per kilo in doubling the birth- weight. . . . 4512 . . . 4243 . . . 3926 . . . 3754 . , . 4304 . . . 4554 5066 Man . 28864 The generalized form of this relationship would be: E = a log x + b where "E" is the energy-consumption, "x" the weight of the animal and "a" and "b" are constants which are the same for all species (excepting man). Doubling of the weight would obviously always add an equal amount to the quotient -, that is, to the total energy- X consumption per kilo. This leads to the differential or velocity- equation : dE E 38 594 THE ANIMAL BODY AS A MACHINE which means that the consumption of energy per unit of tissue-accre- tion increases in proportion to the energy which has already been consumed in reaching the weight to which this unit of tissue is added. When this rate of energy-consumption becomes equal to or less than the Basal or Maintenance-metabolism it is obvious that growth must cease. The more rapidly growth occurs, however, the less energy derived from exogenous metabolism is expended during the time consumed in build- ing up a unit of tissue at a slower rate. This obviously corresponds very well with the facts ascertained by Aron. REFERENCES. GENERAL : Taylor: Digestion and Metabolism, Philadelphia, 1912. Krogh: The Respiratory Exchange in Animals and Man, London, 1916. Lusk: The Science of Nutrition, Philadelphia, 1919. THE LAW OF CONSERVATION OF ENERGY: Atwater and Benedict: Metabolism of Matter and Energy in the Human Body, U. S. Dept. Agric. Bull., 136, 1903. Benedict and Milner: Ibid., Bull., 175, 1907. Benedict and Carpenter: Carnegie Institute of Washington Pub. 123, 1910. Zeller: Arch. f. Anat. und Physiol., Physiol. Abt., 1914, p. 213. Murlin and Lusk: Jour. Biol. Chem., 1915, 22, p. 15. THE PROTEIN- REQUIREMENT: Chittenden: Physiological Economy in Nutrition, New York, 1907. Albertoni and Rossi: Arch. f. exp. Path. u. Pharm., 1908 Supplement, p. 29. Hindhede: Skand. Ai;ch. f. Physiol., 1912 : 27, p. 87; 1913, 28, p. 165; 1913, 30, p. 97. Rubner: Ueber mod erne Ernahrungs reformen, Berlin, 1914. THE NORMAL DIET: Atwater: Storr's Agric. Exp. Station Ann. Kept. No. 9, 1896. Memoirs of the Nat. Acad. of Sciences, U. S. A., 1902, 8, p. 231. Ergeb. d. Physiol., 1904, 3, p. 497. Mombert: Das Nahrungswesen, Jena, 1904. Rubner: Article in von Leyden's Handbuch der Ernahrung, 1903, vol. 1. ENERGY-REQUIREMENTS AND ENERGY-OUTPUT: Rubner: Die Gesetze des Energieverbrauchs bei der Ernahrung, Leipzig, 1902. Voit, E.: Zeit. f. Biol., 1901, 41, p. 113. Friedenthal: Centr. f. Physiol., 1910, 24, p. 321. Dreyer, Ray and Walker: Proc. Roy. Soc. B. 1912, 86, p. 56. Lusk: Jour. Biol, Chem., 1912-13, 13, p. 155. Fry: Quar. Jour. Exp. Physiol., 1913-14, 7, p. 185. Benedict: Jour. Biol. Chem., 1915, 20, p. 263. Dubois: Arch, of Internal Medicine, 1916, 17, p. 887. STARVATION: Benedict: The Influence of Inanition on Metabolism, Carnegie Inst. Pubs., Wash- ington, 1907, No. 77. A Study cf Prolonged Fasting, Ibid., 1915, No. 203. Cathcart: Biochem. Zeit., 1907, 6, p; 109. METABOLISM OF INFANTS: Abderhalden: Zeit. f. physiol. Chen)., 1898-99, 26, p. 487; 1899, 27, p. 408. Heubner: Jahrb. f. Kinderheilkunde, 1905, 61, p. 430. Aron: Biochem. Zeit., 1910, 30, p. 207. Philippine Jour, of Sc., 1911, 6, p. 1. Berl. klin. Wochensch., 1914, 51, p. 972. Murlin: Proc. Soc. Exp. Biol. and Med., 1914, 12, p. 15. Pritchard: The Infant, Nutrition and Management, London, 1914, p. 71. Benedict and Talbot: The Gaseous Metabolism of Infants, Carnegie Inst. of Wash- ington, Pub. 201, 1914. The Physiology of the Newborn Infant, Ibid., 233, 1915. Am. Jour. Diseases of Children, 1914, 8, p. 1. Murlin and Hoobler: Ibid., 1915, 9, p. 81. VEGETARIANISM : Atwater and Langworthy: A Digest of Metabolism Experiments, Washington, 1898. Ostertag: Handbuch der Fleischbeschau, Stuttgart, 1899. Thomas: Arch. f. Anat. u. Physiol., Physiol. Abt,, 1909, p. 219. McCollum and Davis: Jour. Biol. Chem., 1913, 15, p. 167. McCollum, Simmonds and Pitz: Am. Jour. Physiol., 1916, 41, p 333. THE OUTLOOK. The acquisition of knowledge always results in the revelation of wider and yet wider prospects tempting inquiry and inviting explor- ation. To the Pythagoreans life and the universe were fairly simple, a few rules when once discovered would, they felt sure, reduce the seeming chaos to order. In the laws of number lay the simple clue to the whole riddle. To Descartes, two thousand years later in the history of man and of science, how much more complex did the world appear. Yet even he thought that the phenomena of life could be interpreted by geometry and hydrostatics and that emotions arose through oscil- lations of the Pineal gland, originating from the varying pressures of an impinging fluid. But three-quarters of a century later, Newton, incomparably the greatest discoverer of his age, gazed in awe and humility upon the limitless prospect which his labors had revealed: " I do not know what I may appear to the world, but to myself I seem to have been only like a boy playing on the seashore, and diverting myself in now and then finding a smoother pebble or a prettier shell than ordinary, while the great ocean of truth lay all undiscovered before me. ' ' A new ocean of undiscovered truth : that is the revelation which we glean from every fresh achievement of the scientific method, and this is essentially its most inspiring outcome. It is refreshing, from time to time, to pause amid the fruits of our collective labors and gaze upon the widened prospect which lies before us, striving to make out the dim form of truths which are emerging, half- veiled in the mists of the early dawn of knowledge, upon the horizon of our inquiries. In the territory with which we are here most particularly concerned; that territory which lies upon the borderland of life and of atomic affinities, and seeks to illumine the one with the beacon-lights of the other; the unexplored oppresses us with its vastness and entices us with its promise, while the known, the sure ground of fact, comprises only the fringe of our future heritage of knowledge. In the prospect which lies before us certain objectives lie plainly outlined and almost within our grasp, others are less clearly apprehended and others, again, loom gigantic, unformed, terrible in their potentialities for good or for evil, upon the ultimate horizon of our outlook. In the forefront of our prospect lie, patently enough, the vast industrial potentialities of our science, barely touched as yet, but destined in the near future to be a rich field of endeavor, promising inexhaustible resources of wealth and power, the physical foundations of intellectual achievement. The accumulated storehouses of fuel, 596 THE OUTLOOK deposited in the carboniferous era, and now rendered available to us in the form of coal and oil, have merely served, by one of those happy con- junctions of historical circumstance which have rendered possible the spiritual development of man, to tide us over the period of awakening consciousness and undeveloped powers which comprised the age of steam and the industrial revolution of the nineteenth century. Within a period which is relatively brief in the age-long history of man these stores will be exhausted and we must, as we assuredly will, long ere that term arrives, solve the problem of manufacturing illimitable supplies of fuel. Ultimately there is only one way in which this can be done, and that is by transforming the radiant energy of the sun into the potential energy of a falling weight, originally lifted by the heat absorbed in evaporation, or else, as in the utilization of alcohol for motor-fuel, by converting the radiant energy of the sun into the potential chemical energy of a carbohydrate or a related or derived organic compound. The latter method lies almost within our control, the former not so nearly, and hence it is to the understanding and control of the photochemical synthesis of organic compounds that we must look in the main for our future sources of fuel and motive-power. The initial step of photosynthesis having been accomplished, the succeeding stages in the evolution of organic compounds in living organisms are accomplished at low temperatures through the agency of enzymes. We are gathering acquaintance with the nature of these substances and of the circumstances and principles which govern their action, and through their right understanding and employment we will ultimately be enabled to accomplish syntheses which at present are possible only in living organisms or, if imitable in the laboratory may only be achieved at the cost of an expenditure of energy and raw materials far exceeding the value of the product. The further investigation of the oxidative processes which occur in living organisms and underlie luminescence, is undoubtedly destined to supply us with that hitherto elusive ideal, "cold light/' and the remarkable advances in our knowledge of this field during the past few years, assure us that this outcome of biochemical investigation is not now very far from practical realization. The meteoric advance of aviation, from the air-flotation experiments of Langley to the recent flight from London to Australia, has shown us how rapidly in our times practical realization may follow upon the heels of theoretical possibility. The fuller understanding of the nature of enzymatic processes which lies immediately before us will, ere long, lead to the discovery of their chemical nature and composition. Advances have already been made in this direction. Euler has produced an artificial oxidase, and Falk an artificial lipase. It is not at all improbable that the digestive enzymes are not nearly so complex as the earlier investigators imagined and that the synthesizing enzymes are merely the digestive enzymes or modifications of them, acting under differing physical conditions. THE OUTLOOK 597 The synthesis and control of artificial enzymes will revolutionize the science and art of organic synthesis and place in our hands a multitude of inestimably valuable products which have hitherto been regarded as costly rarities, the curiosities of a chemical museum. At the same time, of course, the production of many substances which are already manufactured, or derived from the cultivation of plants or animals, will be very greatly cheapened. But, above all, the artificial produc- tion and the control of enzymes holds out the hope of accomplishing the synthesis of foodstuffs under conditions independent of climatic variations, and in the immediate neighborhood of the great centers of population, thus eliminating for the great majority of humanity the enormous addition to the cost of food-values which is comprised in the expense of transportation. The synthesis of palatable carbo- hydrates and fats, sufficing for a certain proportion of our dietary, when we once acquire control of the enzymes, should not present any insuperable difficulties. The proteins are a far more complex problem because of the diversity of units of which they are composed and the necessity for the provision of each one of them, nor will the synthesis of amino-acids suffice, for while these satisfy merely nutritional requirements they are not palatable and their ingestion in requisite amounts introduces abnormal conditions into the alimentary canal which are not well tolerated. The