MANUAL OF VITAL FUNCTION TESTING METHODS AND THEIR INTERPRETATION WILFRED M. BARTON !TY OF CALIFORNIA Manual of Vital Function Testing Methods and Their Interpretation BY WILFRED M. BARTON, M.D. A ssociate Professor of Medicine, Medical Department, Georgetown University, Attending Physician to Georgetown University Hospital andW asking- ton A sylum Hospital BOSTON: RICHARD G. BADGER TORONTO: THE COPP CLARK CO., LIMITED Copyright, 1916, by Richard G. Badger All Rights Reserved THE GORHAM PRESS, BOSTON, U. S. A. TO DE. GEORGE M. KOBER, DEAN OF THE GEORGETOWN MEDICAL SCHOOL, THIS LITTLE BOOK IS DEDICATED, AS A MODEST TOKEN OF APPRECIATION OF HIS UNSELFISH DEVOTION TO OUR SCHOOL AND HOSPITAL PREFACE THERE should always be some valid reason for writing a new book, especially a medical book. My reason for writing, or perhaps to express it more accurately, for compiling this work, is, that the information that it contains is scattered quite broadly thro a wide and extensive medical literature, that may not be readily accessible to all, and which has never, so far as I know, been collected together in book form. Inasmuch as every progressive physician and sur- geon at the present day is making more or less frequent use of the different functional tests, to determine the efficiency of vital organs, it occurred to me that to collect them all in one volume, together with the neces- sary data whereby they might be intelligibly inter- preted, might prove to be useful, particularly to the busy practitioner. The following work, which is an effort to carry out that idea, includes only the tests for so called vital functions, namely those applied to the liver, kidneys, heart, pancreas, and ductless glands. To go beyond this and take up the functional tests of all the organs, such as the eye, ear, nervous system, etc., would be to exceed the legitimate field of true function testing and to encroach upon the well-trodden fields of general semiology and diagnosis. I hope the book may prove to be useful and con- venient to all who are interested in this fascinating and ever-developing field of clinical pathology. 5 6 Preface It is a matter of which American physicians may well be proud that the most substantial and brilliant progress in the development of tests of organic func- tion, particularly in reference to the kidneys and liver, has been brought about by the assiduous efforts of some of their own fellow countrymen. In the chapter on heart I have received valued assist- ance from my friend and colleague, Dr. Thos. S. Lee. CONTENTS TESTS OF LIVER FUNCTION PAGE GENERAL CONSIDERATIONS 13 FUNCTIONAL TESTS TO DISCOVER DISTURBANCES OF THE GLYCO- GENIC FUNCTION OF THE LIVER 14 The Cane Sugar Test 15 The Glucose Test 16 The Levulose Test 16 The Galactose Test 18 Conclusions Concerning the Carbohydrate or Sugar Tests 18 FUNCTIONAL TESTS TO DETERMINE DISTURBANCE OF THE UREA- GENETIC FUNCTION OF THE LIVER 20 Urea Elimination and Nitrogen Coefficient as Criteria of Liver Function 22 Quantitative Urea Estimation in Urine (Marshall's Method} 24 Total Nitrogen Estimation in Urine (Kjeldahl's Method) 28 Augmentation of Urinary Ammonia as an Index of Urea- genetic Liver Function 30 Estimation of Ammonia Nitrogen in Urine (The Formalin Method) 31 Experimental Provocative Ammoniuria 32 Aminoaciduria as a Criterion of Liver Function .... 33 Experimental Provocative Aminoaciduria .... 33 Estimation of Residual Nitrogen in Blood Serum as an Index of Hepatic Function 34 Summary of the Value of Ureagenetic Tests of Liver Function 35 FUNCTIONAL TESTS TO DETERMINE DISTURBANCE OF THE ANTI- TOXIC FUNCTION OF THE LIVER 36 Methylene Blue Test of Toxopexic Function of the Liver (Chauffard-Castaigne Test) 36 Roche's modification of Methylene Blue Test ... 37 Indicanuria, Spontaneous and Provocative as Means of Testing Integrity of Hepatic Fixation 38 Tests for Urinary Indican 38 7 8 Contents PAGE FUNCTIONAL TESTS TO DETERMINE DISTURBANCE OF THE SAN- GUINOPOIETIC FUNCTION OF THE LIVER 40 Estimation of Blood Coagulation Time as an Index of Liver Function. Wright's Method of Fixing Coagulation Time 41 The Fibrinogen Test of Whipple and Horwitz .... 42 Estimation of Fibrinolysis Time as an Index of Liver Func- tion (Goodpasture' s Test) 43 Estimation of Lipase in the Blood as an Index of Liver Function (Whipple's Test) 44 Lowenhart's Methods of Lipase Estimation ... 45 Ghedini's Test .... 46 Application of Abderhalden's Method to Estimation of Sanguinopoietic Liver Function 46 FUNCTIONAL TESTS TO DETERMINE DISTURBANCE OF THE EXOCRIN- ous OR BILIARY FUNCTION OF THE LIVER 47 Tests for Urobilinogen, Urobilin and Bilirubin in the Urine. Interpretation of Results with Reference to Hepatic Function. The Urobilinogen Test (Ehrlich's Test) . . 51 Tests for Urobilin 54 Tests to Determine the Global capacity of the Liver to Eliminate Foreign Substances 55 Phenoltetrachlorphthalein Test of Liver Function (Rown- tree, Horwitz and Bloomfield Test) 55 TESTS OF KIDNEY FUNCTION GENERAL CONSIDERATIONS 64 URINALYSIS AS A CRITERION OF RENAL FUNCTION .... 70 Estimation of Urinary Water. Experimental Polyuria (Albarran's Method) 72 The Water Tests (Straus-Grunwald Method) 75 The Diuretic Tests (Pharmacological) 75 Estimation of Sodium Chloride as an Index of Renal Func- tion 76 The Sodium Chloride Test. Test of Alimentary Chloruria 79 Sodium Chloride Estimation 79 Estimation of Urinary Nitrogen as an Index of Renal Func- tion 80 Diminished and Delayed Excretion of Urea .... 82 Forced Urea Elimination. Provocative Urea Test of McKaskey 83 Estimation of Urinary Coloring Matter as an Index of Renal Function 84 Estimation of Urinary Diastase as an Index of Renal Func- tion . 85 Contents 9 PAGE STUDY OF THE PHYSICAL AND BIOLOGICAL CHARACTERISTICS OF THE URINE AS CRITERIA OF KIDNEY FUNCTION .... 86 Estimation of Freezing Point of Urine (Cryoscopy) as Index of Renal Function (v. Koranyi's Test} .... 86 Electrical conductivity of the Urine as Index of Renal Function 89 Estimation of Urinary Toxicity as Index of Renal Function 89 STUDIES OF THE BLOOD AS CRITERIA OF RENAL FUNCTION . . 90 Urea and Incoagulable or Rest Nitrogen in the Blood as Indexes of Renal Function 90 Marshall's Method for Determination of Urea in Blood . 94 Estimation of Incoagulable Nitrogen. Morris' Modifica- tion of Hohlveg-Meyer Method; Folin and Denis Method 99 Estimation of Blood Coagulation Time as Index of Renal Function 104 Cryoscopy of Blood as Index of Renal Function .... 104 STUDIES OF THE ELIMINATION OF FOREIGN SUBSTANCES, BY THE KIDNEY AS CRITERIA OF RENAL FUNCTION 105 1. Miscellaneous Substances: The Potassium Iodide Test 105 The Phloridzin Test 107 The Hippuric Acid Test 108 The Lactose Test 108 2. Elimination of Dyes Urinary Chromoscopy. . . . 110 The Methylene Blue Test Ill The Indigo Carmine Test 114 The Phenolsulphonephthalein Test of Rowntree and Geraghty 115 GENERAL SUMMARY OF RENAL FUNCTION TESTS 128 SELECTION AND PRACTICABILITY OF RENAL FUNCTION TESTS . . 138 TESTS OF PANCREATIC FUNCTION GENERAL CONSIDERATIONS 142 TESTS OF PANCREATIC FUNCTION WHICH CONCERN THE EXTERNAL OR DIGESTIVE ACTIVITY OF THE ORGAN 144 Proteid Digestion Tests: Estimation of Undigested Protein in Stool as Means of Determining Pancreatic Hypofunc- tion 146 Schmidt's Cell Nuclei Test 148 Sahli's Glutoid Capsule Test 148 FAT DIGESTION TESTS. DETERMINATION OF EXCESS OF FAT IN THE STOOLS 150 10 Contents PAGE STARCH DIGESTION TESTS 153 IDENTIFICATION OF FERMENTS IN EXCRETA AS EVIDENCE OF PAN- CREATIC FUNCTION 153 Demonstration of Trypsin in Stools 154 Demonstration of Trypsin in Stomach Contents . . . 156 Demonstration of Diastase in Feces 158 Demonstration of Lipase in Stools 160 TESTS FOR PANCREATIC FUNCTION WHICH CONCERN THE INTERNAL OR METABOLIC FUNCTION OF THE ORGAN 161 The Cammidge Reaction 162 Loewi's Pupillary Test 165 Spontaneous and Provocative Glycosuria 166 GENERAL CONCLUSIONS CONCERNING PANCREATIC INSUFFICIENCY TESTS 167 TESTS OF HEART FUNCTION GENERAL CONSIDERATIONS 168 Reaction and Muscular Exertion as Basis for Estimating Cardiac Function 171 The Staircase Case (Selig's Test) 173 The Ergometer Test (Graupner's Test) 175 Mendelsohn's Test 179 Katzenstein's Test 181 Herz's Self-Checking Test 183 Gymnastic Resistance Test 184 The Russian Test 185 The Venous Pressure Test (Schott's Test) .... 185 Cardiac Reflex Estimations in Determining Heart Function 187 Sodium Chloride Elimination and Cardiac Function . . 188 Modern Clinical and Instrumental Methods in Cardio- Pathology; Their Applicability to Estimation of Heart Function 188 Sphygmomanometry. Work- Velocity Ratio . . . 188 Cardiac Efficiency Factor of Tigerstedt 193 Cardiac Strength, Cardiac Weakness Ratio . . . .194 Cardiac Overload Factor of Stone 197 Reontgenoscopy and Cardiac Function 199 Sphygmography and Cardiac Function 199 GENERAL CONCLUSIONS AS TO TESTS OF CARDIAC FUNCTION . . 202 TESTS OF DUCTLESS GLAND FUNCTION GENERAL CONSIDERATIONS 240 The Thyroid Gland 270 Contents HYPERFUNCTION OF THYROID GLAND Hypophysis-Extract Test of Claude, Baudouin and Porak Adrenalin-Mydriasis Test of Loewi Experimental Hyperthyroidism Test Aceto-nitril Test of Reid Hunt Metabolic Tests of Hyperthyroidism Complement-Deviation Test of Hyperthyroidism Abderhalden Test in Hyperthyroidism HYPOFUNCTION OF THYROID GLAND Therapeutic Test of Hypof unction The Parathyroid Glands The Thyrnus Gland The Suprarenal Glands HYPOFTTNCTION OF SUPRARENAL GLANDS Sugar Tolerance and Hypoadrenal Function .... HYPERFUNCTION OF SUPRARENAL GLANDS Adrenalinemia and Hyperadrenalism Adrenalin Glycosuria and Hyperfunction .... Complement-fixation in Suprarenal Disease .... The Hypophysis STATES OF HYPERPITUITARISM 246 Increased Gas Exchange and Hyperpituitarism .... 247 Glycosuria and Hyperpituitarism 248 STATES OF HYPOPITUITARISM 249 INDEX . 251 MANUAL OF VITAL FUNCTION TESTING METHODS CHAPTER I TESTS OF LIVER FUNCTION GENERAL CONSIDERATIONS THE liver cell represents the whole organ in minia- ture. If we possessed an adequate knowledge of all the functions of this cell, we would understand com- pletely the functions of the organ. The functions of the liver cell are numerous and each individual cell performs its quantitative quota of all these functions. The liver is provided with two evacu- ating channels, one internal, by way of the blood (he- patic vein), the other external, by way of the biliary passages. It receives blood by way of the portal vein from the digestive tract and its related organs, and dis- charges into the duodenum its more or less complex excretion. Regarded as a center of elaboration, the liver cell has several important functions, chief of which are: 1. ureagenetic, 2. glycogenetic, 3. lipasic, 4. antitoxic or cytopexic, 5. sanguinopoietic, 6. thermic, 7. ferric. The above mentioned functions are spoken of as en- docrinous. The liver has, however, an exocrinous func- tion which by some is regarded as the most character- istic, namely, the excretion of bile by way of the biliary passages. Tests of hepatic function are directed towards the de- termination of the integrity of one or the other of these phases of activity. 13 14 Manual of Vital Function Testing Methods There is no organ whose functional and clinical ex- amination is fraught with more difficulties than the liver. Slight functional disturbances of the organ are attended by very uncertain symptomatology. When severe or- ganic lesions exist, such as acute yellow atrophy, cirrho- sis, abscess, cancer, etc., modifications in the size of the liver, as well as its consistence, together with symptoms of portal hypertension (ascites) and of bili- ary obstruction (icterus), offer a combination of objec- tive evidence which makes the diagnosis usually clear. But there is a longer or shorter period in all these diseases, usually in the earlier stages during which a condition of hepatic insufficiency (hypohepatism) and more rarely, hepatic hyper function (hyperhepatism) exists, which might be recognized by appropriate tests. The trend of modern investigation is in the direction of the development of practical functional tests of sufficient simplicity to enable the clinician to judge the functional capacity of the liver, before gross organic lesions or indubitable symptoms have appeared. Tests for liver functional capacity may be profitably considered under five headings. 1 The first four are endocrinous, the fifth exocrinous. They are as follows : I. Disturbance of the glycogenic function. II. Dis- turbance of the urcagenetic function. III. Disturbance of the antitoxic function. IV. Disturbance of the hemapoietic function. V. Disturbance of the biliary function. I. FUNCTIONAL TESTS TO DETERMINE DISTURBANCES OF THE GLYCOGENIC FUNCTION OF THE LIVER Normally the liver cells retain in the form of glycogcn almost all the glucose brought to them from the ali- 1 See Les Precedes actuel d'etude de 1'insuffisance hepatique. Gaz. d. hop. Par. 1914, no. 25, p. 408 (Brule, Garban). To this article is appended an extensive bibliography. Tests of Liver Function 15 mentary tract. Under certain conditions of liver in- sufficiency, glucose is not fixed by the cells, and passes immediately into the blood, producing a hyperglycemia, from whence it is excreted in the urine. This fact has been utilized as a basis for testing the glycogenetic integrity of the liver cell. The name of Claude Bernard is closely linked with the history of the physiology of hepatic glycogenesis. Soon after the discovery by Claude Bernard of the role of the liver in carbohydrate metabolism, the use of sugars as tests for hepatic function began. In applying the sugar test, different varieties of sugars have been employed, particularly saccharose (cane sugar), glucose, levulose, and galactose. The first sugar employed for this purpose was saccharose (cane sugar). The sugar tests of hepatic function are four in number, as follows: 1. The Cane Sugar test. 2. The Glucose test. 3. The Levulose test. 4. The Galactose test. 1. The Cane Sugar Test. Colrat, Lepme Test 150 to 200 grams of cane sugar syrup are adminis- tered to the subject in the morning while fasting. The urine is collected every hour or two and examined for sugar with Fehling's solution or other means. The presence of glycosuria renders the test positive. The cane sugar test was considered for a long time the best criterion of liver insufficiency. The clinical results of the test have, however, been contradictory. A rather weighty theoretical objection is the fact that cane sugar must be converted into glucose in the ali- mentary tract before it can be utilized by the organism, and the power of the intestinal juices to produce this 16 Manual of Vital Function Testing Methods conversion is in each case an unknown quantity and therefore a source of error. #. The Glucose Test The patient takes in the morning on an empty stom- ach, 150 grams of pure dextrin-free glucose dissolved in 300 c. c. of water. The ingestion of this amount should not take over a quarter of an hour. The urine is then collected every hour or two for ten hours in separate vessels and tested for sugar, the patient remaining on a milk diet during the time re- quired by the test. Castaigne has advised the following details with a view of perfecting the glucose test. For several days prior to the performance of the test, the subject should be kept on a certain known quantity of carbohydrate. The renal permeability should be investigated and the possibility of spontaneous glycosuria especially after meals eliminated. The results of the glucose test have been rather con- flicting and some investigators have appeared to find glycosuria following the test in apparently healthy sub- jects and its absence in certain cases of hepatic cirrhosis where the liver parenchyma would have been acknowl- edged on general clinical grounds to have been damaged. 3. The Levulose Test. Strauss Test This test was introduced by Strauss 2 in 1901 as a substitute for the saccharose and glucose tests. ' Berl. klin. Wchnschr., 1898, XXXV, p. 398, and 1899, XXXVI, p. 1.59; Deutsch. med. Wchnsch., 1901, XXVII, p. 756, also 1903, XXXIX, p. 1T80. Tests of Liver Function 17 To apply the test 100 grams of levulose are given in the morning on an empty stomach and the urine evacu- ated every four hours thereafter for a day and examined for sugar by the fermentation test or polariscope. Owing to the high price of levulose, honey, which contains a large percentage of it, has been advised as a substitute. A normal person should tolerate 100 grams of levu- lose without levulosuria. The rationale of this test was founded on the experi- mental work of Sachs, 3 who found that frogs whose livers had been removed had a lower tolerance for levu- lose than intact controls. With dextrose and galactose this was not the case. It was contended therefore that there is no mechanism besides the liver capable of handling levulose, while there is such an extra hepatic mechanism in the case of glucose. Immediately after its introduction, this test came Into pretty general use and was commended by Ferra- nini 4 v. Halasz, 5 Bruining, 6 and others, and was con- demned by Landsberg, 7 Churchman, 8 and others. Much was expected from the levulose test because as above stated it was believed that the liver alone is concerned in levulose metabolism. However this may be, experi- ence has apparently failed to substantiate the hopes which the test originally inspired and it is not now be- lieved that the levulose test is essentially superior to other sugar tests of hepatic function. "Zeitschr. f. klin. Med., 1899, XXXVIII., p. 87. *Zeitschr. f. inn. Med., 1902, XXIII, p. 921. 8 Wien. klin. Wchnschr., 1908, XXI, p. 44. "Berl. klin. Wchnschr., 1902, XXXIX, p. 587. 'Deutsch. med. Wchnschr., 1903, XXIX, p. 563. 8 Johns Hopk. Hosp. Bull., 1912, XXIII, p. 10. 18 Manual of Vital Function Testing Methods 4- The Galactose Test. Bauer's Test Forty grams of milk sugar dissolved in 400-500 c.c. of tea are taken in the morning on an empty stomach. The urine is passed every four or five hours thereafter and examined for sugar. In icterus gravis and catarrhal jaundice this test has been reported as giving fairly constant results. Bauer 9 considered the galactose test especially adapted to determining the condition of liver function in catarrhal jaundice. The amount of sugar recovered in the urine after the galactose test was found to be greater in catarrhal jaundice than in obstructive jaun- dice, consequently it was supposed that the test would be of importance in differential diagnosis between the two conditions. This opinion was upheld and confirmed by Bondi and Kb'nig, 10 Riess and Jehn, 11 and Hi- rose. 12 Outside of catarrhal jaundice the results were pro- nounced inconstant by Falk and Saxl, 13 v. Frey, 14 and others. Conclusions concerning the Sugar Tests. It may be said that the investigation of hepatic insufficiency by any or all of the sugar tests, is to be regarded merely as supplementary or complementary to other means of investigation, since the results of these tests alone are not conclusive. Nevertheless the results which may be obtained by their help when associated or corelated with "Wien. med. Wchnschr., 1906, LVI, p. 2557. 10 Wien. med. Wchnschr., 1910, LX, p. 2617. "Deutsch. med. Wchnschr., 1912, XXXVIII. 12 Deutsch. Arch. f. klin. Med., 1912, CVIII, p. 187. 13 Ztsch. f. klin. Med., 1911, LXXIII, p. 131, 325. "Ztschr. f. klin. Med., 1911, LXXII, p. 383. Tests of Liver Function 19 those obtained by other methods are of sufficient value to justify their retention in clinical medicine. It is now understood that the mechanism whereby alimentary glycosuria is produced is more complex than was formerly supposed, and that the liver is not the only organ involved in the process. Other tissues are now known to be concerned in glycofixation and mobil- ization. Furthermore, the individual coefficient of sugar utilization has been found to vary within quite wide limits, 50-350 grams of levulose for example. The co- efficient varies also in the same individual for the dif- ferent sugars so that in reporting results of sugar tests it is deemed expedient to specify the particular kind of sugar used. The unknown factors of intestinal absorption and renal permeability complicate all sugar tests. The sugar tests have been found positive, especially in severe bivenous cirrhosis, in icterus gravis and in cholelithiasis. They are, however, of no prognostic value. A recent comprehensive study of the applicability of carbohydrates as tests for hepatic functional activity has been made by Bloomfield and Horwitz. 15 These authors call attention very properly to the factors which tend to render the sugar tests for hepatic func- tion unreliable. The great theoretical stumbling block in the way of accepting the finding of the carbohydrate tests is the fact that extra-hepatic factors of consider- able importance are concerned in the sugar regulating metabolism. Some of the glands of internal secretion take part in this. Certain lesions of the hypophysis may cause glycogenolysis and glycosuria, while with other hypophyseal lesions the sugar tolerance is in- 15 Johns Hopk. Hosp. Bull., 1913, XXIV, p. 375: a good bibli- ography is appended to this article. 20 Manual of Vital Function Testing Methods creased. The internal secretion of the pancreas is generally considered to exert an inhibitory effect on the mobiliza- tion of glycogen by the liver. The suprarenals have an accelerating effect on the same mechanism. The thy- roid gland also takes part in sugar metabolism, the exact nature of which is unknown. Both the autonomic and sympathetic nerves likewise affect the mobilization of glycogen. These last facts, of course, tend to render the sugar tests less definite and satisfactory. From a practical standpoint it cannot be denied that the sugar tests have certain disadvantages. It is diffi- cult for patients to ingest the large quantities of sugars required without the occurrence in certain cases of nausea, vomiting and diarrhoea. Faulty absorption, intestinal fermentation, portal obstruction with col- lateral circulation, sugar retention due to nephritis, inconstancies in the diet also combine to render the results inaccurate. It will remain for the future, however, to determine whether the sugar tests of hepatic insufficiency can be developed or modified in such a way that the rather numerous objections with which impartial observers are agreed the tests are encumbered, may be eliminated. In such an event these tests, which from an historical standpoint are so interesting, may take a definite place, even though subsidiary, in that important group of tests by which one seeks to obtain an insight into the functional integrity of the liver. II. FUNCTIONAL TESTS TO DETERMINE DISTURBANCES OF THE UREAGENETIC FUNCTION OF THE LIVER All the tests for hepatic function which deal with the ureagenctic activity of the liver are concerned with Tests of Liver Function 21 the question of nitrogen metabolism. The liver plays a large and important part in this metabolism; perhaps not so exclusive a role, however, as was formerly sup- posed. The ureagenetic function tests are, for the most part, merely studies of nitrogen metabolism. They consist mostly of estimations of the amount of nitrogen elimi- nated in the urine with accurate partition of this nitro- gen into different groups, particularly urea and am- monia. The relation between the amount of nitrogen eliminated in these two forms when compared with the total nitrogen eliminated will afford valuable criteria for estimating the ureagenetic functional capacity of the liver. The practical estimation of ureagenetic functional capacity of the liver involves much more complex series of chemical processes than were found to be involved in the investigation of the carbohydrate function by means of the sugar tests. Inasmuch as the first criterion of normal liver function from the standpoint of nitrogen metabolism involves the relation between the amount of urea excreted in the urine and the amount of total nitrogen excreted therein, the investigator must make two separate chemical analyses. First the exact amount of urea excreted must be estimated, and second, the exact amount of total nitrogen eliminated. In discuss- ing these analyses, no attempt will be made to give any data beyond the description of such methods which, on account of their comparative simplicity and established accuracy, have become firmly established in the clinic. The estimation of total nitrogen in the urine by the standard method that of Kjeldahl requires a good deal of time and some technical skill. But it is a method which can be easily performed in any well-equipped laboratory attached to a hospital, and should be car- 22 Manual of Vital Function Testing Methods ried out by a competent person. A rapid and accurate method of quantitative estimation of urea has been lately devised that of Marshall and this method has already become the standard one in the clinic. The estimation of urinary ammonia, or ammonia nitrogen, is another step required in the study of the efficiency of the liver from the standpoint of nitrogen metabolism. Here also it is best for the clinician to familiarize himself with one method, preferably that of Folin, the details of which are further on discussed. The actual administration of ammonium-bearing or amino-acid-containing substances to the patient, with the subsequent examination of the urine to determine the capacity of the liver to convert these substances into urea, is another method of clinical investigation of the liver function. Finally it is conceded that the estima- tion of rest or residual nitrogen in the blood serum may be utilized to determine whether or not the liver cell is capable of producing an adequate urea synthesis of nitrogen products in the body. To recapitulate, we may tabulate the different meth- ods of testing the ureagenetic functional power of the liver under the following heads and in this order they will be discussed: 1. Urea Elimination and Nitrogen Coefficient as Criteria of Ureagenetic Liver Function ; 2. Augmentation of Urinary Ammonia as an Index of Ureagenetic Liver Function ; 3. Amino-aciduria as a Criterion of Ureagenetic Liver Function ; 4. Estima- tion of Residual Nitrogen in the Blood Serum as an Index of Ureagenetic Liver Function. 