synthesis of "protein-sparers" of the type of gelatin, polypeptides which may be utilized with advantage to reduce our protein ration, would doubtless be the first step in this direction. After, all, however, it may well turn out that the most practicable way to synthesize enzymes is to permit organisms to make them for us. Not the complex organisms of the present-day farm, but unicellular organisms which we may cultivate in vats. We have utilized such organisms since the earliest dawn of history to make alcohol and acetic acid for us, and at the present day we utilize unicellular organ- isms, yeasts or bacteria, in the manufacture of bread, of cheese, in the preparation of hides for tanning and other processes of manufacture. This type of industry, which is as yet barely in its infancy, has received a powerful stimulus through the necessities created by the war, and while in the allied countries a special organism was utilized to manu- facture acetone for the preparation of explosives, in Germany yeast was cultivated in media consisting of inorganic salts and glucose, as a means of manufacturing protein. This protein, and the fats, poly- saccharides (glycogen) and vitamines which the yeast-cell also con- tains, might well be employed as a desirable and palatable article for human consumption, but the method in which it was chiefly employed in Germany during the war appears to have been as a concentrated feed, economical of production and transport, for the nourishment of cattle. The gradual replacement of the crude and wasteful, but picturesque and health-giving processes of the farm, interwoven with our remotest origins and endeared to us by innumerable historial associations, by 508 THE OUTLOOK the "sordid" processes of the factory may well seem to many a far from desirable outcome. The scientific investigator, however, like the follower of a religious order, stays not to inquire whether this or that particular consequence of his faith be immediately good or bad in its transient outcome. We cling to the faith that the comprehension of nature will yield ultimate fruits of unalloyed good. The forward march of that comprehension cannot be stayed for the loss of this or that implement of our intellectual youth which must, albeit with poignant regret, be discarded by the way. The ultimate triumph of spiritual over material interests, values, and motives, which is the goal of our understanding, will yield us pleasures upon another plane, as incomprehensible to us, perhaps, as ours are to the primitive savage. Furthermore if the factory is "sordid," that is, after all, not the fault of the knowledge that rendered manufacture possible, but of the de- crepit ideals and stunted imagination of those who utilize our knowledge. The social evils which menace civilization in our day are the indirect outcome it is true of the advances of scientific knowledge, but the responsibility for them rests upon the whole of humanity; they are the visible expression of defective ideals, defective understandings and defective information; they are not of the essence of knowledge, nor does the guilt of their production oppress the soul of the pure seeker after knowledge. An ape knows not how to use fire nor the savage how to use edged tools. Both may hurt themselves with these things, but does it follow then that they are bad or that knowledge of them should be eschewed? Perhaps, after all, the substitution of the factory for the farm may restore, rather than detract from the value of the country to man. Regret it as we may, and long before the factory-synthesis of food- stuffs has begun to be a measurable item in our commerce, the attrac- tivenesss of agriculture as a career is diminishing and has already fallen far below its ancient standard. The restoration of our country- side to untamed nature may serve us after all in good stead, and set free for us the means of enjoying some of the pleasures of primitive man once more, of regaining some of the youth of the world with the intellectual heritage and the securities of an old and complex civil- ization. Returning, for the moment, to more immediately realizable possi- bilities, the utilization of the various products and constituents of living matter, apart from the foodstuffs, is as yet in its infancy. The value of materials arises out of their peculiar suitability for the purposes of man, on the one hand, and their rarity on the other, and the desires and purposes of man are so multifarious in their variety that it may be said that any material possessed of unique physical characteristics will ultimately be found of peculiar utility in satisfying some one or other of our needs. Now among the products of vegetable and animal life, there are numerous substances which are distinguished by their possession of unique physical characteristics. The peculiar properties TJJE OUTLOOK 599 of rubber and of the gums and mucilages, the adhesive quality of gela- tin, the glaze communicated to surfaces by colloids in general and starch and dextrins in particular, and the hard surfaces communicated by the drying oils are already utilized in a multitude of ways in our manu- factures and our daily affairs; but the possibilities held out by the products of life are far frcm being exhausted by these few instances. Among the proteins, for example we find elastin, distinguished by its possession of the rare combination of elasticity and tensile strength without rigidity, spongin exhibiting, although in a different way, a similar combination of qualities, keratin, distinguished by its hardness, insolubility, translucency and ability to take a polish, fibroin distin- guished by its extraordinary tensile strength, lightness and insolubility. These few examples suffice to show us what a variety of physical char- acteristics the various proteins may display, and since these substances do not differ profoundly from one another in structure and compo- sition, we may infer that a relatively slight chemical change may confer upon a protein an entirely new series of physical characteristics. An example of this is afforded by the effect of union with formaldehyde upon the physical characteristics of casein. The proteins are, at present, sparingly employed in the manufac- tures, but casein is used as a substitute for celluloid, and buttons, hair-combs, billiard-balls, and other objects formerly made of ivory or celluloid are now made of casein rendered horny in consistency by treatment with formaldehyde or calcium hydroxide. Casein is further- more utilized as a vehicle for pigments in paints, as a finishing and water-proofing material, and for the manufacture of non-inflammable moving-picture films. The uses of gelatin are manifold and well- known. The employment of the relatively expensive proteins of animal origin in the manufacturing industries, however, is excessively wasteful and cannot continue indefinitely, or expand to very great dimensions. We must seek substitutes for the proteins already used, and new utilities as well, among derivatives of the relatively inexpensive vegetable proteins. The exigencies of the war have, in fact, already called into being a vegetable glue, and a vegetable substitute for casein undoubt- edly merely awaits the seeker. In agriculture, our recent acquisitions of knowledge in the field of growth have already profoundly influenced our practice in the feeding of stock for the market and for breeding purposes. Further advances in^this direction, together with precise knowledge of the time-relations of growth in the various domesticated animals, will ultimately enable us with the utmost precision to define the most economical practice of feeding and the optimal duration of growth for the production of calorific and nitrogenous values. In the growth of perennial crops, also, an exact knowledge of the time-relations of the growth-process will enable us to determine with precision the optimal period of growth which should elapse before cropping. Especially in forestry this knowl- edge will increase the economy of our practice. 600 THE OUTLOOK The biochemical relations between the soil and its bacterial flora on the one hand and the crop on the other is already a flourishing field of investigation, and the results of these inquiries have led to very important improvements in agricultural practice. The further development of this field, and especially the expansion of our knowledge of the metabolism and symbiotic relations of bacteria, will point the way to a multitude of new industrial and agricultural applications. The subject of plant-pathology is also intimately related to biochemistry and the investigation of the biochemical conditions underlying gall- formation, for example, will undoubtedly shed a flood of light upon the essential nature of the internal factors which govern the growth of plants. It is in the practice of medicine, however, that the applications of biochemistry will ultimately come to affect human welfare most directly and profoundly. At the present moment the advances of biochemical knowledge and technique are rapidly furnishing the physician with diagnostic methods of precision, and indications for treatment based upon exact knowledge, where but a few years ago empiricism afforded the sole basis of treatment. The discoveries which lie before us, however, will ultimately transform the scope, and revolutionize the practice of medicine, and the substitution of knowl- edge for empiricism, of science for craftsmanship, as yet barely begun, will not cease until it is complete. The life of man may be regarded from a material point of view as consisting on the one hand of a struggle to obtain nutriment, clothes, and other essentials of existence, and on the other hand a struggle to withstand the deleterious influences of his environment and the imperfections of his own organization. Our environment opposes us with climatic fluctuations and extremes, and with pervading toxic agents, and an ever-present host of parasitic organisms continually seeking, and barely failing in the conquest of our tissues. On the other hand we display the imperfection of our organization in disorders of function and in the culminating disorder of senescent atrophy. Each of these disabilities we are seeking to conquer and in their conquest and control biochemistry must necessarily play a leading if not an absolutely decisive part. Our resistance to toxic agents of environmental or endogenous origin is rendered possible by a peculiar mechanism of adaptation, or " tolerance," which we as yet understand very imperfectly. Its understanding and control will constitute one of the most important among the forthcoming advances of our knowl- edge, and must result, not only in a greatly improved knowledge of the fundamental mechanisms of adaptation, but in throwing a flood of light upon pharmacological science and therapeutic practice. The advances of recent years have demonstrated to us that our resistance to the invasion of parasites is determined by specific chemical agents which our tissues manufacture the various antibodies. The chemical nature of these substances is as yet hardly understood at all, yet this THE OUTLOOK 601 knowledge is fundamental to our control of zymotic diseases. We find that whereas to certain organisms we oppose an impenetrable resistance, to