1. Urea Elimination and Nitrogen Coefficient as Criteria of Liver Function Urea Elimination,- The liver has long been regarded as the chief source of urea, which substance is supposed Tests of Liver Function 23 to be formed from ammonium salts, amino acids and products of nitrogenous catabolism. The urea synthesis is supposed to be the work of the liver cell governed by ferments which it secretes. It has long been recognized that in certain diseases of the liver the percentage of urea eliminated in the urine is lowered. Therefore diminution of urea excretion may sometimes indicate hepatic insufficiency and an estimation of the quantity of urea eliminated is there- fore an available factor in testing the ureagenetic func- tion of the liver cell. The quantity of urea eliminated in the urine by a healthy adult in 24 hours is 25 to 30 grams. In cases of hepatic insufficiency this quantity may fall to 10, 5, 3 or .5 grams or even in icterus gravis. But before one can attribute a diminution of urea elimination to functional disorder of the liver, certain other factors of great importance must be taken into consideration. One of these is the functional capacity of the kidney. It is well understood that urea retention in the blood may be due to defect of kidney permeability. Another important factor is the amount of proteid intake. Before attempting to draw any conclusions with respect to the relation of urea elimination to the ureagenetic functional capacity of the liver it will al- ways be necessary that the individual to be tested shall be placed for a sufficient length of time upon a fixed ration in which the amount of proteid is known and invariable. The Nitrogen Coefficient. The relation of urea ni- trogen to the total nitrogen excreted in the urine is known as the coefficient of nitrogen elimination. This coefficient may be important since under certain circum- stances its diminution may constitute a valuable sign of ureagenetic hepatic insufficiency. 24 Manual of Vital Function Testing Methods The nitrogen coefficient is usually expressed by the following fraction: N. urea N. total the arithmetical relation in other words between the amount of nitrogen eliminated in the urine as urea and the total quantity of nitrogen eliminated. This co- efficient will diminish in proportion to the diminution of urea nitrogen. According to some authors a diminution of the ni- trogen coefficient is absolutely constant in hepatic in- sufficiency. The normal figures of the nitrogen coefficient vary from 85% to 95%. In icterus gravis it has been found reduced to 40 ; likewise in phosphorus poisoning. But just as in estimating the percentage of urea in the urine and considering the same as an index of he- patic function, so also in determining the coefficient of nitrogen elimination for the same purpose the patient must be placed on a fixed and invariable proteid regi- men, though the absolute quantity of proteid taken is negligible. It must also be previously known that there is no deficiency in renal permeability. To determine the co- efficient of nitrogen elimination, two operations must be performed. First, a quantitative estimation of urea must be made, from which the calculation of urea nitro- gen may readily be accomplished. Secondly, the total nitrogen eliminated in the urine must be estimated and this part of the operation is, unfortunately, somewhat difficult and time-consuming. The simplest known means for performing these operations will be given. Quantitative Estimation of Urea in Urine. Several Tests of Liver Function 25 methods are in use for determining quantitatively the amount of urea in urine. One of the most frequently used is the hypobromite method, using the ureometer of Doremus. In this method nitrogen is set free by sodium hypobromite and measured in the apparatus. The re- sults obtained by this method are extremely inaccurate and it is not used, therefore, where absolute results are required. Three other methods which are much more depend- able have been in use for some years in the laboratories. These are: 1. The Morner-Sjoqvist; 16 2. Folin's method ; 17 and 3. Schondorff's method. 18 The details of these methods may be found in any modern text book on laboratory methods. We shall not give a descrip- tion of them here. Marshall's Method of Urea Estimation. Quite re- cently a rapid chemical method for the estimation of urea in urine has been introduced by Marshall of Johns Hopkins. 19 It depends upon the conversion of urea into ammonium carbonate by means of an enzyme prepared from soy bean. This enzyme is called urease because of its facility in effecting this conversion. Urease is found in some bacteria and fungi. The following formula represents the chemical decomposition produced: NH 2 ONH 4 CO +2H 2 O = CO NH 2 ONH 4 The presence of urease in soy bean (glycine hispida) was first observed by Takeuchi in Japan. Its applica- 16 Skand. Arch. f. Phys., 1891, II, p. 438; Zeit. f. phys. Chem., XVII, p. 140. "Zeit. f. phys. Chem., 1901, XXXII, p. 504. "Arch. f. d. Ges. Phys., 1896, LXII, p. 1. 19 Jour, of Biol. Chem., 1913, XIV, no. 3; 1913, XV, no. 3. 26 Manual of Vital Function Testing Methods tion to the quantitative estimation of urea is due as above stated to Marshall. A convenient form of the enzyme urease is now to be found upon the market under the name of urease (Dunning). It is supplied in convenient 25 milligram tablets put up 40 tablets per package by Hynson West- cott & Co., pharmaceutical chemists of Baltimore, Maryland. Urease (Dunning) is a fine, almost white, powder, with little taste or odor, soluble in slightly alkaline water. The apparatus and material required for the estima- tion are as follows : Four 200 c.c. Erlenmeyer flasks with cork stoppers ; one 50 c.c. glass-stoppered burette ; one 5 c.c. bulb pipette; one small glass mortar; 100 c.c. of solution of methyl orange; 1000 c.c. of decinormal solution of HC1; 50 c.c. toluol and a package of urease (Dunning) tablets. Put 1 or 2 c.c. of toluol into each of two Erlenmeyer flasks of 200 c.c. capacity. Into one of the flasks intro- duce exactly 5 c.c. of a specimen of urine and 100 c.c. of distilled water; stopper flask with cork. Crush a urease tablet in a small glass mortar and dissolve in about 5 c.c. of water. Transfer this solution without loss into the other flask containing toluol and rinse mortar with several portions of distilled water until about 100 c.c. have been added to the contents of the second flask. Add 5 c.c. of the urine and stopper with a cork. Each flask is now thoroughly shaken and allowed to stand at room temperature over night or at least 8 hours. If it is necessary to get more rapid estimations two tablets are used instead of one and the mixture digested at 40 C for an hour. The test may indeed be completed in 15 minutes by Tests of Liver Function 27 using only 1 c.c. of urine, two tablets and digesting at 40 Centigrade for 15 minutes. The factor would in this case be 3 instead of .6, as will be seen later by carrying out the longer time limit. After the lapse of the time set the two solutions are titrated to a distinct pink color with decinormal HC1, using methyl orange as indicator. The urease has converted the urea present in the urine into ammonium carbonate. The amount of ammonium carbonate formed by the urease is indicated by the quantity of standard HC1 solution required to exactly neutralize the contents of the flask containing urease minus the quantity required for control specimen. According to the chemical formula representing the conversion of urea into ammonium carbonate (z?. s.) it may be seen that 60 grams of urea are converted (by urease) into 96 grams of ammonium carbonate. This amount (96 grams) of ammonium carbonate would require 72 grams of standard HC1 solution to neu- tralize it. As this quantity of HC1 solution (72 grams) is con- tained in 20,000 c.c. of n / 10 HC1 solution and is equivalent to 60 grams of urea represented by 96 grams of ammonium carbonate, then 1/20000 of the quantity of 1 c.c. of n / 10 HC1 solution will be equivalent of 1/20000 of 60 grams, equal .003 (60-^20000 =.003). Therefore each c.c. of decinormal HC1 solution re- quired to neutralize the enzyme treated specimen minus the number of c.c. required to neutralize the control specimen represents .003 of urea, and as the 5 c.c. speci- men is the 1 /ooo P ar t of a liter, multiply the number of c.c. of n /io HC1 solution in excess of the control re- quirements by the factor .6 (.003X200=.6) to find the urea per liter when estimating the daily output. One part of nitrogen is equivalent to 2.143 parts of 28 Manual of Vital Function Testing Methods urea. Estimation of Total Nitrogen in the Urine. Kjel- dahl's Method. 20 Ten c.c. of urine are carefully meas- ured into a Jena glass, round-bottom flask. Add a few drops of concentrated solution of sulphate of copper, 15 c.c. of H 2 SO 4 and 10 grams of potassium sulphate. The flask is supported in an inclined position to pre- vent loss by spurting. The mouth of the flask is loosely closed by a glass bulb blown on end of a piece of glass tubing. The flask may be conveniently supported by a thick piece of asbestos board with a hole in the center of a size to permit the flame to come in contact only with the portion of flask covered by fluid. Wire gauze protects the flask from direct contact with the flame. The flask should now be gently heated over a Bunsen flame for half an hour. When foaming ceases the flame is raised until the acid begins to boil gently. The whole heating process is carried out under a hood because of sulphurous acid fumes which are given off. When the fluid in the flask is colorless or pale green, oxidation is complete. This requires about two hours. The acid and the oxidizer (CuS0 4 ) convert all nitrogenous mat- ter into ammonium sulphate. The flask is then cooled. The next step is distillation. The same or a larger Jena glass flask may be used, but preferably a copper apparatus which will not break. Two hundred c.c. of water are added and enough 30% NaOH solution to make the mixture strongly alkaline. Ammonia is set free by the action of the alkali. This is distilled over in 80 c.c. of decinormal H 2 SO 4 , which has been accu- rately measured into a flask. The flask containing the original solution is heated until about 2/3 of it have passed over and there is considerable bumping from 80 Zeit. f. Chem., 1883, XXII, p. 3T8. Tests of Liver Function 29 the separation of sodium sulphate. This usually re- quires about thirty minutes. Bumping may be dimin- ished by adding fragments of pumice, granulated zinc or talcum powder at the beginning of distillation. A simple form of apparatus can be extemporized in the laboratory. [See Illustration.] The tube D contains a few glass beads and some of the H 2 SO 4 is poured over these to prevent the escape of any ammonia. A few drops of methyl orange are added to the pearls and the flask C to indicate alkalin- ity, in which event more acid is to be added promptly. D is not necessary if a Liebig condenser is used. The tube B prevents the alkaline fluid in A from spurting over into E. The tubes E and B are made of broken pipettes of 50 or 100 c.c. capacity. The decinormal acid into which the ammonia has 30 Manual of Vital Function Testing Methods condensed is titrated with n / 10 NaOH. Methyl orange is used as an indicator. Subtract the number of c.c. of decinormal NaOH used from the number of c.c. of acid taken and the remainder will give the amount of ammonia distilled over, for every c.c. of acid neutralized by the ammonia is equivalent to so much decinormal ammonia. Decinormal ammonia contains 1.4 grams of nitrogen to the liter or .0014 gram per one c.c. The amount of nitrogen can therefore be determined by multiplying the number of c.c. of acid neutralized by .0014. This gives the nitrogen in grams for 10 c.c. of urine used. The description of the method given above is taken from Wood's Chemical Diagnosis, N. Y., 1909, p. 408. 2. Augmentation of Urinary Ammonia as an Index of Ureagenetic Liver Function Ammonia, amino acids and carbonates constitute the last intermediaries in the metabolic processes by which the body proteids are catabolized into urea. As before stated, the liver has been regarded as the chief factor in this conversion. If the functional capacity of the liver is deficient, the amount of nitrogen eliminated in the form of am- monia will be increased. Normally, .7 gram of am- monia are excreted by the urine in 24 hours. In hepatic insufficiency the amount may be doubled or trebled. The relation of ammonia nitrogen to total nitrogen in the urine is normally 3 or 4 parts per hundred. Under pathological conditions, however, it may rise to 30 parts per 100 and such an increase may, under cer- tain circumstances, indicate hepatic insufficiency. In states of acidosis the ammonia nitrogen is also increased. Tests of Liver Function 31 It is often useful to make a comparison between the amount of nitrogen excreted as ammonia and the nitro- gen eliminated as urea. This is done as follows : The molecular weight of ammonia NH 3 is 17, the nitrogen fraction of ammonia is, therefore, 14 /i7- NH 2 The molecular weight of urea CO is 60. NH 2 The nitrogen fraction is 28 / 6 o o r 7 /i 5 . The amount of ammonia in grams in a given sample is estimated by the formalin method (v. .), and 14 /i7 of this represents the ammonia nitrogen. The urea is calculated in grams in the same sample of urine, by Marshall's method, and T / 15 of the weight is nitrogen. Under normal conditions the ammonia nitrogen is about 1 / 20 of the urea nitrogen. Estimation of Ammonia Nitrogen in the Urine. The Formalin Method. The estimation of acidity is the first stage in the estimation of ammonia nitrogen. Pro- ceed as follows : Measure out 25 c.c. of urine into a beaker and dilute with about double the volume of distilled water. Add 2 or 3 drops of phenolphthalein. Run in n/ 10 NaOH from a burette until a faint permanent pink color is produced. Note the number of c.c. of NaOH used. Measure about 10 c.c. of commercial (40%) formalin into a second beaker. Add phenolphthalein. Neutral- ize exactly with n / 10 NaOH. Add the neutral formalin to the neutral urine. The pink color disappears. Run in n / 10 NaOH until the pink color returns. Note the number of c.c. of NaOH used. The result is calculated in terms of n / 10 NaOH for the acidity. That is to say the acidity of the urine is given as the number of c.c. of n /io NaOH required to neutralize 100 c.c. of urine to phenolphthalein. Thus if 10 c.c. of soda were used in 32 Manual of Vital Function Testing Methods the first titration to neutralize 25 c.c. of urine, the acid- ity of the urine is 10 / 25 X1QO : =40. The ammonia re- sult should be expressed in grams of ammonia per 24 hours. The number of c.c. of soda used in the second titration of the urine is the equivalent of the number of c.c. of ammonia present in the 25 c.c. of urine. Sup- posing the number of c.c. of soda used in the second titration to have been 10, then 10 c.c. n / 10 NaOH= 10 c.c. n/ 10 NH 3 = 10 X -0017 gm. NH 3 . Therefore the ammonia passed in the 24 hours = 10 X .0017 X 24 hours urine in c.c. 25 The reaction depends upon the combination of the am- monium salts with formaldehyde to form urotropinc and the consequent liberation of the acids previously combined with ammonia. Experimental Provocative Ammoniuria. This test is based upon the fact that normally, the liver trans- forms all ammonia transported to it into urea. If there is pathological alteration of the liver cell, this am- monia will not be transformed and the quantity of ammonia in the urine increases. Before applying the test of provocative ammoniuria in a given case the total ammonia excretion in 24 hours should be estimated over two days. Meanwhile the patient is put upon a fixed regimen. In the morning, after having urinated, the subject is given 6 grams of ammonium acetate. The urine for the next 24 hours is collected and the ammonium content estimated (v. s.) which can be compared with that found prior to the test. The value of this test is variously estimated. Some have concluded that any considerable increase of am- monia in the urine after the ingcstion will always in- Tests of Liver Function 33 dicate an impairment of the functional integrity of the liver cell. It is only claimed to be of value when posi- tive. Others, on the contrary, have insisted that in spite of very advanced disease of the liver the ammonium salts ingested continue to be transformed into urea. 3. Aminoaciduria as a Criterion of Ureagenetic Function The existence of considerable quantities of amino acids, leucine and tyrosine, in the urine in liver diseases was noted in 1866 by Frerichs. In 1907 Glaessner 21 showed that in disease of the liver there is usually an increase in the relation of amino nitrogen to total nitro- gen in the urine. Normally this relation, or so called coefficient, of aminoaciduria has been shown to vary from .5 to 3.5 per 100. In diseases of the liver (chole- lithiasis, cirrhosis), the ratio may rise to 11 or even 13 per 100. Labbe and Bith have estimated the amount of amino acids in the blood serum and have found it increased in certain liver diseases. Hyperaminoaciduria, according to Brille and Gar- ban, 22 is not necessarily an indication of liver disease. It may occur during states of rapid emaciation, cach- exia, pneumonia, typhoid fever and diabetes compli- cated with acidosis. These authors agree, however, that in all these states it may not be improbable that the appearance of am- inoaciduria is dependent upon a disturbance of the function of the liver cell. Provocative Aminoaciduria. The principal sub- stances which have been used are glycocol, alanin, as- a Zeit. f. Exper. Path. Therap., 1907, IV, p. 336. 22 Gaz. d. Hop., 1914, LXXXVII, p. 405. 34 Manual of Vital Function Testing Methods paraginic acid, and commercial peptone. These amino acids, also peptone, have been employed by different experimenters. 23 The patient is given certain quanti- ties of these substances and the amount of amino-acid excreted in the urine carefully measured. If the proper care has been taken in the days which precede the test to establish the normal amino nitrogen coefficient for the individual and to keep the subject under observation upon a fixed and invariable diet, then provocative aminoaciduria tests may be of value. They will frequently show a marked increase of the coefficient after the ingestion of the amino-acids (or peptones) as compared with the coefficient prior to the test, and the marked increase according to some authors will only occur when the liver parenchyma is diseased. The insuperable difficulty in these methods of esti- mating liver function arises from the fact that the esti- mation of amino nitrogen in urine and blood requires very complicated chemical manipulations which effectu- ally prevent their introduction into clinical medicine. 4- Estimation of Residual Nitrogen in Blood Serum as an Index of Hepatic Function. Chauffard Brodin Test 24 The quantity of residual nitrogen in blood serum is obtained by subtracting the urea nitrogen from the total nitrogen estimated in dealbuminized serum. Re- sidual nitrogen is made up of ammonia, amino-acid, uric acid, etc. It has been contended that the elabora- tion of residual nitrogen products is exclusively de- ""Ztschr. f. klin. Med., 1910, LXXI, p. 261; 1911, LXXIII, p. 325. 24 Jour. Biol. Chem., 1912, XII, p. 301; Jour. Amer. Chem. Soc., 1913, XXV, p. 1567, Tests of Liver Function 35 pendent upon the liver. In normal persons the re- sidual nitrogen is always below 10 grams per liter of serum. In many acute and chronic diseases of the liver, the residual nitrogen has been found above 10 grams, the amount being in proportion to the grav- ity of the hepatic lesion. The diet and condition of the kidney are negligible. The estimation of the amount of residual or rest nitrogen in the blood serum has come to be regarded as of more importance as a test of kidney than of liver function. For this reason a more complete account of the method and its interpretation will be found later under that head. General Summary of the Value of Ureagenetic Func- tion Tests. In the first place it must be admitted that some of these ureagenetic tests are complicated to such an extent that they cannot be carried out without the assistance of an expert chemist, thus diminishing their applicability to clinical work. Further than this it must likewise be admitted that many physiological causes of error exist which militate against a too strict interpretation of results. Other factors besides the liver collaborate in nitrogen metab- olism. Notwithstanding these valid objections it must be admitted that the study of nitrogen has given some useful results and it is to be hoped that future investi- gations may bring a greater degree of order from the now more or less confused and contradictory material which comprises our stock of knowledge today concern- ing the true relation of the liver to nitrogenous metab- olism. When this day arrives the clinician will be better able to interpret the results obtained from ureagenetic tests than he is at the present time. 36 Manual of Vital Function Testing Methods in. FUNCTIONAL TESTS TO DETERMINE DISTURBANCE OF THE ANTITOXIC FUNCTION OF THE LIVER Poisons which obtain access to the organism thro the portal circulation are fixed and destroyed under normal circumstances by the liver cells. It is natural, therefore, that this important function should be em- ployed as a basis for testing the integrity of the organ. The first efforts made in this direction consisted in estimating the toxicity of urine and blood serum. An increase of toxicity of these bodies, it was held, in- dicates diminished toxicopexic power of the liver paren- chyma. Tests of this character are carried out upon lower animals. The urine to be tested being injected intravenously into rabbits according to Bouchard's 25 method, and blood serum or urine into the cerebrum of rabbits according to Widal's method. The numerous sources of error and the technical complications sur- rounding these two methods have prevented their intro- duction into clinical medicine and they need not be described. The methods which are in use to determine the anti- toxic functional power of the liver are two in number. They are as follows: 1. The Methylene Blue Test of Chauffard and Castaigne. 2. Estimation of Indica- nuria, Spontaneous and Provocative. 1. Methylene Blue Test of Toxopexic Function of the Liver. Chauffard, Castaigne Test 26 When the liver cell is unable to properly arrest and fix poisons, it reacts to the passage of methylene blue 28 See also Estimation of Urinary Toxicity as a Test of Renal Function. ""Presse M6d., 1898, April 23; Jour, de Physiol. Path., 1899, May. See also Babaliantz, These de Geneve, 1912. Tests of Liver Function 37 through its parenchyma by an intermittent elimina- tion. Test. Inject 1 c.c. of 5% solution of methylene blue subcutaneously. Collect and examine the urine in half an hour, then every hour. Normally at the end of half an hour after injection the urine becomes colored, the color rising to maximum in 3 or 4 hours, disappearing in about 50 hours. If the elimination when it commences instead of being continuous occurs in cycles, that is intermittently, the test is positive and may indicate hepatic insufficiency. It must be acknowledged that the value of this test is greatly diminished by the fact that the state of renal permeability must be considered, since this will have a predominant influence upon the quantity and rapidity of elimination. Where this factor is known the test may be of value. Roche's Modification of Chauffard's Methylene Blue Test. In this test the methylene blue is taken inter- nally instead of being given hypodermically. .002 gm. of methylene blue is swallowed at eight o'clock in the morning on an empty stomach. The substance is given in capsule. The urine is collected every four hours in separate vessels. If the urine of the second recipient is clearly colored it will denote inability of the liver to retain the pigment and consequent hepatic insuf- ficiency. Normally this amount of methylene blue should be completely arrested and fixed by the liver so that no coloring matter appears in the urine after its administration. If the test is positive, the urine, especially that passed four to eight hours after ad- ministration, will be colored green. Unfortunately the condition of the kidney must also be reckoned Avith in applying this test, as failure of 38 Manual of Vital Function Testing Methods elimination of the dye may be due to renal impermea- bility. The same remarks with respect to renal permea- bility apply in interpreting the Roche modification as when the dye is given by hypodermic injection. It can never be known a priori whether a failure of elimi- nation is due to renal insufficiency or to normal fixa- tion of the dye by the liver cell. In other words, the state of the kidney function must be previously known or ascertained. 2. Indicanuria Spontaneous and Provocative as a Means of Testing the Integrity of Hepatic Fixa- tion Spontaneous Indicanuria. It has been urged that the normal liver is always capable of arresting and destroying indican which is formed in the intestine as a result of putrefaction of albuminoids. Therefore the spontaneous presence of indican in the urine has been held to be evidence of hepatic insufficiency. Provocative Indicanuria .001 gm. of indol is in- gested in the morning on an empty stomach. The urine is collected every four hours and examined for indican. The individual to be tested should be put on a milk diet for a few days before the substance is ingested. TESTS FOR INDICAN IN THE URINE Qualitative.- The principle involved is to decom- pose the sodium or potassium compound of indoxyl sulphuric acid present in the urine by strong HC1 and oxidizing this compound. Obermayer's reagent is usually employed. This con- sists of strong HC1, sp. gr. 1.19, to which is added two parts per thousand of ferric chloride. The liquid is Tests of Liver Function 39 fuming yellow and keeps indefinitely. Precipitate the specimen of urine to be tested with a small amount of lead acetate or subacetate, avoiding excess, and filter. This removes pigments. 15 c.c. of filtered urine are mixed with equal quantity of Ober- mayer's reagent and 2 c.c. of chloroform added. Cork or cap the tube with rubber, and slowly invert. The chloroform takes on a blue color whose depth will roughly show the amount of indican present. Quantitative. Strauss' 2T method is a good one. Twenty c.c. of urine are mixed with 5 c.c. of 20% lead acetate solution and filtered. Ten c.c. of filtrate (cor- responding to 8 c.c. urine) are placed in a small gradu- ated separatory funnel and mixed with 10 c.c. of Ober- mayer's reagent (v. s.). Five c.c. of chloroform are added, the tube corked and gently shaken. This is repeated in two minutes. Pour the chloroform from the tube. Add 5 c.c. of chloroform and repeat the extraction. Continue until added chloroform remains colorless. Two c.c. of united chloroform extracts are put in a small test tube of same diameter as tube containing standard solution. Chloroform is added drop by drop until colors are matched, against white background. The standard solution is made by dissolving .001 gm. C.P. indigotin (Kahlbaum) in 1000 c.c. of chloroform. Portion is sealed in test tube and kept in the dark. If the total amount of chloroform used for extrac- tion is equal to a and the amount of chloroform used to dilute the 2 c.c. to the color of the standard tube equals x, the total amount of chloroform necessary to dilute all the chloroform used in extraction equals 27 Deutsch. med. Wchnsch., 1902, p. 299. 