others our resistance is very slight. Our acquired resistance, result- ing from infection or artificial immunization, varies between the same extremes. The transient or inappreciable immunity conferred by immunization in many diseases lays us open continually to their inroads with resulting loss of life and efficiency which have been displayed upon a gigantic scale in the recent world-wide scourge of influenza. The erection of defenses against such plagues, and the common infections of the respiratory or alimentary tracts which are responsible, in the aggregate, for so much loss of effort, time, life and efficiency in the world, w r ill never be possible until we understand the underlying chemical reasons why resistance, natural or acquired, to this disease should be high and permanent and to that, slight and transient, and our understanding of this will in turn depend upon the acquirement of knowledge of the actual chemical nature of the anti- bodies and the precise nature of the processes involved in their inter- action with the tissues or toxins of the invading parasite. The study of these substances and reactions is proceeding apace, and a clear and full understanding of the mechanisms of immunity, while perhaps as yet remote, will unquestionably be acquired. The conquest of zymotic disease has begun, many of the bitterest scourges of the middle ages have disappeared from our lives never to return, and one by one our parasitic enemies are being deprived of power to mar or destroy our lives. Our disorders of function are gradually becoming understood, chlorosis and gout are disappearing, myxedema may be prevented, such conditions as cretinism and asthma are being traced to avoidable origins, diabetes is coming under control, and while cancer still exercises its ravages almost uncurbed that dark problem too now presents some openings which the forth- coming advances of our knowledge of the chemistry of growth will undoubtedly enable us to convert into means of its eradication or prevention; for the problems of pathological growth are fundamentally identical with the problems of normal growth, and the information which sheds light upon the one type of growth will reveal the origin of the other. Senescence alone remains untouched, the final triumph of nature over the human desire to live; but if we can once rid ourselves of the suggestive influence of age-long experience and view the phenomenon impersonally, as the culmination of a definite, understandable and therefore controllable process, we. will perceive that this too must ultimately, fall under the sway of human intellect. The indefinite prolongation of his own life is the manifest destiny of man, and the progress already achieved is certainly not less than that which had been made toward our conquest of the air when Leonardo da Vinci so confidently, and as it then seemed so futilely, predicted that man would ultimately fly. 602 THE OUTLOOK The goal of the biological sciences has been stated by J. Loeb to be the artificial creation of living matter. To this, too, we dare not ascribe impossibility, but its attainment seems at present to be almost certainly more distant than any of the objectives we have hitherto reviewed; for our increasing knowledge of life-phenomena reveals to us more and more clearly that the processes of life are wrapped up, not merely with a peculiar admixture of unstable chemical compounds, but also with a definite architectural arrangement of these compounds. The simplest living organism with which we are acquainted possesses a definite structure, and even supposing our knowledge of the chemistry of life to have become so exhaustive as to permit the precise imitation of the chemical constitution of living matter, its structural constitution would still remain an incentive to investigation and an obstacle, but not an insuperable one, to the attainment of our ultimate goal. The slow, hesitating, clinging grasp of science, like that of the many- ten tacled denizens of the sea, cannot be loosened or evaded. Through many trials and failures, let the superficial appearance which hides the precious truth be as polished and impenetrable-seeming as it may, a flaw will be found, a foothold gained, and atom by atom, through centuries if need be, the very heart of mystery is unveiled. There is not, nor ever can be in our universe, anything which directly or in- directly can be made to assail the senses of man, that his intellect cannot ultimately fit into the supreme architecture of the mind, and there is not, nor ever can be, one thing which the intellect of man fully comprehends which he cannot in some measure appropriate and employ for the direction of his own destinies. But in what way will we employ these powers? That, indeed, is a riddle to which science can furnish no solution; its answer lies hidden from our senses, in the deepest recesses of the moral nature of man; but the responsibility for the choice, whatever it may be, rests not with the scientific discoverer, save only in the degree to which he shares our common humanity. INDEX OF AUTHORS. The numbers in heavy type refer to the bibliographies. A B ABDERHALDEN, E., absorption of proteins, 253; amino-acids in blood, 242; ammo- acid metabolism, 242; amino-N deter- mination, 146; chemical composition of blood, 337, 364; chemical identity of animal proteins, 331; composition of milk, 590; enzyme specificity, 220, 226; infant-metabolism, 594; iron in foodstuffs, 52 ; iron therapy in anemia, | 356; localization of protein synthesis, i 245; polypeptides in urine, 554; pyrrole I grouping, 488; time-relations in hydro 1- j ysis, 210 Abel, J., absorption of proteins, 253; ni- trogenous waste products, 565; vivi- diffusion, 243 Achalme, 227 Acree, 227 Adami, fluid crystals, 102; senescence, 519 Adamson, 309 Aders, 146 Adler, 519 v. Adlung, 561, 566 Albertoni, 594 Albu, 52 Aldrich, 499, 519 Allen, degeneration of islets of Langer- hans, 406; depancreatization, 401; dia- betes, 51, 403, 416 Alsberg, 92 Anderson, 105 Anistchakov, 94, 105 Armsby, 92 Armstrong, 75, 92, 222, 226, 227 Aron, energy-consumption in growth, 592; infant-metabolism, 594; starva- tion-metabolism, 502 Arrhenius, digestion and absorption, 253 ; enzyme action, 226, 425; hydrolysis of ethyl acetate, 211; quantitative secre- tion of gastric juice, 251; transmission of bacterial spores by cosmic dust, 282, 435 Ascoli, 551, 552, 566 Atkins, 261, 283 Atwater, calorific value of diet, 582; conservation of energy, 594; normal diet, 594 ; respiration-calorimeter, 571 ; wastage on different diets, 584 Auer, 333 BACH, 413, 416 Baeyer, 435 Bailey, 105 Bain, 520 Baker, 283 Bancroft, F. W., 333, 450 Bancroft, W. D., 309 Bang, 105 Barcroft, 303, 309, 352 Barger, 186, 200 Barney, 520 Bartell, 306 Batelli, 416 Baumann, 384 Bayliss, 214, 226 Bean, 536 Beatty, 146 Bell, 536 Bendix, 75 Benedict, calorimetry, 594 ; factors deter- mining total metabolism, 589 ; ratio of basal metabolism to body-surface, 588; specific stimulators of metabolism, 589; starvation - metabolism, 416, 594 Bennett, 520 Berg, 226 Bergell, 226 Bernard, Claude, glycosuria, 400; pan- creas, 232; saline cathartics, 315; sugar- content of liver, 86 Bernheim, 536 Berninzone, 227 Bernstein, 442, 445 Berthelot, 186 Bertrand, 186, 412, 413 Bethe, 523 Beutner, 309 Biach, 105 Biddle, 227 Biedermann, 312 Biedl, 389 Biehler, 146 Bigland, 309 Birchard, 150, 153, 172 Blaauw, 430, 431 Blackman, 445 Blair, Bell, 377, 390 Blake, 310 Blasel, 156, 172 Blish, 384 604 INDEX OF AUTHORS Bloor, absorption of fats, 253; absorp- tion of fatty acids, 235; fat-metabol- ism, 416; fats in diabetes, 403, 406 Bohr, 352 Bolhner, 146 Bolin, 413 Bosworth, 105 Botazzi, one-sided permeability in kid- neys, 291; osmotic pressure of sea- water and tissue-fluids, 262, 283 Boveri, 462 Bowditch, 438, 518 Bradley, 223, 361 Briggs, 364 Browder, 470, 505, 520 Brown, A. J., 209 Brown, A. P., 356, 359, 364 Brown, H. T.. 437 Brunner, 441 de Bruyn, 65 Bryan/ 535 Buck, 342, 364 Buckmaster, 362 Bugarsky, 172 v. Bunge, 20; hematogen, 45; hippuric- acid synthesis in tissues, 32, 556; min- eral requirement in foodstuffs, 36, 52 Burge, 414, 416 Burnett, 106, 333; catalase, 414, 146; catalyzers in cancer, 520; influence of temperature on muscle-stimulation, 428; sea-water glycosuria, 271 Burton, 309 Biitschli, cell-division, 466; death, 513; structure of protoplasm, 308 Butterfield, 359, 364 Byk, 435 Cohen, 419, 445 Cohnheim, 238 Conklin, 462, 470 Cook, 316, 333 Cooke, 309, 390 I Corson-White, 106, 505, 520 ! Cottrell, 273 Cramer, 502, 519 ! Cremer, 227 I Croft-Hill, 222, 227 Curtms, 139 Gushing, 400, 494, 498, 519 Cushny, 200 Cutler, 520 Czapek, 445 DAKIN, arginase, 544, 565; hydrolysis mandelic acid esters, 219, 226; nitro- genous waste products, 566; oxida- tions, 416 Dale, 186 Davis, 52, 92, 519, 594 Delage, 450 Delprat, 520 Denis, 94, 241, 253, 390, 566 Descartes, 595 Donaldson, 518 Dore"e, 105 Douglas, 389 Drechsel, 545 Dreyer, 590, 594 Dubin, 566 Dubois, 414, 416 Du Bois, 512, 594 Ducceschi, 283 Dumanski, 309 Duval, 522, 535 CAJAL, 522 Cameron, 153, 536 Cannon, chemical correlation of circu- lation and digestion, 390; pylpric sphincter. 372; suprarenal function, 371, 390 Carlson, 401, 421, 428 Carpenter, 594 Carrel, 387, 520 Cathcart, absorption of proteins, 253; anti-enzymes, 227 ; starvation, 416, 594 Cattell, 390 Chalatov, 94 Chick, 426 Child, 486, 503, 519 Chittenden, 579, 580, 594 Chodat, 413, 416 Chun, 426 Clapp, 146 Clark, 520 Clark, E. B., 75 Clark, G. W., 364, 455, 470 Clark, W. M., 274, 283 Clausen, 420 EBBINGHAUS, 529, 532, 535, 533 Ebstein, 75 Effront, 226 Eijkman, 191 Ellis, 105 Engelmann, 434, 440 Epathy, 523 Erdheim, 387 Erlanger, 253 Euler, 226, 346, 413, 416, 596 Ewald, 421 Exner, 524, 534 FALK, 596 Fano, 283 Faraday, 568 Farkas, 283 Fick, 393 INDEX OF AUTHORS G05 Findlay, 390 Fine, 416 Fischer, E., ammo-acids, 132; amino-N determination, 133, 146; enzyme speci- ficity, 226; lock-and-key hypothesis, 219; mutarotation, 72; peptide for- mation, 139-142, 147; peptide hydrol- ysis, 220; structure of hexoses, 55; synthesis of glycerose, 53 Fischer, M. H., 308, 309 Fitz, 564, 566 Fitzgerald, 332 Fleischer, 519 Folin, absorption of amino-acids, 241; absorption of proteins, 253; atophan, 553; chemical correlation of circula- tion, 390 ; nitrogenous waste products, 566; origin of creatinine, 545; sulphur excretion, 560; uric-acid elimination, 549; uric-acid reagent, 191, 199, 549; vitamines, 200 Fourneau, 140 Fraenkel, 283 Fraser, 105 Friedenthal, cephalization-factor and life-duration, 517, 519; energy-require- ment and output, 594; indicator- method, 275, 283 ; protoplasmic tissues and body -surface, 590 Fry, 594 Fiihner, 369 Fulk, 253 Funk, 191, 200, 519 v. Fiirth, cholesterol, 97, 105; iodothy- rin, 384; melanins, 413; myosin and myogen, 398 G GABRIELT, 283 Galeotti, 164, 420 Gamgee, 351, 364 Gantor, 427, 445 Gardner, 94, 105, 492 Garrod, alcaptonuria, 559; cystinuria, 562-566; urinary pigments, 566; pen- tosuria, 75 Gay, 227, 332, 334 Gies, 226 Gilbert, 536 Givens, 52, 551, 566 Glikin, 105 Gobau, 526, 535 Godlewski, 390, 470 Goodspeed, 426 Gortner, chemical identity of fibrins, 331, 334; humin substances, 384 Graham, 302 Gramentzki, 215 Gray, 390 Grimaux, 139 Grund, 75 Gudernatsch, 486, 519 Gudzent, 552 i Guest, 146 Guggenheim, 200 Guldberg, 162, 203 Gtirber, 