40 Manual of Vital Function Testing Methods The total number of c.c. used in the extraction and in the dilution of the extraction mixture represents, therefore, a bulk containing .001 gm. of indigo. In normal urines 5-10 c.c. of chloroform are usually all that is required to extract the whole amount. To obtain the results in milligrams it must be con- sidered that the amount of indigo extracted was from 8 c.c. of urine. IV. FUNCTIONAL TESTS TO DETERMINE DISTURBANCES OF THE SANGUINOPOIETIC FUNCTION OF THE LIVER CELL Some results of practical value have already come from tests of hepatic function based upon the haemic activities of the liver. The whole question of the rela- tion of the liver to the biology of the blood is very com- plex and not completely understood at the present time ; but it is generally agreed that the liver is inti- mately concerned in the elaboration of some of the constituents of the blood, particularly fibrinogen and certain of the ferments. The haemic tests so far proposed have dealt with two physiological aspects of the relation between the liver and blood : first coagulability and second the presence of miscellaneous ferments. If the liver is in reality the principal source for the elaboration of fibrinogen any notable diminution of this substance in the blood will indicate a diminution of the func- tional integrity of the liver cells. With diminished fibrinogen and perhaps also fibrin ferment, a delay or deficiency in the coagulability of the blood will ensue. One test of liver function will therefore consist in estimating the coagulability of the blood. With respect to the tests for ferments in the blood Tests of Liver Function 41 it may be said that the estimation of lipase is so far the most important. The liver under normal circum- stances inhibits the formation of this ferment so that hepatic insufficiency results in an actual increase in the amount of lypolytic enzyme in the blood. The other ferment tests of hepatic function are ap- parently too subtle and uncertain to be of any con- siderable practical value. The functional tests which are in use at the present day to determine the sanguinopoietic capacity of the liver are as follows: 1. Estimation of blood coagula- tion time, and estimation of fibrinogen. 2. Estimation of fibrinolysis time. 3. Estimation of the amount of lipase in the blood. 1. Estimation of Blood Coagulation Time as an Index of Liver Function Prolongation of coagulation time of the blood is said to be present when the liver is physiologically de- fective and consequently the estimation of coagulation time has been proposed as a simple and effective means of determining hepatic insufficiency. Unfortunately while the relation of normal hepatic function to normal coagulation of the blood is un- doubtedly intimate and important, it is also true that so many other factors besides the liver enter into the physiology of blood coagulation as to materially lessen its importance as a basis for functional estimation. Coagulation time of the blood has also been sug- gested by Tettinger and Bachrach as a means of test- ing renal function. Wright's Method of Estimating Coagulation Time. The necessary apparatus consists of a series of cap- illary tubes, elastic bands, a beaker, a jug of hot and 42 Manual of Vital Function Testing Methods a jug of cold water, a watch with a second hand and a thermometer. The capillary tubes are of the same caliber and are provided with a 5 c. mm. mark. The procedure is as follows: Clean the patient's thumb with ether. Wrap a piece of elastic tubing round the thumb from the base nearly to the tip. Puncture the tip of the thumb with a sterile surgical needle. Draw up blood to the mark on the pipette. It is not essential to obtain the exact quantity of blood, slight variations in the amount being of less importance than rapid manipulation. Note the exact time by the watch. Stretch a flat elastic band over the ends of the tube to prevent entrance of water. Stand the tube in the beaker filled with water at 37 C. Stir the water oc- casionally with the thermometer and keep the tem- perature constant by adding hot or cold water. Prepare three or four more capillary tubes in the same way, numbering each tube and taking the time of each. At the end of three minutes take out the first tube and blow out the blood. Give the second tube 3}/o minutes and if the blood is still fluid give the third tube four minutes and so on. The tube from which the blood fails to be expelled by blowing gives the co- agulation time. The normal time for blood coagulation is 31/2 minutes. The Fibrinogen Test. Whipple Hormtz. 28 20 c.c. of oxalated blood plasma are heated to 59 C. for 20 minutes. Fibrinogen is precipitated. The precipitate is isolated by centrifugation, washed with water, alcohol and ether, dried at 120 C. and weighed. A rough esti- mate of the amount of fibrinogcn in the blood is made by clotting a little plasma with calcium, testing the 28 Jour. Exp. Med., 1911, XIII, p. 136; also Johns Hopk. Hosp. Bull., 1913, XXIV, p. 207, 343. Tests of Liver Function 43 toughness of the clot with a glass rod. The quantita- tive or weighing method is, however, better. Normally fibrinogen exists in the plasma in the pro- portion of .30 to .40 gm. per 100 c.c. In case of liver deterioration, alteration or injury the amount will be found diminished. In some cases of cirrhosis the fibri- nogen content has been found very low (.05 gm. or less.) #. Estimation of Fibrinolysis Time as an Index of Hepatic Function. Goodpasture Test 29 Very recently, Goodpasture has called attention to the interesting and important fact that in chronic, atrophic, hepatic cirrhosis the blood possesses the power of completely digesting the clot in a few hours at body temperature. The clot in normal blood remains undigested for days and sometimes even for weeks. Goodpasture be- lieves that the dissolution of the clot is due to an enzyme. The activity of this enzyme is destroyed by heat and inhibited by normal serum. The fibrinogen content of the blood in his reported cases (four in number) was below normal. He suggests premature digestion of clot durante vivo may account for the frequency of spontaneous hemorrhage in atrophic cirrhosis. The Goodpasture test will, in all likelihood, be found of great value in estimating hepatic insuf- ficiency in chronic cirrhosis. It will be interesting to observe what the test for fibrinolysis will show in early or latent cases of cirrhosis as well as in hepatic con- ditions generally. Technic of Goodpasture Test. Blood is drawn from an arm vein by means of the usual technic. The coagu- 29 Johns Hopk. Hosp. Bull., 1914, XXV, p. 330. 44 Manual of Vital Function Testing Methods lation time is estimated.* A portion of blood is drawn into 1 Jo solution of sodium oxalate to prevent clotting of the specimen. This last is centrifuged and 20 c.c. of supernatant plasma is used to determine fibrinogen content if this is desired. Otherwise the entire oxalated plasma is used for tests of coagulation time and fibrin- olysis. In testing the oxalated plasma for coagulation time, use 1 c-c. of plasma -)- 1 gtt. CaCl 2 (1%). The original clot from the drawn blood and speci- mens of clotted oxalate plasma are placed in the ther- mostat at 37. They are examined every hour. If the test is positive, the blood clot liquefies and is dissolved in 31/2 to 5 hours. In negative (normal) cases there is no digestion of the clot for several days, even where no aseptic pre- cautions are taken. 3. Estimation of Blood Lipase as an Index of Liver Function. Whipple's Test 30 There exists normally in the blood a lipolytic fer- ment lipase. The percentage of lipase in normal blood is remarkably uniform. In certain diseases of the liver this ferment has been found to increase in amount indi- cating that under normal circumstances the liver in- hibits its formation. Any considerable increase, there- fore, of lipase in the blood has been held to indicate hepatic insufficiency. Whipple, in collaboration with Mason and Peightal, found that after acute injury of the liver from chloroform there was always found an in- * For other methods than Wright's consult modern laboratory manuals. 30 Johns Hopk. Hosp. Bull., 1913, XXIV, p. 207; ibid., 343; ibid., 357. Tests of Liver Function 45 crease in the lipase of serum or plasma. Sometimes this rise amounted to 12 c.c. of 1 / 10 normal acid. Inas- much as the content of lipase may increase five to eight times the normal under certain conditions of hepatic disease it was naturally suggested as a test of hepatic insufficiency. The value of the test is chiefly qualitative rather than quantitative. It has been found of especially positive value in suspected eclampsia, chloroform poisoning, yel- low atrophy and cholangitis. In cirrhosis of the liver there may be a subnormal lipase. Tests for Lipase in the Blood. Lowerihart's 31 Method of Estimating Lipase Is Usually Employed. Blood serum is collected in four tubes and a little toluene, .3 c.c., is added to prevent microbic contami- nation. Each tube contains 1 c.c. plasma or serum diluted with 4 c.c. of distilled water. Two of these tubes are used as controls. To the others a little ethyl butyrate (butyric ether), .26 c.c., is added. The four tubes are shaken, corked and put in the thermostat at 38 for 18 to 24 hours. At the end of this time the two control tubes are ex- amined for their normal alkalinity by means of deci- normal acid solution. The other two tubes are titrated for free butyric acid by decinormal alkali solution. The total lipolytic activity is measured by adding the two figures, since the butyric acid formed had to first neutralize the normal alkalinity of the serum. The exact method is as follows : After incubation the tubes are cooled in ice water, 3 drops of azolitmin added and then titrated in pairs to a neutral reaction, using Vio normal acid and alkali. The two control tubes usually show the blood alkalinity to be .1 c.c. Vio normal acid and the butyrate tubes show 81 Amer. Jour. Physiol., 1902, VI, p. 331. 46 Manual of Vital Function Testing Methods the acid production to be .1 to .2 c.c. above the neutral point. This means that the total lipolytic activity is .2 to .3 c.c. Vio normal solution, that the plasma lipase has split up the ethyl butyrate to this amount. Normal plasma lipase is then in terms of Vio normal acid equal to .20 to .30 c.c. Ghedini's Test.^ 2 This consists in an estimation of the power of the blood serum to convert glycogen into glucose. A reducing ferment supposed to be formed by the liver cells effects the conversion. The Test. Blood serum to be examined is added to a solution of glycogen. Scrum from a known normal person is similarly treated and used as a control. To both tubes is added a little sodium hydroxide, then potassium sulphocyanide, and the solutions fil- tered and examined with the polariscope. If there is hepatic insufficiency the rotatory power of the serum will be less than the normal control. The factors upon which this test are founded are rather subtle and imperfectly understood ; indeed it is by no means accepted that the liver is the unique or even the most important source of the ferment which affects the conversion of glycogen into glucose. 4- Application of Abderhalden's 33 Method to Estima- tion of Sanguinopoietic Functions of the Liver Recently it has been held that destruction of liver parenchyma by disease (autolysis) gives rise to the presence of an excess of protcolytic ferments in the blood serum and this fact has formed a basis for test- 32 Gazz. degli ospcdali, Milan, Jan. 12. 33 See Breitmann, Zentrbl. f. innere Med., XXIV, 1913, p. 857. Tests of Liver Function 47 ing the functional capacity of the liver. The technic of the method is too complicated to admit of its use in clinical practice and the results ob- tained are as yet too meagre to justify any conclusions as to its value. V. FUNCTIONAL, TESTS TO DETERMINE DISTURBANCE OF THE EXOCRINOUS OR BILIARY FUNCTION OF THE LIVER Bile is partly a secretion and party an excretion, for it not only plays a role in certain digestive processes, notably the splitting or absorption of fats, but it also contains certain waste products which escape in the feces. It is important to remember, however, that cer- tain constituents secreted and excreted in the bile are reabsorbed by the intestine and carried back by the blood stream to the liver. Perfectly fresh hepatic bile, in contradistinction to bile in the gall bladder, contains one pigment only, namely, bilirubin, and from this pigment others are gradually formed by processes of oxidation. Fresh human bile from the hepatic duct is golden yellow in color but becomes olive brown to grass green after re- maining in the gall bladder and cystic duct, because of the presence of biliverdin, which is an oxidation product of bilirubin. It is now universally accepted that the bile pigment bilirubin originates from the disintegrated hemoglobin of the red blood corpuscles. When these break up their contained pigment is carried to and fixed by the liver, where it is converted into an iron-free pigment, bilirubin. It appears that the power of transforming blood pigment into bilirubin is not exclusively the property 48 Manual of Vital Function Testing Methods of the liver cell, since in old blood extravasions any- where in the body a substance, haematoidin, is found which is chemically identical with bilirubin. Under ordinary physiological conditions, however, the liver seems to be the only place in the body where bilirubin is formed. It is not known whether the actual dis- integration of red corpuscles is confined to the liver, but certainly this organ has the unique power of fixing the hemoglobin set free by haemolysis, retaining its iron and