328 HALDANE, 389 Halliburton, crystalline form of hemo- globin, 360, 364 ; myosin clot, 398 Hamburger, 260, 266, 283 Hammarsten, blood-coagulation, 343; plasma-clotting, 344; water in tissues, 255 Handovsky, 172 Hanriot, 227 Hanson, 227, 341, 364 Harden, 227 Hardy, inversion of precipitating ion, 159, 163; protein complexes, 171, 330; structure of gels, 298-301, 309; union of acids and bases with proteins, 172 ; viscosity, 309 Hari, 539, 565 Harris, 433, 445 Hart, 519 Harter, 535 Hartmann, 520 Harvey, artificial fertilization, 470; bio- luminescence, 414, 416; surface-layer of cells, 309 Haskins, 545, 565 Hasselbalch, 283 Hedblom, 92 Hedin, anti-enzymes, 227; hematocrit method, 266, 283 Heidenhain, 290, 362 Hekma, 364 Henderson, alkali-reserve, 283 ; fitness of environment, 282; neutralizing-power of acids, 277, 283 ; respiration, 389 Henriques, 242 Herter, 556 Hertwig, membrane-formation with chlo- roform, 451 ; temperature-coefficient of development, 423 Hertz, 316, 333 Heubner, absorption-spectrum of hemo- globin, 359, 364; heat-production in infants, 591, 594 Hewlett, 253 Hildebrandt, 227 Hill, 303, 309 Hindhede, 594 Hinkins, 227 Hirschfeld, 156, 172 Hirschstein, 164 Hoagland, 273, 283, 416, 556, 566 Hocker, 306 Hoeber, 262, 274, 283 Hofmeister, dehydration in protein coag- ulation, 165; protein assimilation, 238; swelling of protein jellies, 309 ; union of protein-groups, 172 606 INDEX OF AUTHORS Hoobler, 594 v. Hoogenhuyze, creatine-content of mus- cle, 399, 416; origin of creatinine, 545, 566 Hooper, 52 Hope, 550, 565 Hopkins, accessory foodstuffs, 193; cal- cium in foodstuffs, 52; conjugated ex- creta, 566 ; endogenous catalyzers, 478; growth-substrates, 519; intestinal putrefaction, 560; pure proteins in growth and maintenance, 490; uric- acid output on purine-ffee diet, 550, 565 Hoppe-Seyler, lecithin-content of em- bryonic tissue, 463; volumes of plasma and corpuscles in blood, 336 Horsford, 327 Howell, blood-coagulation, 346-348; fibrin-structure, 348-349; hemophilia, 347; kephalin, 344; vasomotor theory of sleep, 530 Hoyt, 433, 445 Hiifner, 359 Hunt, 385 Hunter, allantoin, 551, 566; protein-pro- tein compounds, 170, 172; uricolytic index, 551 Hurwitz, 341, 364 IMBERT, 441, 445 Izar, 551, 566 JACOBS, 75, 183 James, 535 Johansson, 539, 565 Johnson, 519 Jolles, 566 Jona, 262, 283 Jones, H. C., 165 Jones, W., 146, 182, 200 Joslin, 403, 407, 416 Jost, 445 KANITZ, influence of reaction on enzymes, 226; temperature-coefficient of heart- beat, 420, 445 Kastle, enzymatic synthesis, 227 ; oxidiz- ing enzymes, 416 Katz, 309, 361 Kellner, 395 Kendall, 384, 387, 390, 489 King, 519 Kleiner, 405 Klugine, 251 v. Knafn-Lenz, 309, 459 Knoop, 66, 416 Knudsen, 253 Kober, 433, 445 Koch, 119 Kocher, 390 Koelker, 210 Koeppe, cytolytic agents, 451; hemato- crit, 266; origin of acid secretions, 327 ; osmotic pressure of cell-contents, 283 Konig, 381 Koranyi, 528, 535 v. Korosy, 309 Kossel, acid-combining capacity of sal- mine, 153; amino-acids, 132; amino-N determination, 146; arginase, 544; ni- trogenous waste products, 565 Krause, 547 Krogh, 422, 423, 547, 565, 594 Kiilz, 392 Kunkel, 52 Kurijama, 92 Ktister, 364 LA FORGE, 92 Lamson, 253 Lander, 105 Landois, 388 Landsteiner, 38 Lane-Claypon, 390 Langley, 596 Langworthy, 584, 594 Lapicque, 52 Laqueur, 368, 389 Lavoisier, 567 Leathes, 332 Leavenworth, 155, 172 Le Bel, 54 Lee, 439 Lehmann, 102 Lepine, 522, 535 Levine, amino-N determination, 146; amino-sugars, 75, 92; chondroitinsul- phuric acid, 92; glucosides, 75; glu- cothionic acids, 91; dinucleotid link- age, 182; nucleic acids, 200; pentoses, 75; phospholipins, 119; thymus nucleic acid, 183 Levites, 172 Levy, 283 Lewis, 166 Lewkowitsch, 119 Liebermann, 172 Liebig, 315, 488, 569 Liesegang, 309 Lifschutz, 105 Lillie, F. R., artificial fertilization, 470; twin pregnancy in cattle, 377, 390 Lillie, R. 8., antagonistic salt action, 333 ; artificial fertilization, 470 ; membrane- formation, 459; muscular contraction, 445; osmotic pressure of proteins, 303, 309 INDEX OF AUTHORS 607 Linder, 158, 163 Lipman, 321, 333 Loeb, J., antagonistic salt action, 318, 322, 333; artificial fertilization, 309, 446, 470; Bunsen-Roscoe law, 431;! crystal-form of hemoglobin, 359, 364; i cytolysis, 459; cytolytic power of ! foreign blood, 453, 470 ; effect of lack of oxygen on development, 460; general physiology, 283; growth, 518; heliotropism, 429-30, 445 ; immortality ! of unicellular animals, 514; influence of reaction on life phenomena and enzymes, 225, 281; memory, 535; oxi- dation in sea-urchin eggs, 459, 461; parthenogenetic frog, 450; protoplas- mic streaming, 467; ratio of sodium to calcium in tissues, 313; rhythmic contraction in jellyfish, 313; Ringer i and Locke's solution, 268; saline cathar- ! tics, 315; salt stimulation, 311; selec- j tive action of tissues, 329; senescence, I 519; swelling in tissues and jellies, 307, j 309 ; synthesis of nuclear material, 463, 470 ; time-relations of voluntary move- ! ment, 528; twin formation, 470 Loeb, L., coagulation of blood, 364; j growth-catalyzers, 519 ; placenta! out- growth, 378, 390; toxicity of white light, 432; wound -healing, 520 Loeb, W., 437, 445 Loevenhart, 227 Loewy, 554 London, digestion of proteins, 241; gas- tric digestion, 247; selective action of intestinal epithelium, 584 Lopez-Suarez, 92 Lubs, 283 Lucas, 427, 428, 445 Ludeking, 309 Luden, 101, 106, 511, 520 Lundsgaard, 283 Lunin, 52 Lusk, carbonaceous waste products, 565 ; dextrose-nitrogen ratio, 403; energy- < requirement and output, 594 ; lactic- ! acid output in phosphorus poisoning, 398; metabolism, 416, 594; specific! dynamic action of proteins, 578 Liithje, 402 Lyman, 553 M McCLENDON, 294, 309, 470 McCollum, essential dietary constitu- ents, 489; growth-substrates, 519; hippuric-acid excretion, 556, 566; in- organic foodstuffs, 52; symbiosis, 92; vegetarianism, 594 McCord, 500, 519 McDougal, 442, 445 McKendrick, 475, 478, 482 McLean, phospholipins, 119, 345, 349 McQuarrie, 341, 364 Macallum, A. B., iron in foodstuffs, 52; kidney-development in protoverte- brates, 270; mineral constituents of serum and sea-water, 269, 283 Macallum, A. B. (Jr.), 191, 200, 519 Macallum, J. B., effect of calcium remov- al on tissues, 333 ; emulsions and sur- face-tension, 309; saline cathartics, 316 Macallum, W. G., 387, 390 Maclean, 119 Macleod, absorption of carbohydrates, 253; chemical regulation of circula- tion, 389; diabetes, 416; equilibrium between ammonium carbamate and carbonate, 545; glucohemia from splanchnic stimulation, 371; glucose metabolism in depancreatization, 405; nitrogenous waste products, 565 Madsen, 213 Magnus-Levy, 409 Maly, 327 Manacelne, 536 Mandel, 91, 92, 398 Mansfield, 416 Marchlewski, 355, 364 Marcuse, 392 Marie, 494, 497 Marshall, 253, 279, 390 Martin, 364, 426, 519, 540 Matthaei, 429, 445 Matula, 156, 172 Maudsley, 524, 535 Mawson, 395 Maxwell, C., 445 Maxwell, S. S., 200, 388, 390, 426, 445, 499 May, 119, 200 Mellanby, 416, 545, 566 Meltzer, 333, 405 Mendel, amino-N content of proteins, 146; dietary essentials, 490, 492, 513; growth substrates, 519; nitrogenous waste products, 566; parenteral ad- ministration of proteins, 239; protein absorption, 253 ; starvation-metabo- lism, 416, 502; symbiosis, 92 Mendenhall, 390 Merckx, 333 v. Mering, 401 Mesernitzky, 463 Metchnikoff, 514, 519 Meyer, G., 75, 253 Meyer, Hans, 293 Meyer, K. F., 341, 364 Meyer, Max, 523 Meyers, 200, 416 Michaelis, 283, 308 Miescher, 463, 470 Milner, 594 Minkowski, 401, 543 Minot, 518, 519, 566 Mitchell, 519 INDEX OF AUTHORS Miyake, 536 Moll, 536 Mombert, 594 Moore, A. R., 309, 470 Moore, B., 309, 424 Moore, T. E., 428 Morawitz, 344, 346, 364 Morgan, 446 Mofgenroth, 245 Morgulis, 92 Morner, 146 Morris, 437 Morse, 416 Mosso, 523, 535 Muirhead, 565 Munk, 236, 522, 535 Murlin, 591, 594 NAGELI, 330 Nathanson, 309 Nencki, ammonia retention by liver, 544; chemistry of hemoglobin, 364 ; urea ex- cretion with Eck fistula, 542 Neubauer, 559 Neuberg, 75 Newcomer, 364 Newton, 595 Nicolaier, 553 v. Noorden, 416 Du Nouay, 520 Nuttal, 92, 331, 334 OCHORWICZ, 536 Osborne, amino-N determinations, 146; copper compounds of edestin, 155; growth on deficient diets, 492, 513; growth-substrates, 519 ; inorganic food- stuffs, 52; oligodynamic action, 330; origin of acid secretions, 328, 334 ; pure proteins in growth and maintenance, 490; starvation, 502; union of acids and bases with proteins, 172 Osier, 337 Osterhout, action of anesthetics on pro- toplasm, 326; antagonistic salt action, 318, 320; electrical conductivity of tissues, 294, 322 Ostertag, 579, 594 Ostwald, William, 166 Ostwald, 518, 536 Oswald, 384 Ota, 165 Overton, isotonic solutions, 264; narcosis, 293, 309; one-sided permeability, 289 ! PALITZSCH, 281 Palladin, 445 Panzer, 104 Parkin, 437, 445 Pasteur, 54, 435 Paton, 388, 389, 390 Patrick, 536 Patten, 146 Pauli, acid-combining capacity of pro- teins, 156, 172; coagulation of pro- teins, 164; electrolyte-free egg-albumin, 164; swelling protein jellies, 309 Pawlow, ammonia retention by liver, 544; digestion, 390; enterokinase, 375; pancreatic secretion, 374; reflex stimu- lation of gastric glands, 372; secretion acid gastric juice, 328; urea excretion, 542 Pearce, 389, 405 Pearl, 519 Pembrey, 389, 539, 565 Pfliiger, 402, 416 Philip, 283 Piccinini, 420 Pickering, 166, 343 Picton, 158, 163 Pieron, 532, 535, 536 Pitz, 594 Plimmer, 147, 463, 470 Poiseuille, 315 Porter, 518 Pottevin, 223 Poynting, 166 Pregl, 554 Pribram, 146 Priestley, 436, 445, 567 Prince, 522, 536 Pritchard, 594 Procter, 305, 309 Q QUINCKE, 308 RAMSDEN, 308 Ray, 106, 519, 520, 594 Rayleigh, 285, 445 Read, B. E., 200 Read, J. M., 472, 519 Reichert, 356, 360, 364 Reid, 303, 309 Reiss, 364 Rhode, 385, 390 Richet, 587 Righetti, 364 Ringer, 311, 411 Roaf, 309, 353 Roberts, 518 INDEX OF AUTHORS 609 Robertson, anti-enzymes, 227; antigenic properties compound proteins, 332, 334 ; calcium removal and stimulation, 333 ; catalyzers in cancer, 520 ; chemi- cal mechanics of cell-division, 470; cholesterol and carcinoma, 106; crys- stal-form hemoglobin, 364; emulsions and surface-tension, 309; enzymatic synthesis, 226, 227; factors determin- ing gestation-period, 390; forgetting, 536; general characteristics of pro- teins, 147; growth, 519, 520; healing of wounds, 520; influence of reaction on enzymes, 226; influence of tempera- ture on life-processes, 445 ; membrane- forming agent in blood, 470; memory, 535; muscular contraction, 445; neu- trality of tissues, 283 ; phospholipins in developing sea-urchin eggs, 463, 470; precipitation and coagulation, 172; protein compounds, 172; rate of ex- traction of colloids, 536 ; refractometric method for globulin-albumin ratio, 340; respiration, 389; serum-proteins, 364; tethelin, 119; viscosity, 309 Rockwood, 239, 253 Rogoff , 390 Rohmann, 164 Rohonyi, 301, 309 Romanes, 313 Rona, 308 Rose, 105, 416, 551, 566 Rosenberg, 359, 364 Rosenheim, 119 Rossi, 594 Rowe, 364 Rowntree, 200, 253, 283 Rubner, calorific output, 513; calorific value of proteins, 570; energy-con- sumption in growth, 593; energy- requirement and output, 594; equical- orific quantities of fats and carbohy- drates, 575; isodynamic value of food- stuffs, 396; metabolism per unit body- surface, 588; metabolism of small animals, 587; normal diet, 594; pro- tein requirement, 594 ; senescence, 519 ; specific dynamic action, 578; total metabolism of different animals, 587 SABBATINI, 342, 364 Sackur, 295, 309 Saiki, 227, 519 Salkowski, 561 Sansum, 253, 361, 416 Saundby, 519 Saxon, 106, 505, 520 Schafer, endocrine organs, 200, 389, 519; microscopic structure of muscle, 442; retardation of growth, 499 39 Scheele, 107 . Schimmelbusch, 348 Schlesinger, 316, 333 Schmidt, A., 343, 345 Schmidt, C. L. A., action of tethelin, 119, 200; antigenic properties of com- pound proteins, 332, 334; influence of reaction on enzymes, 226; globin- deuteralbumose compound, 170, 172 ; globulin-albumin ratio in protein im- munity, 341; modification of Cottrell H electrode, 273, 283 ; serum-proteins, 364; tables pn, H+ and OH~ values, 283; taurine metabolism, 561, 566 Schmiedeberg, 32, 315, 556 Schneider, 389 Schonbein, 412 Schottelius, 92 v. Schroeder, 309, 544 Schultz, 158, 160, 163, 361 Schumm, 359, 364 Schiitzenberger, 138 Scott, 389, 463, 470 Seidell, 193, 200 Sellards, 283 Shaffer, 394, 416 Sharp, 283 Sherman, 47 Sherwin, 566 Shorter,- 309 Sidis, 522, 536 Simmonds, 594 Sjoqvist, 212 Skraup, 146 Smith, 499, 519, 535 Snyder, 420 Sollmann, 200 Sorenson, formol titration,564; indicator- method, 275, 283 Soret, 350, 433 Spain, 520 Starkenstein, 105 Starling, digestion and metabolism, 389, 390 ; origin of lymph, 363, 364 ; osmotic pressure of protein solutions, 302 Stehle, 52 Steinach, 376, 390 Stepp, 519 Stern, 416 Stewart, 336, 389, 390 Stockard, 470 Stockholm, 385, 390 Stokes, 351 Stratz, 519 Sweet, 106, 505, 520 Swift, 536 v. Szily, 283 TALBOT, 594 Tashiro, 427 610 INDEX OF AUTHORS Taylor, amino-N determinations, 146; digestion and metabolism, 416, 594; enzymatic synthesis, 227; fermenta- tion, 226; protamine synthesis, 224; protein requirement, 580; purine me- tabolism, 551, 566; solubility of urates, 552; station of equilibrium in enzy- matic hydrolysis, 223; time-relations in hydrolysis, 210 Thierfelder, 92 Thomas, 584, 594 Thompson, 364, 519 Thomson, 284 Tottingham, 200 Toyama, 364 Tranter, 364 Turner, 253 U AF UGGLAS, 170, 172 Uhlenhuth, 359, 519 Underbill, 253 Usher, 436, 445 VAN SLYKE, absorption of proteint, 253; acidosis, 564, 566; alkali-reserve of blood, 279, 283; ammo-acid equilib- rium in tissues, 561 ; amino-N determi- nation, 144; free amino-groups in pro- tein molecule, 150, 153; hydrolysis of proteins, 147; union of groups in proteins, 172 Van't Hof, enzymatic synthesis, 222 Vernon, 155 Verploegh, 399, 416, 545, 566 Verzar, 368, 389 Vincent, 389 da Vinci, Leonardo, 601 Voegtlin, 200, 390 Volt, C., 518 Vqit, E., growth, 518; heat-production in resting animals, 588, 594; protein assimilation, 238; protein metabolism in work, 394; standard requirement of proteins, 579; total metabolism, 589 de Vries, 263 W WAAGE, 162, 203 Wacker, 106, 511, 520 Wagner, 315 Waksman, 119 ! Walbum, 213 i Walker, 594 I Waller, 438 ! Walters, 210 i Warburg, 460, 461, 565 I Wasteneys, 423, 430, 431, 459, 461, 463, 470 Watson, 561, 566 i Waynick, 333 Wells, 253 Weinland, 227 '- Weir Mitchell, 51 Wells, 200, 240, 364 Weltmann, 105 West, 119 Whetham, 160, 172 Whipple, absorption of proteins, 253; fibrinogen content of blood in phos- phorus- and chloroform-poisoning, 348; iron as foodstuffs, 52; proteose intoxi- cation, 560 Wichmann, 361 Wiedermann, 309 Wiessmann, 514 Wilder, 253, 416 i Willcock, 490, 519 Williams, 192, 193, 200 Wilson, 309, 390 i Windaus, 66 ! Winterstein, 368, 369 i Wislicenus, 393 | Wolf, 441 Woodruff, 519 Woodyatt, 253, 416 Wooldridge, 343, 344, 364 Wright, 344 Wuertz, 331, 334 Wulzen, 499, 519 YOUNG, 227 ZALESKI, 364 Zeller, 576, 594 Ziehen, 522 Zsigmondy, 361 Zuntz, 396 INDEX OF SUBJECTS. of fats, 232, 234 -spectrum of blood, 350 of water from intestine, 252 Accelerative factor in growth, 480 Accessory foodstuffs, 193 hydroaromatic derivatives, 95 Acetic acid, 108 in butyric-acid oxidation, 411 in diabetic urine, 405 in membrane-formation, 448 Acetonitrile, 385 Acetyl choline, 197 number of fats, 110 /3-Acetyl-propionic acid, 174 Achroodextrin, 86 Acid albuminate, 130 number of fats, 110 secretions, origin of, 327 Acidosis, 279, 564 ammonia, utilization in, 545 in children, 409 in diabetes, 276, 405 on fat-diet, 576 on flesh- diet, 586 urinary ammonia in, 276 Acids, production of, in brain during activity, 526 Acree's reaction for proteins, 143 Acromegalic gigantism, 496 Acromegaly, 494, 511 tethelin in, 1 18 Acrpse, 54 Action-current in muscle, rate of con- duction of, 428 Addison's disease, 369 Adenase, 177 Adenine, 174, 178 in antineuritic substance, 193 formula, etc., 176 mononucleotid, deaminization in tissues, 231 Adenine-uracil dinucleotid, 181 Adenosine, 178 Adrenaline, 369, 489, 514 effect of, on blood-pressure, 187 effects of administration of, 370 formula, etc., 197 tests for, 198 Adrenin, 197 Aerobic bacteria, oxygen-requirement, 434 Agalina, 426 Agar jelly, spongy structure of, 298 Agar-agar, 84 Agmatine, physiological action of, 187 Alanine, 134, 398 0-alanine, 189 Albino rabbits, 343 Albumin in blood-serum, 339 in milk, 591 Albuminoids, 127 Albumins, 126, 136 Albuminuria, 342 Albumoses, 132 Alcaptonuria, 558 Alcohol in protein coagulation, 122, 169 respiratory quotient, 538 Alcohol-soluble proteins, 136 Alcoholase, 412 Alcohols, monatomic, permeability of blood corpuscles for, 268 Aldoses, 57 distinction of, from ketoses, 62 Alexin, 454 fixation, 333 Algin, 84 Alimentary glycosuria, 72, 229, 400 Alkali administration and uric-acid removal, 553 albuminate, 130 Alkali-reserve of blood, 273, 279 Alkaline-earth chlorides, in sensitization of eggs to serum, 217 Alkalinity, critical, in tryptic digestion, 217 i Alkaloidal reagents, 122 Alkalosis in parathyroidectomy, 387 Allantoin, 550 Allen's paradoxical law, 408 Alloxan, 175 Alloxantin, 199 Alveolar air, 279, 367 Ambard formula, 564 Ambergris, 101 Amblystoma, thymus administration and tetany, 389 | Ambrine, 101 ! Ameba, protoplasmic streaming in, 444 Amines derived from amino-acids, 184 produced by bacterial action, 185 Amino-acetic acid, formula of, 134 synthesis of, by living tissues, 490 612 INDEX OF SUBJECTS Amino-acid equilibrium in tissues and circulation, 561 radicals in proteins, determination of, 144 lacking in certain proteins, 490 Amino-acids, 121, 131-138 absorption and assimilation of, 240- 246 amines derived from, 184-186 assimilation-limit of, 244 in blood, 337 formula? of, 134-135 heat-output of, 578 proportions of, in vegetable pro- teins, 584 in protein digestion, 240 ultraviolet spectrum of, 433 a>-Amino-acids, 189 Amino-benzoic acid, detoxication of ultraviolet light by, 433 7-Amino-butyric acid, 189 Aminp-ethyl alcohol, 196 a-Amino-glutaric acid, 135 a-Amino-5-guanidine-valerianic acid, 135 a-Amino-iso-caprpic acid, 134 a-Amino-iso-valerianic acid, 134 a-Amino-j8-methyl-j8-ethylpropionic aci d, 134 o:-Amino-normal-caproic acid, 134 Amino-polysaccharides, 88 a-Amino-propionic acid, 134 Amino-succinic acid, 135 Amino-tyrosine, test for, 199 6-Amino-valerianic acid, 189 Ammonia, output of, in acidosis, 545 after parathyroidectomy, 387 as source of urea, 544 uric-acid synthesis from, in birds, I 550 in urine, 557, 564 in acidosis, 276, 576 Ammonium carbamate, 545 carbonate, transformation to urea in | liver, 544 formate, transformation to urea in liver, 544 purpurate, 175 salts, permeability of blood-cor- puscles for, 267 Amnesia, 523, 531 Amphibia, descent of birds from, 262 Amphibian blood, clotting of, 345 Amphoteric acids, 121, 138 character of proteins, 151 Amygdalin, 76, 89 Amylase, 228 in pancreatic juice, 229 Amylin, 86 Amylodextrin, 86 Amyloid, 83 Analysis of proteins, 144 Anaphylactic shock, 188, 239, 361 fat-infiltration of tissues in, 286 methyl-guanidine in urine in, 194 Anaphylaxis after protein ingestion, 240 Anemia, blood-count in, 336 iron therapy of, 356 Anesthetic action of magnesium salts,310 Anesthetics, effects of, on protoplasm, 326 Animal fats, essentiality of, in diet, 585 proteins, calorific value of, 579 Annelids, artificial parthenogenesis of, 449 Antagonistic salt action, 318 origin of, 321 Anterior lobe of pituitary, 199, 499, 505 Antibodies, 239, 331, 600 enzymes a^, 226 in identification of proteins, 224 Anti-enzymes, 226 Antigenic properties of compound pro- teins, 332 proteins, 130 Antigens, 331 Antimony sulphide, colloidal, 158 Antipepsin in intestinal worms, 226 Antiprothrombin, 348 nature of, 349 Antipyrin, effect of, on globulin-albumin ratio, 342 Antiscorbutics, 193 Antithrombin, 347 nature of, 349 in uterine secretions, 378 Antitoxins in diphtheria, 339 Antitrypsin in intestinal worms, 226 Apis mellifica, beeswax from, 112 Apnea, 366, 367 Arabinose, 64 formula, 70 Arabitol, 64 Araboric acid, 64 Arachnidce, 129 hemocyanin in, 350 Arbacia, artificial parthenogenesis in, 446 Arenicola, heliotropism of, 430 Arginase, 544 Arginine, determination of, 145 formula, 135 relation of, to creatine, 195, 388, 548 separation of, 133 as urea precursor, 543 Ariolimax columbianus, nerve-conduction in, 426 Aromatic oxyacids, 558 Arsenic as a foodstuff, 49 compounds, effect of, on metabol- ism, 311 sulphide, 158 Arteriosclerosis, 237 Artificial lipase, 596 parthenogenesis, 446 improved method, 449 Artificially fed infants, 591 Aspartic acid, 135 Aspergillus oryzce, diastase from, 215 Asphyxia, 366 Assimilation-limit of ammo-acids, 244 of carbohydrates, 402 Association in memory, 531 INDEX OF SUBJECTS 613 Asthma, 188, 240 Atophan, 553 Atropine, effect of, on pancreatic secre- tion, 373 in poisoning by choline and neurine, 196 Atwater-Rosa calorimeter, 571 Autocatalysis in oxidation of linseed oil, 111 Autocatalytic formula, 528 application of, to central ner- vous phenomena, 527 Autocatalyzed monomolecular reaction, 475, 477 reactions, 439, 524 curve of, 475 Autodestruction of enzymes, 425 Autohydrolysis, 131 Autolysis, 177 Aveno saliva, heliotropic curvature, 430 temperature-coefficient of, 428 B BACILLUS aminophilus intestinalis, de- carboxylization of amino-acids by, 186 cholerce, relative permeability in, 265 prodigiosus, proteolytic enzymes in, 206, 214 subtilis, antagonistic salt effects of, 321 Bacteria, growth of, 475, 478, 482 curves of, 474 production of nitrogenous bases by, 184 protein metabolism of, 395 Bacterial spores, Arrhenius' theory of origin of life through, 435 resistance of, 282 Balanced solutions, 318 Balanus eburneus, calcium necessary for motility, 325 Barium chloride as a purgative, 316 Basal metabolism in growth, 594 influence of temperature on, 422 ratio of, to body surface, 588, 590 in sea-urchin eggs, 459 Basedow's disease, 386 Beeswax, 112 Benzene administration, globulin-albu- min ratio in, 341 membrane-formation by, 451 oxidation of, in diabetes, 404 Benzidine reaction, 362, 413 Benzoic acid in urine, 412, 556 Beri-beri, 191 Betaines, 189-193 formula, 189 Bicarbonates in blood, 277 neutralizing-power of, 278 Bile, 231 r Bile, channel of sulphur excretion, 560 -concretions, 100 osmotic pressure of, 261 -pigments in bile-concretions, 100 in urine, 105 -salts, 102-105, 233 absorption of, 237 as cholagogues, 105 as cytolytic agents, 451, 453 effect of, on surface tension, 207, 284 Bilirubin, 563 Biological individuality of blood, 356 of tissues, 330 values of proteins, 584 Bioluminescence, 414 Birds, osmotic pressure of blood-sera, 261 Birotatiqn, 72 Birth-weight, energy consumed in doub- ling, 593 Biuret-reaction, 149 with pituitrin, 199 with proteins, 123 Blastula stage in sea-urchin eggs, 462 