converting it into bilirubin. Under normal conditions bilirubin does not appear in the feces, but in its place is found a reduction prod- uct which is known as urobilin (stercobilin). The reduc- tion of bilirubin to urobilin takes place in the intestine from nascent hydrogen liberated by bacterial action. Part of this urobilin formed in the intestine escapes in the stools. Another portion is reabsorbed by the in- testinal mucosa as a protochrome, urobilinogen. Part of this substance is eliminated in the feccs. The re- mainder passes back thro the portal circulation to the liver, where it is changed back into bilirubin to be again reexcreted by the bile when the cycle is re- peated. A very minute amount of the urobilinogen absorbed by the bowel escapes in the urine. Under normal con- ditions this quantity is very small. Soon after urobilinogen is eliminated by the urine, it changes to urobilin so that the urobilin is not present in freshly passed urine but only appears from the break- ing down of urobilinogen by light and air. Urobilin was recognized as a constitutent of patho- logical urines long before urobilinogcn, from which it is formed by oxidation, was discovered. Urobilinuria and urobilinogenuria may therefore be regarded as practically identical and due to the same Tests of Liver Function 49 substance in different forms. The pathological causes of both are the same. It has been known for a long time, however, that urobilin appears in the urine in a comparatively large number of pathological conditions, among which may be mentioned the infectious diseases, particularly ma- laria and pneumonia, cirrhosis of the liver, lead poison- ing, decompensated heart disease, pernicious anaemia, pulmonary infarction and visceral hemorrhages. Like- wise certain drugs and poisons are known to produce urobilinuria. In obstructive jaundice, it often appears in the urine before bilirubin and may, in fact, alternate with this substance. Urines which contain much uro- bilin present a dark yellow color which may be imparted to the foam on shaking. They are thus quite like or- dinary icteric urines. The hepatic conditions in which urobilin appears in the urine fall into two groups: 1. Mechanical inter- ference with biliary flow in the ducts. 2. Insufficiency of the liver cell. In the first group, if the obstruction is absolute as in some cases of stone, no bile reaches the intestine and no urobilin is formed, hence no uro- bilinogen appears in the blood or urine. If the ob- struction remains complete the bile pigment bilirubin begins to be absorbed by the blood and excreted in the urine. If the occlusion of the ducts is only partial, some bile reaches the intestine, urobilin is formed, ab- sorbed and may appear as urobilinogen in the urine. On removal of the obstruction the full flow of bile in the intestine is resumed. The functional activity of the liver is sufficient to eliminate the urobilinogen brought to it by the blood from the intestine and hence there is no call for its elimination by the kidney and it becomes reduced to a mere trace in the urine or com- pletely disappears therefrom. 50 Manual of Vital Function Testing Methods As a sign of liver insufficiency urobilinogenuria may be absolute or relative. Absolute when the liver paren- chyma is totally unable to eliminate urobilinogen and allows it to get into the blood; relative when an ex- cessive breaking down of red corpuscles anywhere in the body overwhelms the liver with pigment which the cells are unable completely to convert into bilirubin. In other words, if the liver is insufficient it will be un- able to excrete increased amounts of urobilinogen whether derived from intestinal urobilin or from blood pigments. In either case there will be an increase or accumulation of urobilinogen in the blood and it will appear in measurable amounts in the urine. Icterus with the appearance in the urine of normal bile pigment (bilirubin) may be regarded as a further step in such a pathological process. Here the defect of excretory function of the liver is greater because of actual inflammatory obstruction of the biliary tract. Jaundice, as is well known, is the result of the absorp- tion into the blood of the bile pigment bilirubin. Jaun- dice, whether obstructive or toxemic, is always due to some lesion of the biliary excretory passages by which the flow of bile into the intestine is diminished or pre- vented and is always accompanied by some inflam- matory state of the biliary tubules. Icterus, when well marked, becomes clinically quite evident from the discoloration of tissue which results. The demonstra- tion of bilirubin in the urine is then superfluous. In such an event, icterus becomes an element in the semi- ology of hepatic disease and has to be evaluated with other symptoms in making a diagnosis. The presence of small quantities of bile pigment (bilirubin) in the urine is, however, an earlier sign of exocrinous hepatic insufficiency than icterus. For this reason the tests for bile pigment in the urine become Tests of Liver Function 51 tests to a certain extent at least of hepatic disease and perhaps insufficiency. To summarize the above facts in their relation to the estimation of the external secretory function of the liver it may be said that disturbances of this func- tion are identified by tests which disclose the presence in the urine of the three substances above mentioned, namely, urobilinogen, urobilin and bilirubin ; the tests for these substances will therefore be given together with special data bearing upon their individual sig- nificance. There is, however, another way by which the total power of the liver to carry on its external secretory function may be judged. This consists theoretically, in finding a substance which if injected into the circula- tion will be eliminated exclusively by the liver. It was only in recent years that such a substance was dis- covered, phenoltetrachlorphthalein. Tests to determine the status of the excretory func- tional power of the liver may, therefore, be properly divided into two categories : 1. Tests for urobilinogen, urobilin and bilirubin in the urine with the interpretation of results in reference to hepatic insufficiency. 2. Tests to determine the global capacity of the liver to eliminate foreign sub- stances. In this category there is but one test so far devised, namely that of phenoltetrachlorphthalein. 1. Tests for Urobilinogen, Urobilin and Bilirubin in the Urine. Interpretation of Results with Refer- ence to Hepatic Insufficiency The Urobilinogen Test. Ehrlich's Test. 34 The Ben- zaldehyde Reaction. While investigating the aniline 34 Wien. med. Wchnschr., 1901, XV; Munch, med. Wchnschr., 1901, XV. 52 Manual of Vital Function Testing Methods dyes for their effects upon trypanosomes, Ehrlich found that the addition of paradimethylamidobenzaldehyde to certain fresh urines produced a bright red coloration. This was in 1901. In 1903 Pappenheim 35 called atten- tion to the fact that the reaction occurred only in those urines which, on standing, gave the reaction of urobilin. In the same year Neubauer 36 demonstrated that the reaction was due to urobilinogen, a colorless chromogen which gradually becomes converted into urobilin. Technic. The test is very simple. Add to fresh urine in a test tube several drops of Ehrlich's reagent. The reagent is as follows : Paradimethylaminobenzaldehyde ... 8 gms. Concentrated hydrochloric acid .... 80 gms. Distilled water 200 gms. If urobilinogen is present a red color appears. The color reaction in the cold urine is of pathological sig- nificance only when a distinct scarlet color is obtained. There are a few sources of possible error. The in- gestion of hexamethylamine or antipyrine may cause the same reaction. The presence of acetone in the urine must also be excluded as it produces a similar coloration. If the reaction persists after free purgation it is more significant. The reaction is not constant in all conditions in which it has been found because the liver may be able to excrete enough urobilinogen to prevent its appearance in the blood and urine. Urobilinogen has been found in the urine in many pathological conditions, chief of which are cirrhosis of the liver, cholangitis, infectious diseases, heart diseases 80 Berl. klin. Wchnschr., 1903, II, p. 42. 36 Sitz. d. Gesell. f. Morph. u. Physiol., Munich, 1903, July, H. ii. Tests of Liver Function 53 in the stage of decompensation, pernicious anaemia, pul- monary infarction and visceral hemorrhage. Ehrlich's test has been highly recommended as a clin- ical method for determining hepatic insufficiency by Miiller, Bauer, Neubauer, Hilderbrand and others. It has been regarded by some observers as an adequate and infallible criterion of hepatic function. Some observers have claimed for it a prognostic significance. All of these contentions have been found to overstate the facts. The presence of urobilinogen in considerable quan- tity in the urine may indicate that there is a partial interruption in the biliary excretion and that some of the intestinal urobilin absorbed into the blood is not being thrown off by the liver. The primary cause may be either a disorder of the liver cells or a congestion or toxemic obstruction of the biliary channels. The discovery of a persistent urobilinogenuria should therefore point to a careful study of the hepatic func- tions by all known means. If hepatic insufficiency can be ruled out the only remaining explanation is that excessive destruction of the blood corpuscles is taking place somewhere in the body. Recently rather strong criticisms of Ehrlich's test have appeared. Wilbur and Addis 37 in 1913 stated that they do not believe it constitutes a reliable criterion of hepatic function. This opinion has been reiterated by Chesney, Marshall, and Rowntree, 38 last year. All these authors maintain that observations on a single or 24-hour specimen of urine have no significance. That great variations appear from day to day and that only when repeated tests are made covering a period of two weeks, controlled by studies of urobilin content in the feces, can the results be accepted. 87 Arch, of Int. Med., 1913, Feb., p. 235. 38 Jour. Amer. Med. Assn., 1914, LXXIII, p. 1533. 54 Manual of Vital Function Testing Methods The simplicity of Ehrlich's test will insure for it a permanent, if not paramount, place. It must always be remembered that only a persistent and well-marked urobilinogenuria is to be regarded clinically as sig- nificant. A well-marked and lasting reaction appears to in- dicate one of two things, an hepatic insufficiency or excessive hemolysis. There is nothing in the test itself to enable a differentiation between the two to be made. The test is therefore of some importance when regarded only as a corroborative sign of insufficiency of the excretory function of the liver. Tests for Urobilin. First add to the urine a few drops of 10% solution of zinc chloride and enough am- monia to dissolve the precipitate. Filter into a test tube and hold same against a dark background ; a green fluorescence denotes urobilin. Equal quantities of 1% solution of zinc acetate and urine may be used in the first part of the test. The fluorescence may be made more visible by concentrating light on the tube with a lens. Another and perhaps more delicate method of de- tecting urobilin is to extract 50 c.c. of urine with 50 c.c. of pure ether. Pour off the ether into a tube and evap- orate. Dissolve the brown residue in a little strong alcohol. The solution will be pale yellow with a green fluorescence if urobilin is present. A third method is to acidify 20 c.c. of urine with several drops of HC1. Shake gently with 5 c.c. of amyl alcohol. This extracts the pigment and shows a bright green fluorescence when treated with an alcoholic solu- tion of zinc chloride and a little ammonia. By trans- mitted light the amyl alcohol is a faint pink shade. The most rapid method of testing for urobilin is by Tests of Liver Function 55 the use of the pocket spectroscope. If the urine is dark it should be diluted. The characteristic spectrum band is seen between the green and blue, between the lines C and F. Tests for Bilirubin. The tests usually employed are those of Gmelin, Rosenbach, Huppert and Smith. Gmelin's Test. The urine is treated with sufficient concentrated HNO 3 containing a trace of nitrous acid, sufficient to form a layer beneath. If bilirubin is pres- ent there will be a play of colors at the zone of contact from yellow through green, blue, violet, red and orange. The green will lie nearest the urine and the orange in the upper acid. Rosenbach' 's Test. Filter the urine through Swedish filter paper ; apply a drop of HNO 3 containing a trace of nitrous acid upon the paper. The play of colors above mentioned under Gmelin's test will appear. Huppert' s Test. Precipitate the urine with barium chloride and ammonia. The precipitate is washed with water. Wash the precipitate with alcohol into alcohol acidulated with sulphuric acid. Boil for a time. If bilirubin is present an emerald green color will appear. Smith's Test. A small amount of urine is placed in a test tube and overlaid with a few c.c. of tincture of iodine diluted with alcohol 1 :10. If bilirubin is present a distinct emerald green ring will develop at the zone of contact. 2. Tests to Determine the Global Capacity of the Liver to Eliminate Foreign Substances The Phenoltetrachlorphthalein Test of Liver Func- tion. Rowntree, Horwitz, and Bloomfield Test. Phe- 56 Manual of Vital Function Testing Methods noltetrachlorphthalein was originally studied pharma- cologically by Abel and Rowntree 39 in 1909. It was proposed in 1913 as a test for functional capacity of the liver by Rowntree, Horwitz, and Bloomfield. (Johns Hopk. Hosp. Bull., 1913, XXIV, p. 327.) Important experimental work was done on the drug in 1913 to determine its behaviour in different liver injuries, by Whipple, Mason and Peightal, 40 and by Whipple, Peightal and Clark. 41 Great interest is attached to the phenoltetrachlor- phthalein test for liver function ; first, because it bids fair to become the most satisfactory test for the pur- pose yet devised, and, secondly, because all the work upon it has been done in America. Professors Orndorff and Black 42 of Cornell Univer- sity were the first to make the substance in 1909. The pharmacological investigations of Abel and Rowntree included a study of phenolphthalein together with the new synthetic phthaleins, with special reference to their behaviour as purgatives. They found that phenolphtha- lein and its halogen substitution products, of which phenoltetrachlorphthalein is one, do not differ greatly in their physiological action. They are nonirritant to mucous membranes and subcutaneously when injected in oil. They are of low toxicity and possess no bacteri- cidal action. Both phthaleins are laxative when given by mouth, subcutaneously or intravenously. When an oily solu- tion of phenoltetrachlorphthalein (.4 gm.) is injected under the skin of dogs or human beings a laxative action is induced which continues from 4 to 6 days. When the 80 Jour. Pharmacol. and Expcr. Therap., 1909, I, p. 231. "Johns Hopk. Bull., 1913, XXIV, p. 207. "Johns Hopk. Bull., 1913, XXIV, p. 343. Amer. Chem. Jour., XLI, 1909, p. 349. Tests of Liver Function 57 tetrachlorphthalein is given subcutaneously it escapes from the body exclusively in the bile. When the same substance is given by mouth it is not absorbed. After subcutaneous administration of phenoltetrachlorphtha- lein the drug escapes dissolved in the bile and becomes later absorbed by the mucous membrane of the large intestine. These facts form the physiological basis for the phe- noltetrachlorphthalein test. The drug is eliminated ex- clusively, or practically so, in the bile, and since this excretion can be hurried through by purgatives, no time will be given for its absorption, and thus the actual amount of the drug eliminated may be found. The experimental work done by Whipple, Mason and Peightal, and by Whipple, Peightal and Clark has established the fact that there is a striking parallelism in animal observations between the amount of experi- mental liver injury produced and the amount of phtha- lein eliminated. Their method of determining this fact was as follows : Female dogs were used because of ease of catheteriza- tion. The dogs were given intravenously .1 gm. of the phthalein when weighing between 10-20 pounds ; .2 gm. when weighing over 20 pounds. The injection was given in the forenoon and 200-300 c.c. of water administered by stomach tube. After 5 or 6 hours the urine was col- lected and magnesium sulphate and croton oil given to produce several semi-fluid stools. The feces were col- lected next morning. The total feces were then diluted to 1 to 2 liters. The mixture was made alkaline with 5-10 c.c. of 40% solution of sodium hydroxide and shaken until uniform. One tenth of this quantity was taken and diluted to 500 c.c. with water, 3 to 4 c.c. of 40% solution sodium hydroxide added and the mixture thoroughly shaken. Of this second solution 100 c.c. 58 Manual of Vital Function Testing Methods were precipitated with 5 c.c. of saturated solution of basic lead acetate. After a few seconds a curdy pre- cipitate fell. The solution was made up to 200 c.c. with water containing 4 c.c. of 40 c /o solution sodium hydroxide. On standing, the supernatant fluid showed a clear phthalein color. This was filtered and the clear solution read off in a colorimeter against a standard solution .01 gm. phthalein to the liter. In the hands of its authors, this method gave pretty uniform results; on normal dogs the amount excreted being 35 to 50% with .1 gm. injection and 40 to 50% with .2 gm. injection. The drug did not appear in the urine. About 10-15% of the phthalein injected was lost from the time of the injection to that of its being poured out by the bile into the intestine. This shows that the liver is quite specifically concerned in the elim- ination of the substance, phenoltetrachlorphthalein. Whipple and his collaborators found that when the liver parenchyma is artificially injured, as by chloro- form, phosphorus or hydrazinc, there is a very notable drop in the output of the substance in the feces down to 20 or 10% or even a mere trace. The phthalein then begins to be excreted by the urine. When the hepatic lesion improves, the phthalein output in the bile in- creases toward normal. The normal output of phe- noltetrachlorphthalein in dogs is 45% of the amount injected. The drop in phthalein output is always pro- portional to the extent of liver injury. If the injury is grave enough to produce death, the phthalein output falls to zero. In some instances as the effects of the hepatic injury are spontaneously repaired, there may be an actual increase of phthalein output even above normal, a hypersecretion of phthalein as it were. Besides injury to the liver parenchyma by poisons, the above investigators found that severe circulatory Tests of Liver Function 59 disturbances artificially produced are followed by a drop in the phthalein output. Actual destruction of liver parenchyma produces similar results. In experimental obstructive icterus there is, of course, no phthalein output, since none of the drug reaches the intestine. In experimental hematogenous icterus there is no modification of phthalein output. The experimental studies so far performed indicate that the phenoltetrachlorphthalein will be valuable from a quantitative as well as a qualitative standpoint in the estimation of insufficiencies of liver function. The clinical application of phenoltetrachlorphthalein as a test for hepatic function was first worked out by Rowntree, Horwitz and Bloomfield. We owe to Rowntree the suggestion that the specific- ity of the liver in excreting phenoltetrachlorphthalein analogous to that of the kidney towards phenolsulphon- phthalein would indicate that estimations in man of the quantity of dye excreted by the liver after an in- travenous injection ought to afford a practical clinical method of determining the functional capacity of the liver. It will thus be seen that the phenoltetrachlorphthalein test of Rowntree, Horwitz and Bloomfield is founded upon rational theoretical considerations and that it was subjected to a very rigid experimental investigation. Technic of Phenoltetrachlorphthalein Test. An aqueous solution of the disodium salt is used. It is pre- pared by placing 2.5 gms. of phenoltetrachlorphtha- lein in a 200 c.c. Erlenmeyer flask with 5 c.c. of 2 / n NaOH solution and 45 c.c. of freshly distilled water. This is boiled under a reflux condenser for 20 minutes. The solution is filtered into a 100 c.c. flask. This solu- tion is of 5% strength and is approximately isotonic 60 Manual of Vital Function Testing Methods with blood. It is intensely purplish red in color. Since the phthalein is precipitated by CO 2 in the atmosphere it will not keep more than a few days, hence requires to be freshly prepared for use. The patient is given two compound cathartic pills the night before the test is applied. In making the test 8 c.c. of the solution which will contain about 400 milligrams of tetrachlorphthalein are measured. It has been found that this amount is never followed in normal persons with the excretion of any dye in the urine and is sufficient to produce an intense color in the final preparation of the feces. The 8 c.c. is given intravenously as follows, of course under strict aseptic precautions : A funnel with properly connected intravenous system is filled with freshly distilled water or salt solution and the flow into the vein is started. When this is well established, the phthalein solution is added. Fifty to 100 c.c. of water are used and the phthalein solution is washed in with freshly distilled water until the fluid entering the vein is clear. About a quarter of an hour is required for the injection. After the injection, the patient is given another pur- gative, usually two compound cathartic pills, and this dose is repeated the following morning if the bowels are not running freely. The stools are collected for 48 hours in a covered vessel. The urine is collected for 24 hours. The quantitative determination of the amount of phthalein passed is made as follows: The total feces collected are put in a wide mouth 2 liter bottle diluted with water to 1 or 1.5 liters, according to quantity, and the whole put in a shaking machine and well agi- tated for from 5 to 20 minutes. One-tenth of the total amount is immediately poured Tests of Liver Function 61 off in a 1-liter flask. To this is added 5 c.c. of 40% NaOH, which makes the mixture a dirty red color. The flask is stoppered and thoroughly shaken. One hun- dred (100) c.c. of the contents of this flask are placed in a 200 c.c. flask, to which is added 5 c.c. of saturated basic lead acetate. This decolorizes the mixture and throws down a heavy precipitate which leaves a color- less supernatant liquid. Five (5) c.c. of 40% solution of NaOH are added, which produces the phthalein color. More hydroxide solution may perhaps be needed to bring out the full color, but excess should not be used. The contents of the flask are then made up to 200 c.c., shaken, and the solution allowed to stand five minutes. The supernatant fluid is clear and some can be poured off for colorimetric examination. The read- ing is made in a colorimeter similar to the one used in testing kidney permeability with phenolsulphonphtha- lein. The comparison solution is made by taking .4 c.c. of the original solution used for injection and diluting it up to 1 liter plus sufficient NaOH to make the deepest color. The per cent, of dye eliminated can be read off on the instrument. If the reading is low be sure that the maximum color has been developed by adding a little NaOH again to the 200 c.c. dilution above mentioned. NaOH must be carefully added because excess will tend to render solu- tions yellowish red instead of pure purple red. In case the quality of color is unsatisfactory the authors of the test recommend the following procedure : After the addition of about 10 c.c. of 40% NaOH dilute the feces mixture up to 1 liter. Take one-tenth of this and add 5 c.c. of sodium hydroxide and water up to a liter. One hundred c.c. of this is put in a 200 c.c. flask and to it is added 5-10 c.c. or more of the following solution : 62 Manual of Vital Function Testing Methods CaCl 2 90 gms. Cone. NH 4 OH 10 c.c. Water 50 c.c. This brings out a good quality of color. Dilute up to 200 c.c. and allow to stand covered for some hours, per- haps even 24. The supernatant liquid is then tested in the colorimeter with the standard solution as above. The lower limit of normal output is 20%. A year's experience with the phenoltetrachlorphtha- lein test of liver function has prompted a recent com- munication from Chesney, Marshall, and Rowntree (Jour. Amer. Med. Assn., 1914, LXIII, p. 1533) in which these authors conclude that outspoken changes in the liver can, in most cases, be demonstrated by the test. It was found positive in advanced cirrhosis, in passive congestion (cardiac liver) and in cancer and syphilis involving the liver. These authors recommend the association with the phthalein test, of estimation of nitrogen partition in the blood and urine, and fibrinogen estimation in the blood serum. They make the important general observation that the information to be derived from tests of the liver function does not compare in reliability with that ap- plied to the kidney. Similar views have likewise been expressed by other writers. The reason would appear to be plain. We do not as yet understand the functions of the liver regarded as a unit or dissociatively as we do those of the kidney. The symptomatology of hepatic insufficiency is not understood to an equal extent with that of the kidney. However this may be, sufficient progress has been made to afford ample congratulations for the work of the past and an optimistic outlook for Tests of Liver Fwnction 63 future developments. Krumbhar 43 has recently stated as a result of his researches and investigations that the phenoltetrachlor- phthalein test of Rowntree, Horwitz, and Bloomfield promises a greater value than all other tests so far devised for estimating the functional capacity of the liver. 