Blood, absorption spectrum of, 350 alkali reserve of, 273, 279 amino-acids in, 337 ammonia in, 337 benzidine reaction for, 362 bicarbonates in, 277 biological individuality of, 356 chemical composition of, 337 detection of, 361 coagulation of, 342, 348 composition of, 335 cytolytic power of foreign, 453 defibrinated, 336 glucose content of, 337 guaiacum test for, 362 hemin test for, 362 phosphates in, 277 photographic spectrum of, 350 identification of species of, 362 platelets, 337, 344 protein salts in, 277 serum albumin, 339 specific gravity of, 337 titratable alkalinity of, 273 urea in, 337 Blood-pressure, effect of amines on, 186 Blood-serum, proteins in, 339 Bodily heat, 568 surface and basal metabolism, 588, 590 temperature, regulation of, 540 Boiling point of water, elevation of, by dissolved substances, 259 Bolina, 426 Brain, phospholipine, 114 ratio of weight of; to body weight, 517 Bright's disease, 96 i British infants, growth of, 476 Bromelin, 214 a-Brom-iso-capronyl chloride, 141 614 INDEX OF SUBJECTS a-Bromopropionyl chloride, 141 Bronchioles, ergamine effects of, 188 Brucine, 181 Buffer-solutions, 279 Bunsen-Roscoe law, 430 Burns, effect of, on suprarenal cortex, 369 Butter-fat, 409 Butyric acid, 108 in artificial parthenogenesis,440 in membrane-formation, 451 7-n-Butyro-bet aine, 1 90 CACHBXIA strumipriva, 382 Cadaverine, 562 physiological action of, 187 Cadmium sulphide, 158 Caffeine, 176, 563 Calcium carbonate in bile-concretions, 100 chloride in blood-coagulation, 342 inhibition of membrane-forma- tion, 459 effect of removal of, from tissues, 312, 314 excretion, 43 as a foodstuff, 40 ions in blood-coagulation, 342 in metabolism, 387 in menstrual fluid, 377 in milk-clotting, 249 in modified milk, 42 precipitants as nerve stimulators, 374 in blood coagulation, 342 as cathartics and diuretics, 314 salts as catalyzer in adrenaline test, 198 Callianira, 426 Calorie, 391 Calorific requirement and surface-law, 587 value of diet, 582 values of foodstuffs, 569 Canalization-hypothesis, 524 Cancer, 515, 601 death-rate, 581 Cane-sugar, 76. in artificial parthenogenesis, 446 hydrolysis of, 439 photosynthesis of, 437 toleration of, in cliabetes, 404 Cannabis sativa, edestin from, 126 Capillary electrometer, 443 forces, 535 Capric acid in fat metabolism, 410 Caprine formula, 134 Caproic acid in fat-metabolism, 410 Caprylic acid in fat-metabolism, 410 Caramel, 59 Carbamic acid, 544 Carbohydrate esters, 91 Carbohydrate radical in nucleic acids, 173 Carbohydrates, 53, 568 calorific value of, 569 digestion of, 228 energy liberated by, 396 intermediate metabolism of, 391-399 oxidation of, 436 photosynthesis of, 434 source of acetone bodies in urine, 405 Carbon dioxide, assimilation of, by plants, 434 rate of, 437 temperature-coefficient of, 429 determination of, in bloo.d, 279 as fatigue-product in muscles, 524 output, 539 production of, in germinating seeds, 420 respiratory control by, 367, 524 as waste product, 537 monoxide hemoglobin, 352 poisoning, 398 Carbonaceous waste products, 537-541 Carboxethyl-glycyl-glycine ester, 140 Carcinoma, 504 cholesterol content of tissues in, 511 deficiency of cholesterol derivatives in, 101 effect of cholesterol on growth of, 99 of tethelin, 507 spontaneous development of, 511 Carica papaya, proteolytic enzyme in, 214 Carnaiiba wax, 1 13 Carnitine, 395 formula, 190 Carnosine, 189 Cartilage, effect of, on solubility of urates, 552 Casein, 129, 157 anhydrous, 167 combining weight of, 154 compounds, 164 conversion of, to paracasein, 345 effect of introduction of, into circu- lation, 239 electrical conductance of, in solu- tions, 157 as foodstuff, 492 formate, coagulation and precipita- tion of, 168 glutamic acid in, 136 in milk, 591 separation of, in sour milk, 40 rate of solution of, by alkali solu- tions, 535 uses of, 599 Castor oil, 111 Castration, 376 Catalase, 414 Catalysis, mechanism of, 203 Catalyzers of growth, 478, 493, 503 Cataphoresis in protein solutions, 157 INDEX OF SUBJECTS 615 Catechol, 197 -group, essentiality in diet, 480 oxidation of, by enzymes, 414 Cathartics, 314 Cell-contents, osmotic pressure of, 263 division, chemical mechanics of, 466 drawings of, 468 Cellular elements of blood, 335 Celluloses, 82-84 in diet, 231 Central nervous phenomena, autocata- lytic nature of, 527 Cephalization-f actor, 517 . Cerebellar excitation, 315 Cerebronic acid, 91 Cerebrosides, 91 galactose in, 72 hydrolysis of, 197 Ceriodaphnia, heart-beat in, 420 Cerosin, 86 Cetyl alcohol, 112 palmitate, 236 Chlamydomonas pisiformis, heliotropism, 430 Chara, protoplasmic streaming in, 445 Characterization of proteins, 144-147 Chauvenet's criterion, 432 Chemical mechanics of cell- division, 466 of muscular contraction, 438 Children, nutrition of, 590 Chinese wax, 1 12 Chitin, 88 Chloracetyl glycyl-glycine ester, 140 group in polypeptide synthesis, 141 Chloral hydrate, effect of, on fertilized eggs, 460 on pancreatic secretion, 3 74 narcosis, 294 Chlorine in foodstuffs, 49 Chloroform, effect of, on globulin-albu- min ratio, 342 emulsions, 288 membrane-formation, 451 -poisoning, fibrinogen-content of blood in, 348 Chlorophyll, 47, 429 carbon-dioxide assimilation of, 434 fate of, in alimentary canal, 356 formaldehyde synthesis in vitro, 436 relationship of, to hematin, 355 Chloroplasts, photosynthesis of, 436 Chlorosis, 43, 601 Cholagogues, 105 Choleic acid, 104 Cholesterol, 90 absorption of, 237 in bile-concretions, 100 crystal-form, 98 in diabetic blood, 406, 409 in diet, 94, 492 effect of, on development of sea- urchin eggs, 465 on growth of carcinoma, 504, 511 on growth of mice, 505 Cholesterol esters, 101 saponification of, by lipase, 237 in suprarenal cortex, 369 formula, 97 as a growth catalyzer, 500 Cholic acid, 93, 102-104 i Choline in cell division, 468 formula, etc., 196 in nuclear synthesis, 469 oleate, 468 Chondroitin, 92, 129 sulphuric acid, 91, 562 in chondroproteins, 129 Chondroproteins, 129 Chromoproteins, 129 Chyme, 228 discharge of, into intestine, 372 Chymosin, 249 Cinchonine, effect of, on creatine con- tent of muscle, 399 Cipollina's test, 68 Circulatory system, chemical regulation of, 368-371 I Clemmys marmorata, temperature coeffi- cient of heart beat, 420 Clotting of bird and amphibian blood, 345 of blood, 342 effect of, on heat production in infants, 591 Clupeine, 170 Coagulated proteins, 131 Coagulating agents, effect of, on per- meability, 295 Coagulation, 131 of blood, 337, 342, 348 effect of, on structure, 300 of proteins, 121, 158, 164 reactions, 121-122 ! Coagulative power of salts, 164 Cobalt chloride, 166 ! Cod-liver oil, 110 assimilation of, 236 Cold light, 596 Collagen, 127 Collodion gel, sponge structure of, 298 ! Colloids, precipitation, 158 i Color blindness, 348 reactions of proteins, 122 Colostrum, 381 Columnar epithelium, 290 Conduction of stimuli, 426 Conductivity, effect of temperature on, 418 Conjugated excreta, 554-558 glucuronic acids, 554 proteins, 128-130 sulphates, 559 Connective tissue in growth, 510 .proteins of, 245 I Conservation of energy, 567, 571 of matter, law of, 32, 567 Copper ferrocyanide membranes, 257 Coprosterol, 98 Corpora lutea, 377, 378, 493 616 INDEX OF SUBJECTS ( lotion-seed oil, 111 Crcatine, 194, 388, 395, 515 estimation of, 548 formula, 388 muscle content of, in work, 399 in muscles of various animals, 194 stimulation of nerve cells by, 428 in urine, 547 Creatinine, 195, 545-547 formula, 545 output of, in inosite administration, 96 tests for, 548 Creosote, membrane formation by, 451 Cresol sulphuric acid in urine, 556 Cretinism, 382, 601 Cruciferce, glucosides in, 89 Crustacea, chitin in, 89 hemqcyanin in, 129, 350 Cryoscopic method, 260 Crystal habit, 357 Crystalline serum albumin, 339 Ctenophores, 426 Cuorin, 116, 349 Cupric bromide, dehydration of, by salts, 166 caseinate, 167 chloride, dehydration of, 166 effect of, on permeability of paramecium, 295 Curare, 194, 312 effect of, on lymph flow, 363 Curve of forgetting, 535 of growth, carcinoma, in tethelin rats, 507 in cholesterol-fed mice, 505 in tethelin-fed mice, 506 Cyanides, effect of, on cell oxidations,460 on central nervous system, 501 Cyclamen, glucosides in, 90 Cyclodus gigas, carbon dioxide output, 540 Cycloses, 95-96 Cypridina, photogenin in, 414 Cystine, 561-562 detoxication of ultraviolet light by, 433 essentiality for tissue accretion, 490 estimation of, 145 formula, 134 in keratin, 127 separation of, 133 test for, 562 in urine, 562 Cystinuria, 562 Cytidine, 178 Cytolysis, 451, 458 in sea urchin egg, 452 Cytolytic agents in luminescence, 415 Cytosine, 174-175 DEAMINIZATION, 184, 244 effect of thyroid secretion, 385, 486 Dcaminized gelatin, 155 proteins, 124 Death-rate, 581 Decarboxylization, 373 by bacteria and fungi, 184 Decomposition products of nucleic acids. 173 Defibrinated blood, 336 Dehydration in protein coagulation, 158, 165, 167 Dendrites, 522 * Dendrostoma, blood of, as cytolytic agent, 463 i Desoxycholic acid, 104 Deuteroproteose, 130 Development, effect of temperature on, 426 reversal of, 460 Dextrins, 82 i Dextrose-nitrogen ratio, 403 Diabetes, 399-412, 601 acidosis in, 276 blood-fat in, 236 fat tolerance in, 576 inosite in urine, 96 insipidus, 563 mellitus, 399 spontaneous, 402 ; Diabetic coma, 405, 408 puncture, 400 Diacetyl, 195 Diamines, physiological action of, 187 | Diamino acids, 389 carboxylic acids, 133 hydroxy-monocarboxylic acids, 133 mpnophosphatids, 114 trioxydodecanic acids, 133 a-to-Diamino caproic acid, 135 | Diastase, 78, 206, 215 synthetic action of, 223 Diazobenzene sulphonic acid, 199 oi-5-Dibromovaleryl chloride, 141 Dichroism, 351 in hematin solution, 354 Dicystein, 134 Diet, essential constituents of, 488 normal, 582 Dietary, effect of, on bodily dimensions, 485 fads, 51 Diffusion, effect of temperature on, 418" Digestion, 377 and absorption, time and mass rela- tions, 250-252 of carbohydrates, 228-232 of fats, 232-237 of proteins, 238-249 Digitalis purpurea, glucosides in, 89, 90 j Digitonin, 90 j Diglycerides, 107 i Diglycyl-glycine, 141 i Dihydrophenols, 199 Diketo-piperazine, 143, 169 Dilaurate of isomannitol, 235 Dimethyl-guanidine, 388, 395 INDEX OF SUBJECTS 617 Dipeptides, 132, 142 Diphtheria, 339 antitoxin, serum sickness, 240 Disaccharides, 53, 76-81 enzymatic hydrolysis of, 218 Distilled water as cytolytic agent, 451 Diurates, 549 Diuresis, caused by adrenaline injection, 370 by calcium precipitants, 314 Diuretics, 563 Dolphins, spermaceti from, 112 Drying oils, autocatalysis in, 440 Ductless glands, 376 Dulcitol, 63 Duodenum, reaction of contents of, 374 Duration of life, 518 effect of tethelin on, 516 temperature coefficient of, 424 Dysmenorrhea, 378 Dyspituitarism, 498 Dyspnea, 277, 366 Dystrophia-adiposo-genitalis, 497 ECK'S fistula, 542 Eclampsia, 395 creatinine output in, 195 Edema, 308 Edestin, 126, 150, 152 copper compounds of, 155 effect of introduction of, into circu- lation, 239 formation of acid by, in solution, 328 Efficiency of surface tension engine, 441 Egg albumin, 143, 159 digestion of, by pepsin, 212 electrolyte free, 164 osmotic pressure of solution of, 303 precipitation of, by silver ni- trate, 164 recrystallization of, 361 Eggs, hypersensitivity to proteins of, 240 osmotic pressure of contents of, 261 in vegetarian diet, 585 Ehrlich's reaction, 69, 117 Elasmobranchii, osmotic pressure of serum, 262 Elastic tissue, composition of, 245 Elasticity of cell membranes, 264 Elastin, 121, 127, 143 properties of, 599 Electrical conductivity of blood, 336 of protein solutions, 296 of sea urchin eggs, 294 potential changes in muscular con- traction, 443 sign of ions in precipitation of col- loids, 158 Electrolyte-free albumin, 164 Electrolytic dissociation, 260 Electronegative colloids, 159 Electronegative protein, 159 Electropositive protein, 159 Electrostatic tension in gelatin swelling, 306 Elodea canadensis, toxicity of formalde- hyde for, 435 Embryonic tissues, water content of, 502 Emotional glycosuria, 371 states, effect of, on suprarenals, 371" Emphysema on ergamine administra- tion, 188 Emulsification of fat in normal cells, 286 by pancreatic juice, 233 in water, 286 Emulsin, 78, 89, 222 Emulsion, fat, 107 structure of protoplasm, 284 water in oil, 287 Emydura macquarice, osmotic pressure of serum, 262 Emys europea, osmotic pressure of blood serum, 262 temperature coefficient of heart beat, 420 Endocrine organs, 376 relationship of, to growth, 493 Endogenous catalyzers, 478, 482, 502, 503, 509, 589, 590 metabolism, 482, 490 creatinine in, 546 of purines, 549 of sulphur, 560 End-product of hydrolysis, 131 Energy equivalent of growth, 592 transformations in living organisms, 417-445 Enterokinase, 346, 375 Environment, fitness of, 282 influence of, on growth, 484-488 Enzymatic hydrolyses, influence of reac- tion on, 217 Enzyme reactions, temperature coeffi- cient of, 425 Enzymes, 201-226 autodestruction of, 418 inactivation of, 216 influence of temperature on, 213-217 oxidizing, 412-414 quantitative relationships of, 207 reaction, influence of, 217-218 specificity of, 218 synthesis by, 221 thermolability of, 346 Eosin, inactivation of enzymes by, 216 as photochemical sensitizer, 432 Epinephrin, 197 Epithelial cells, radial symmetry in, 292 tissues, growth of, 508 Equilibria in thermodynamical equa- tions, 265 Equilibrium, 503, 513 constant, 353 effect of temperature on, 417 Equisetum, salt antagonism in, 320 Erepsin, 184, 238, 248, 375 618 INDEX OP SUBJECTS Ergamine, 187 relation of, to pituitary extract, 199 Ergot, 187 Ergotoxine, 187 Erucic acid, 235 Erythritol, 267 Erythrocytes, 335 Ether, effect of, on globulin-albumin ratio, 342 Ethereal sulphates, 559 Ethyl acetate, hydrolysis of, 211 membrane formation by, 448 alcohol, effect of, on pancreatic secretion, 374 butyrate, enzymatic synthesis of, 223 hydrolysis of, 221 Ethylene glycol, permeability of blood corpuscles for, 268 Eudendrium, heliotropism, 430-43 1 Euglena viridis, heliotropism, 430 Euglobulin, 339 Exercise, effect of, on heat production; 591 Exogenous metabolism, 482, 485, 494 in growth, 592 of proteins, 580 of purines, 549 of sulphates, 560 thyroid control, 581 urea in, 546 Exophthalmic goiter, 386 External phase of gels, 300 F FATIGUE in memory, 521 muscular, 398, 439 products of nerve centers, 524 in sleep, 530, 532 Fat soluble A, 489 solvents as cytolytic agents, 451 effect of, on cells, 294 Fats, 107-111, 568 absorption of, 232, 250 calorific value of, 569 carbohydrates and, isodynamic val- ues of, 575 digestion of, 232-236 effect of exclusion of, from diet, 403 on pancreatic secretion, 373 emulsification of, in digestion, 233 essentiality in diet, 489 in genesis of acidosis, 405 intermediate metabolism of, 399 respiratory quotient of, 538 as source of muscular energy, 396 Fatty acids, 108 effect of, on surface tension, 284 in olive oil, 467 Fehling's method of sugar estimation, 58 solution, action of, mode of, 413 on nucleosides, 178 Ferric chloride reaction for aceto-acetic acid, 405 for adrenaline, 198 Fertilization, 446 membrane, 447 causative agents of, 294, 450 Fever, temperature coefficient of heart- beat in, 422 i Fibrils, 441 Fibrin, 335, 343 chemical nature of, 348 crystals, 349 Fibrinogen, 336, 343 action of thrombin on, 345 chemical nature of, 348 migration of, toward electrodes, 157 source of, 348 Fibroblasts, 509 Fibroin, 121, 127, 143 properties of, 599 Fibrous tissues, glycocoll content of, 245 Field of consciousness, 531 Fischer's amino-acid method, results of, 146 Fishes, osmotic pressure of blood serum of, 262 Fistuke in digestion studies, 241 Fluid crystals, 102 Fluorine as foodstuff, 49 Foaming in protein solutions, prevention ,of, 274 Foods, definition of, 33 requirement in infants, 591 Foodstuffs, 485 classification of, 33 in growth, 471, 482 as substrates of growth, 488 Forgetting, velocity of, 533 Formaldehyde, action of, on casein, 599 in carbohydrate synthesis, 435 Formic acid, 108, 174 in anaerobic decarboxylization, 184 solution of casein in, 167 Formol titration, 564 Free amino-groups, 149 in unhydrolyzed proteins, 150 carboxyl-groups in proteins, 153 Martin, 377 Freezing-point, lowering of, by dis- solved substances, 259-260 Frohlich's disease, 498 Fructose, 57 photosynthesis of, 437 d-Fructose, 77 Fucus, artificial parthenogenesis in, 450 as source of polysaccharides, 84 Fundulus, eggs, development in balanced solutions, 318 formation of monstrosities, 470 temperature coefficient of heart-beat, 421 Fungi, phytosterols in, 100 production of nitrogenous bases by, 184 INDEX OF SUBJECTS 619 Funnel-shaped pores, 292 Furfurol, 62 G GALACTIN, 86 Galactose, 79-80, 230 in diabetes, 404 distribution in animal kingdom, 72 d-Galactose, 79, 230 Galanthus nivalis, photosynthesis in, 437 Gallois's reaction, 96 Ganassini's reaction, 549 Gas chain, 154 Gastric digestion, 248, 251 juice, amylase in, 228 lactase in, 228 lipolytic action of, 232 quantity excreted, 250 secretion of, 371 Gelatin, 127, 143 in chloroform emulsions, 288 dietary deficiencies of, 577 as foodstuff, 491 gels, velocity of diffusion of crystal- loids through, 296 structure of, 299 effect of coagulation on, 300 glycine content of, 136 indican output on diet of, 555 liquefaction of, by trypsin, 213 properties of, 599 as a protein sparer, 491 swelling of, 305 Gelatinization, 298 Generative organs, chemical correlation of, 376-380 Gestation, period of, 380 Gigantism, 494 tethelin in, 118 Gliadin, 127, 156 amino-groups lacking in, 490 dietary deficiencies of, 577 digestion of, 241 peptides in hydrolysis of, 143 selective absorption of ammo-acids of, 246 Globin, 129, 350 caseinate, antigenic properties of, 332 compound with casein, 170 ultraviolet spectrum of, 351 Globulin-albumin ratio in blood, 339, 340 Globulins, 126, 136, 159 Glucohemia, 79, 229, 399, 402 in diabetic puncture, 400 following intravenous injection of adrenaline, 370 Gluconic acid, 404 Glucoproteins, 129 Glucosamine, 66 in chondroitin, 129 formula for, 69 Glucose, 86 in blood, 337 formation of, from propionic acid in tissues, 411 identification of, in urine, 402 photosynthesis of, 437 in urine, 229 a-5-Glucose, 74, 238 0-5-Glucose, 74 5-Glucose, 71, 77, 230 Glucose-glycogen equilibrium, 400 Glucosides, 76, 89-91 analogy of, to nucleosides, 178 enzymatic hydrolysis of, 218 Glucothionic acid, 91 Glucuronates, 554 Glucuronic acid, 62, 66-67 in chondroitin, 129 formula, 404 naphtho-resorcinol reaction of, 67 origin of, in urine, 555 Glutamic acid, 135 Glutelins, 126 Glutenin, 126 Glycerol, permeability of blood corpus- cles for, 268 solutions, viscosity of, 296 Glycerophosphoric acid, 237 Glycerose, 53 Glycine, anhydride, 139 crystal form, 137 formula, 134 Glycocholic acid, 237 Glycocoll, from hydrolysis of glycocholic apid, 103 in fibrous tissues, 245 synthesis of, by living tissues, 490 in urine, 554 as vehicle of excretion, 556 Glycogen, 71, 82, 86-88, 229, 230 content of heart, 501 distribution of, in body, 88 in muscular work, 391 synthesis of, by diastase, 223 Glycogen-glucose equilibrium, 400 Glycoleucine, 134 Glycosuria, 70, 79, 399 adrenaline, 370 alimentary, 72 emotional, 371 from extirpation of mammary glands, 230 from magnesium chloride in blood, 318 phloridzin, 398 sea-water, 271 Glycyl-glycine, 140, 156 chloride, 139 Goiter, 386 Gonionenus, rhythmic contractions in, 3 13 Gout, 601 uric acid in, 552 Graafian follicle, 377 Grape sugar, 71 620 INDEX OF SUBJECTS Gray matter, insensitivity of, to calcium precipitants, 315 Growth catalyzers, 503 effect of, on parenchyma, 516 curve in female white mice, 512 in normal and pituitary fed mice, 499 cycles, 472-475 effect of cholesterol on, 98 energy equivalent of, 592 guinea-pig, man, tumors, 472 impeding factors in, 478 process of, general characteristics of, 471-484 influence of race and sex on, 484-488 regenerative, 472 relationship of endocrine organs in, 493-500 substrates, 483-493 Guaiaconic acid, 362 Guaiacum test, 413 tincture, 412 Guanase, 177 Guanidine, 177, 194 tetany, 389 Guanine, 174, 176 from thymus nucleic acid, 389 Guanosine, 178-180 Guanylic acid, 174, 178, 180 Guinea-pig, growth of, 472 Gums, 82, 599 HAMMARSTEN'S reaction, 103-104 Heart beat, temperature coefficient of, v 420 ganglion, temperature effects on, 421 in inanition, 501 Heat coagulation of proteins, 122 of combustion, 443, 569 evolution, Lavoisier's work on, 568 production in infants, 591 in resting animals, 588 Heavy metals, oligodynamic action of 328, 330 salts, effect of, on protoplasm, 311 in protein precipitation, 122 Hehner, number of fats, 110 Heliotropism, 429-432 Heller's test, 122 Hematin, 126, 129, 350, 354, 355 Hematocrit, 266 Hematogen, 45 Hematoidin, 355 Hematoporphyrin, 45, 355 Hemin, crystals, etc., 354-355 test, 362 Hemochromogen, 355 Hemocyanin, 129, 350 Hemoglobin, 43, 129, 343 absorption spectrum of, 351 Hemoglobin, casemate, 170 chemistry of, 350 coagulation of, temperature effects of, 426 compounds with oxygen, 352 crystal form, 356 crystals, how obtained, 356 iron content of, 350 in luminescence of pyrogallol, 415 migration of, in electric field, 157 molecular weight of, 353 in nutrition, 488 osmotic pressure of, 302, 303, 353 oxygen saturation of, in respiration, 367 as oxygenase, 413 recrystallization of, 361 Hemolysins, 331 Hemplysis, 90, 266 inhibition of, by proteins, 456 Hemolytic agents, membrane formation by, 451 Hemophilia, 347 Hemopyrrole, 355 Hempseed, edestin from, 126 Hensen's line, 442 Hepatic cirrhosis, 348 Hexoses, 53, 55, 62, 174 Hexylamine, blood-pressure effects of, 186 Hibernating animals, respiratory quo- tient of, 539 Hippuric acid, synthesis of, in kidney tissue, 32 in urine, 412, 556 Hirudin, 343, 349 Histidine, 66, 188, 189 crystal form of, 137 determination of, 145 formula, 135 separation of, 133 Histones, 126, 136 Homarus, composition of blood serum of, 269 Homogentisic acid, 558 Homoptera, wax production by, 112 Honeycomb structure of gels, 300 Hopkins-Cole reaction, 122, 124 Hordein, 127 Hormones, 335 Horse serum, crystalline serum albumin from, 339 Human growth, 473, 475 milk, composition of, 590, 591 Humin, 82 substances, 62, 66 relation of, to iodothyrin, 384 Hyaloplasm, 442 Hydremic plethora, 363 Hydrochloric acid of gastric juice, 228, 328 membrane formation by, 451 specificity of, in pepsin hydro- lyses, 217 Hydrocyanic acid, action of, on aldoses,65 INDEX OF SUBJECTS 621 Hydrogen electrode, 154, 273 ions, as cytolytic agent, 452 in membrane formation, 450 peroxide, 362, 412 Hydrolysis, 131 in development of tissue, 460 in living tissue, 365 of proteins, products of, 130, 155 temperature effect on, 417 velocity of, 201-223 Hydrolyzing enzymes, 201-226 synthetic action of, 221 /3-Hydroxy-a-amino propionic acid, 134 Hydroxyaromatic derivatives, metabol- ism of, in diabetes, 409 Hydroxybutyric acid in diabetic urine, 405 in oxidation of fats, 408 Hydroxyhexahydrophthalic acid, 104 Hydroxyl ions, concentration of, 273 as cytolytic agents, 452 a-Hydroxypyridine, 192 Hydroxy-a-pyrrolidine carboxylic acid, 135 Hymenoptera, wax production by, 1 12 Hypaphorine, 190 Hyperpituitarism, glycosuria in, 400 Hyperpriea, 366 Hyperthyroidism, 386 Hypertonic sea water, 461 in artificial parthenogene- sis, 446, 449 solution, 264, 266 Hypnosis, 522 definition of, 531 Hypnotoxin, 532 Hypophysectomy, 498 Hypopituitarism, sugar tolerance in, 400 Hypotheses in science, 22-44 Hypotonic solutions, 266, 459 Hypoxanthine, 176, 177, 179 enzymatic oxidation of, 412 formula, 549 in oocytin, 455 Hysteresis in reversible gels, 299 ICTERUS, 105 Identical twins, 469 Iminazole ring, 66 Iminazolyl group, essentiality of, in diet, 489 radical, 176 in tethelin, 117 /3-Iminazolyl-a-aminopropionic acid, 135 jS-Iminazolylethylamine, 381 in intestinal mucosa, 373 Immunity, 188 Inactivation of enzymes, 216 Incubation, osmotic pressure of eggs dur- ing, 261 India rubber, inosite in leaves of, 96 sponge structure of, 298 Indican, 554 Indicator method, 275 Indigo from indican, 555 Indole, 124, 186 group, essentiality of, in diet, 489 jS-Indole-a-aminopropionic acid, 135 Indolethylamine, physiological action of, 187 Indoxyl, 67 Indoxylglucuronic acid, 67 Infantile growth cycle, 475 Infants, extra-uterine growth of, 476 Infraproteins, 130 synthesis of, 224 Infundibulum of pituitary, 199 Inhibitive factor in growth, 480 Inorganic environment, 310-327 salts, action of, .on protoplasm, 311 precipitation and coagulation of proteins of, 158 sulphates, 559 Inosinic acid, 174, 178, 179 Inosite, 93, 95, 117 oxidation of, in diabetes, 404 Insecta, chitin in exoskeleton, 88 rhythmic contraction of, in intes- tines, 313 Insoluble serum globulin, 339 Intermediate metabolism of carbohy- drates, 391-399 of fats, 399-412 Internal phase of a gel, 300 secretion, 489 work, muscle tonus in, 392 Interstitial cells of testes, 376 Intestine, absorption of, 250 Intestinal epithelium, one-sided permea- bility of, 291 selective absorption by, 246 putrefaction, 560, 563 on flesh diet, 586 stasis, 555 worms, anti-enzymes in tissues of, 226 Intravascular clotting, 343 Inulin, 71, 82, 86 in diabetes, 404 Inulinase, 86 Inversion of sugar, 77 Invertase, 78, 204, 207, 215 absence of, from digestive juice, 229 specificity of, 219 time relations in action of, 209 Iodine in marine algse, 329 number of fats, 110 reaction for adrenaline, 198 in thyroid, 50, 329, 384 lodothyrin, 384 lonization of proteins, 157, 296 Iron in anemia, 44 content of foods, 47 as foodstuff, 43 in nucleoproteins, 129 in oxidizing enzymes, 413 Irreversible coagulation, 131 622 INDEX OF SUBJECTS Islets, of Langerhans, 401 Isobutylamine, effect of, on blood-pres- sure, 186 Isodynamic foodstuffs, 396 values of, 575 Isolactose, 76, 80 synthesis of, 223 Isoleucine, 134 Isomaltose, 76, 79, 222 Isomannitol dilaurate, absorption of, 235 Isomorphism in hemoglobin crystals, 357 Isomorphous salts, physiological action of, 310 Isotonic solutions, 263 JAFFE'S reaction, 195, 548 Japan wax, 112 Jaundice, 105 Jecorin, 117, 349 Jellies, 298 KEPHALIN, 116, 344 in blood coagulation, 345 saponification of, 196 thromplastic powers of, 346 Kephir yeast, 79, 223 Kerasin, 91 Keratin, 121, 127, 509 composition of, 245 cystine content of, 136 properties of, 599 Ketone structure of sugars, 58 Ketoses, 62 Kidney, development of, in protoverte- brates, 270 one-sided permeability of, 291 Krause's membranes, 442 LACCASE, 346, 412 function o/ manganese in, 413 synthetic, 413 Lactalbumin, 492 Lactase, 78, 79, 223 absence of, from digestive juices, 229 in gastric juice of calf, 228 Lactic acid, 397 in carbohydrate oxidation, 438 effect of, on peptic hydrolysis, 217 fatigue product of, in muscle, 217 in muscle fatigue, 439 oxidation of, in diabetes, 404 of fats and sugars, 399 of glycogen, 397 in respiratory control, 524 Lactic acid in urine, 541 of hibernating animals, 539 (8-Lactic acid, 411 Lactone structure of sugars, 72-74 Lactose, 72, 76, 79, 223 in diabetes, 404 possible forms of, 80 Laminaria, Osterhout's experiments with, 323 Lanoline, 101 non-absorption of, 235, 237 Large intestine, absorption from, 252 Latent period in muscle stimulation, 428 Laurie acid, 235 Lavosin, 86 Law of conservation of energy, 567-575 of mass action, 203 Least squares, method of, in biochemis- try, 26 Lecithin, 90, 115 absorption and digestion of, 237 distribution of, in cell, 284, 286 formula, 113 as growth catalyzer, 500 in nuclear synthesis, 463, 469 retardation of development by, 464 saponification of, 196 source of methylamine in putrefac- tion, 185 Lecithoproteins, 129 Leucine, action of ultraviolet light on, 433 crystal form of, 137 formula, 134 Leucyl-diglycyl-glycine, 142 Leucyl-glycyl-glycine, 141 Leukocytes, 335 Leukocytosis, 341 Levulinic acid, 62, 174 Levulose, 77, 230 in diabetes, 404 distribution of, 71 in urine, 229 Lichenin, 86 Lichens, 86 Lieberman-Burchard reaction, 99 Liesegang rings, 301 Life duration, effect of temperature on, 426 relation of, to cephalization fac- tor, 517 processes, influence of light on, 429 of temperature on, 417 Lifschutz's reaction, 100 Light, influence of, on life processes, 429 Lignification, 84 Lignin, 84 Lignoceric acid, 91 Limulus, composition of blood serum of, 269 effect of temperature on heart gang- lion of, 421 i Linseed oil, 111 JLipase, 107, 115, 234 in gastric juice, 232 INDEX OF SUBJECTS 623 Lipase in pancreatic juice, 233 specificity of, 219 synthetic action of, 223 Lipemia, 237 in diabetes, 406 Lipman's capillary electrometer, 443 Lipoid theory of narcosis, 294 Lipoids, 113 distribution of, in cell, 286 non-antigenic, 332 in suprarenal cortex, 369 surface tension and, 284 Lithium as foodstuff, 49 urate, 553 Liver, deaminization in, 244 extirpation of, effect on uric acid excretion, 543 oxidizing enzymes of, 412 as source of fibrinogen, 348 urea formation in, 542-543 Living matter, methods of study of, 19 Local anesthetics, effect of adrenaline on, 370 Lock and key hypothesis, 219 Locke's solution, 268 Longevity, 518 Lucerne, laccase from, 412 Lupinus luteus, galactin in, 86 Lymph as distributing agent, 335 flow, 363 origin and composition of, 362 osmotic pressure of, 261 Lymphagogues, 363 Lysine, 562 determination of, 145 deficiency of, 491 essentiality of, for tissue accretion, 490 formula, 135 nitrogen, 150 separation of, 133 M MAGNESIUM as foodstuff, 49 formation of monstrosities, 470 purgative action of, 317 in tissue fluids, 269 salts, anesthetic action of, 310 glycosuria in, 399 Maintenance metabolism, 594 Malignant tumors, growth of, 472 Maltase, 78 in pancreatic juice, 229 Maltose, 76, 78, 86 . in photosynthesis, 437 possible forms of, 80 synthesis of, by emulsin, 222 tolerance of, in diabetes, 404 Mammary glands in castrated animals, 377 effect of pituitary extract on, 199 Mammary glands, relation of, to growth of embryo, 379 Mandelic acid ester, hydrolysis of, 219 Manganese in laccase, 412 Mannitol, 63 oxidation of, in diabetes, 404 Mannosaccharic acid, 64 Mannose, 438 Manometer for osmotic pressure meas- urement, 258 Mass law, 162, 203 Master reaction, 478 in food assimilation, 48 in growth process, 483 Meat consumed by different countries. 579 Mechanical work, 574, 576 Medicago saliva, laccase of, 346, 413 Medicine, relation of, to biochemistry, 600 Medulla oblongata, respiratory control by, 365 Melanins, 413 Melibiose, 81 Membrane formation, 448, 450, 458 effect of, on cell oxidations, 461 Memory, 521-524 time relations of, 529 Memory trace, 526, 530 fading of, 532 Menstrual fluid, 377 Mercapturic acid, 562 Mercuric chloride, adrenaline test of, 198 Metabolic rate, 500, 501, 509, 589 of infants, 591 of nervous tissues, 517 in old age, 512 Metabolism, basal, 422, 459, 588, 590, 594 carbohydrate, 391 chemical regulation of, 381-389 of children, 592 fat, 399 intermediate, 391, 399 maintenance, 594 stimulation, of, by exertion, 589 Metamorphosis, 499 influence of thyroid on, 486 Metastases in carcinoma, cholesterol effect of, 504 Metazoa, growth in, 477 Methane in excretions, 541 Methemoglobin, 351 Methyl glucosides, hydrolysis of, 218 glyoxaline, 66 guanidine, 388, 395 formula, 194 tetany, 389 oleate, synthesis of, 223 orange, 271 pyrrole, 355 oi-Methyl-d-glucoside, 73 0-Methyl-d-glucoside, 73- Methylacetate, hydrolysis of, 439, 475 Milk, clotting of, 249 624 INDEX OF SUBJECTS Milk, coagulation of, by rennet, 213 composition of, 590 formation of, in mammary glands, 236 guaiac test for, 362 osmotic pressure of, 261 in vegetarian diet, 585 Millon's reaction, 122, 123 Mineral acids in protein coagulation, 122 constituents of tissue fluids, 268-271 requirements of organism, 34 Mistletoe, inosite in, 96 Molecular concentration, estimation of, 260 solutions, first use of, in physiology, 310 Molisch test, 58 Mollusca, artificial parthenogenesis in, 449 hemocyanin in, 350 Monoamino-dicarboxylic acids, 132 Monoamino-phosphatids, 114 Monoglycerides, 107 Monomolecular chemical reaction, 475 logarithmic formula, 251 Mononucleotids, 178 absorption of, 231 Monosaccharides, 53 distribution of, in living tissues, 69-72 Monourates, 548 Monstrosities, 469-470 Moore's test for sugar, 58 Motility in Balanus larva, calcium neces- sary for, 325 Mucic acid, 64 formula, 404 Mucilages, vegetable, 82 Mucins, 129 glucosamin in, 69 Mucoids, 129 glucosamin in, 69 Multirotation, 72 Murex, ammonium purpurate in, 175 Murexide test, 549 Muscle element, diagram of, 444 fatigue, products of, 524 plasma, 398 Muscular activity in infants, 592 contraction, 391 chemical mechanics of, 438 reactions underlying, 428 exertion, stimulation of metabolism \ in, 589 tissues, glucose consumption by, in children, 592 work, 568 carbon dioxide output in, 539 respiratory quotient in, 538 tissue glycogen in, 391 Mutarotation, 72 Mutton tallow, assimilation of, 235 Mylius' reaction, 103 Myogen. 398 fibrin, 398 Myogenic contractions in skeletal mus- cle, 312 Myqneural junction of sympathetics, stimulation of, 369 Myosin, 398 fibrin, 398 Myricyl alcohol, 112 Myristic acid, 410 Myrosin, 89 Myxedema, 382, 386, 486 N NAPHTHO-RESORCINOL reaction, 67 Narcotics, lipoid theory of, 293 Naunyn plan, 407 Negative phase of intravascular clotting, 343 Nephritis, cholesterol deposition in, 237 globulin-albumin ratio in, 341 urea excretion in, 542 Nerve cells, chemical changes in, 428 passage of impulse in, 523 centers, fatigue products of, 524 fibers, rate of conduction of, 523 impulse, effect of temperature on, 426 Nervous system, growth catalyzers in 517 in inanition, 501 tissues, 514 growth relationships of, 500 Neuberg-Rauchwerger's reaction, 100, 104 Neurine, 116, 196 Neurokeratin, 127 Neurons, 522 in cerebral cortex, stimulation of, by creatine, 195 Neutral fat in circulation, 234 in diabetic blood, 406 as source of acetone bodies, 409 salts in protein coagulation, 122 sulphur, 560 output of, after taurine admin- istration, 561 Neutrality of tissue fluids, 271 Neutralizing power of blood, 277 of saliva, 279 Newborn infant, heat production by, 591 Nicotinic acid, 191, 192 Ninhydrin reaction, 123 Nitriles, 65 Nitrogenous bases, 173-200 derived from guanidine, 194 from hydrolysis of nucleic acid, 174-184 from phospholipins, 196 in internal secretions, 197-200 production of, by bacteria and fungi, 184 metabolism 580 poisons, 514