43 N. Y. Med. Jour., 1914, c. 719. CHAPTER II TESTS OF KIDNEY FUNCTION GENERAL CONSIDERATIONS Historical. In 1830 Hahn noticed after ingestion of turpentine in gouty persons that the substance failed to render the urine odorous as it was known to do in healthy persons. In 1837 Rayer noticed the same thing with regard to asparagus. Clinicians for a long time have known that many persons with nephritis can not take mercury, salicylates, iodides, bromides and various other drugs without rap- idly showing signs of intolerance. Todd wrote upon this subject in 1857 and Roberts in 1865. Duckworth and Bouchard in 1873 showed experimentally that many drugs which normally pass quite readily through the kidney, fail to do so in nephritis. The first practical application of these facts was made by Achard and Castaigne, who introduced methyl- ene blue in 1897 as a direct test of the functional capacity of the kidneys. Physiological. As well expressed by Blum the fun- damental function of the kidney is its osmoregula- tory power; its power to constantly maintain at an unvarying point the molecular concentration of the blood. This it does by removing a series of substances whose accumulation in the organism would eventually 64 Tests of Kidney Function 65 produce serious and even fatal consequences. These substances are removed in the urine. Two general theories of urinary secretion have existed, side by side, for several decades. They are known eponymically by their originators and although they have been added to or subtracted from in details by a host of subsequent workers they yet stand as op- posing schools of physiological interpretation. These schools are known as that of Ludwig on the one hand and that of Bowman-Heidenhain on the other. According to Ludwig, the elimination of urine is a simple process of physical filtration and diffusion. The anatomical structure of the glomcrulus and the physio- logical conditions existing therein, appearing to favor the idea of filtration, Ludwig believed that water passes through the epithelium of the capillary wall and the glomerular epithelium as through a filter, carrying with it sodium chloride and other inorganic salts and urea, and that the diluted urine in its passage through the uriniferous tubules becomes concentrated through loss of water by diffusion into the more concentrated blood and lymph. According to the other theory, that of Bowman- Heidenhain, the elimination of urine is fundamentally a secretory act and not fundamentally a physical act. It assumes that the glomerulus secretes water and inor- ganic salts while the epithelial cells of the uriniferous tubules secrete urea and the other specific constituents of the urine. We shall not attempt here either a historical or phys- iological review of these theories. It may be said that the majority of physiologists adhere to the more con- servative and vitulistic hypothesis of Bowman-Heiden- hain. The grounds for this belief, and, indeed, all of the facts bearing upon both sides of this now classical 66 Manual of Vital Function Testing Methods controversy, are properly to be found in any modern text book of physiology. 1 It is pretty generally agreed that whether by filtra- tion or secretion, water leaves the kidney through the glomerulus. Beyond this generally accepted fact there is so little unanimity of opinion as to the exact place where sodium chloride urea and the other solid constitu- ents of the urine are eliminated, that it is impossible to make any categorical statement with reference thereto. Many attempts have been made to divide up the total kidney function into categories or topical functions and to locate these functions anatomically in parts of the glomerulo-tubal structure. But it cannot be pretended at the present time that any such differentiation has been proven, certainly not to such an extent as to justify deductions of great practical importance. This question will, however, be more fully elaborated later. The composition of the urine is far from simple. Its chief constituents, apparently, are water, sodium chlo- ride and urea. But besides these substances, urine con- tains purine bodies (uric acid, xanthin, hypoxanthin), creatinin, oxalates, glycuronates, phosphates, sul- phates, various oxy-nitrogenous and fatty acids, and the pigments urochrome and urobilin. Nearly all of the nitrogen excreted from the body is supposed to pass through the kidney. The total amount of nitrogen eliminated in the urine in 24 hours is conse- quently regarded as the most important index of proteid metabolism. The actual estimate of total urinary nitro- gen is usually done by the method of Kjeldahl (described under Liver Tests, q. r.). The total weight of nitrogen in the urine multiplied by 6.25 gives the amount of protein broken down in the body, since nitrogen forms 16% of the weight of 1 See Howell, Halliburton, etc. Tests of Kidney Function 67 the protein molecule. The total amount of nitrogen eliminated in 24 hours by a normal adult is between 14 and 18 grams, which corresponds to 88-117 grams of protein. The total nitrogen eliminated in the urine is divided as follows: 1. Urea nitrogen; this averages 87.5% of the total. 2. Ammonia nitrogen; this averages 4.3% of the total. 3. Creatinin nitrogen, 3.6%. 4. Purin nitrogen, variable. Urea occurs in the urine as its chief nitrogenous constituent (about 2% ). Since a normal adult secretes 1500 to 1700 c.c. of urine in 24 hours the amount of urea eliminated will vary from 30 to 34 grams. Urea, of course, is not manufactured by the kidneys, but is merely eliminated by them. Urea is a normal con- stituent of blood existing in that fluid in quantities varying from .035 to .153%. If the kidneys are re- moved or become impassible to urea this substance accumulates in the blood. Sodium chloride is the chief inorganic constituent of urine, amounting to about 15 grams per day in a nor- mal adult. Under pathological conditions a variety of sub- stances organic and inorganic may appear in the urine, whose search and identification is a part of the routine analysis conducted for clinical purposes. A consideration of these substances belongs, of course, to the domain of general clinical pathology. The question of how far an ordinary urinary examina- tion can serve to reveal the functional capacity of the kidney will be taken up presently. The kidney functionates normally at a point below its maximum capacity, retaining unused a certain amount of functional energy which constitutes its re- serve. When this reserve becomes exhausted the func- 68 Manual of Vital Function Testing Methods tional capacity of the organ will be irremediably dam- aged unless it can recuperate. When uremia or edema have appeared the amount of functional incapacity of the kidney has become considerable. No special test of function is required to discover it, perhaps, but even under such conditions, it may be extremely useful to determine just how far, in any given case, the deprecia- tion of functional integrity has gone. Classification. There are various general plans by which the functional state of the kidney may be in- vestigated. In the first place we may determine how far the kid- ney is able to eliminate increased amounts of its normal constituents, such as water, salt and urea. In the second place we may select substances foreign to the organism, but which are eliminated by the kid- ney, and determine the rate and quantity of their excre- tion. Iodide of potassium, lactose, phenolsulphon- phthalein, phloridzin are examples of such substances. Thirdly, the study of the blood will constitute an- other avenue of approach to the problem of estimating the function of the kidney, because one important result of renal insufficiency will be the accumulation of sub- stances in the blood which should be eliminated in the urine. Among methods of this type may be mentioned partitive estimations of nitrogen in the blood, particu- larly incoagulable nitrogen. Such examinations will often disclose an abnormal degree of accumulation or retention of such products in the blood if a condition of renal impermeability or insufficiency exists. Thus it will be seen that all tests for kidney function are based upon the broad principle that any deprecia- tion of renal activity will be reflected in the urine on the one hand and the blood upon the other. The urine will contain less of its normal constituents than normally Tests of Kidney Function 69 and less of any substance artificially eliminated by the kidney, while the blood will show the effects of renal inadequacy by disclosing an accumulation in the plasma of substances which are normally excreted continuously in adequate amounts. All tests for renal function so far devised may be satisfactorily divided into the three following cate- gories : 1. The urine as an index of renal function (a) Urinalysis, (b) Physical and Biological characteristics. 2. The Blood as an index of renal function. 3. Elimi- nation of foreign substances by the kidney as an index of renal function. In the following synopsis, we may see how the various tests which have come to be used can be distributed among the three classes : I. Urinalysis as an index of renal function. A. Urinalysis. 1. Estimation of water : experimental polyuria. 2. Estimation of sodium chloride : experimental chloruria. 3. Estimation of urinary nitrogen ; urea, etc. 4. Estimation of urinary coloring matter. 5. Estimation of urinary diastase. B. Physical and biological characteristics. 1. Cryoscopy of the urine. 2. Electrical conductivity of the urine. 3. Estimation of urinary toxicity. IT. Studies of the blood as indices of renal function. 1. Estimation of blood urea and of incoagu- lable (residual) nitrogen in blood. 2. Estimation of coagulation time. 3. Cryoscopy of the blood. 70 Manual of Vital Function Testing Methods III. Studies of elimination of foreign substances by the kidney as criteria of function. A. Miscellaneous. 1. Potassium Iodide. 2. Phloridzin. 3. Hippuric Acid. 4. Lactose. B. Dyes or colors: experimental urinary chromos- copy. 1. Methylene blue. 2. Indico carmine. 3. Phenolsulphonephthalein. I. THE STUDY OF UUINALYSIS AS AN INDEX OP RENAL FUNCTION A . Urinalysis The urine represents, as it were, the concrete results, almost the total results of renal activity. Inasmuch as the entire urinary output of the kidney for any given period of time may be readily collected it would seem natural to assume that a chemical analysis would throw all the light that is necessary upon the problem of renal function ; but as a matter of fact, while it is true that chemical analysis of the urine provides an adequate insight into the amount of salts, of water and of urea secreted by the kidney, it is not true that urinalyses are sufficient to determine the functional capacity of the organ. Gross anatomical and physiological disturb- ances are often thus discovered and extremely important information is thus derived concerning the diagnosis of diseases of the kidney and urinary organs. But this is a very different proposition from deter- Tests of Kidney Function 71 mining thereby the functional capacity or incapacity of the kidney, in the absence of evidence of gross organic lesion. Even under apparently normal circumstances the actual amount of the different urinary constituents excreted may vary considerably. For example the chlorides may be eliminated in excess and nitrogen re- tained or vice versa. But such variations are not neces- sarily dependent on anatomical lesions of the kidney or even upon any disturbance of renal permeability. If, in a given 24-hour specimen of urine, the figures representing the elimination of the different important constituents depart from the usual normal, we cannot draw any absolute conclusions from this fact alone as to whether the functional capacity of the kidney is below or above normal. One reason for this is that the chemical constitution of the urine is not dependent alone upon kidney functional power but it is influenced by a large number of extremely important extrarenal factors, among which may be mentioned the intake of food and fluids, the condition of the nervous system and other organs, etc. It is a well-known fact that before any important conclusions as to nitrogen metabolism can be drawn from the chemical constitution of the urine, these factors must be taken into consideration, and if they are ade- quately considered the task becomes complicated by many necessary experimental refinements. The whole question of body metabolism must be taken up. The intake and output in every direction must be measured. But even after this is done and it is demonstrated from urinalysis that there is a deficit in nitrogen excretion, the kidney function may be perfect and the nitrogen simply retained in the tissues. Variations in nitrogen elimination occur under so many different conditions that interpretation is often 72 Manual of Vital Function Testing Methods difficult or impossible. The same may be said with regard to the output of other constituents of the urine. Concerning the application of urinalysis to the inter- pretation of kidney function it may be said that if the figures are all consistently and invariably normal, the kidney function is apt to be good; and if there is a persistent and considerable departure from normal the kidney function may be deficient. But in order to be of real value, the tests, particularly those regarding the nitrogen excretion, must often as- sume the proportion of metabolism experiments, which makes them impractical for clinical use. Fortunately for the purposes of clinical medicine the physician will not be called upon to consider the in- tricate problems of nitrogen metabolism in his investi- gations of renal function. His desire will be to know the capacity of the kidney to do its work and fortu- nately this object may be accomplished without recourse to extremely elaborate and technical processes. We are justified in expecting that under an average regimen the kidneys will eliminate somewhere near the average amounts of the urinary constituents. But knowing how many and how variable the extrarcnal fac- tors are which influence the absolute quantities of uri- nary constituents eliminated, it will become apparent that the results of a urinalysis, no matter how complete, will require to be supplemented by other and better means of determining the functional capacity of the kidney. 1 . Estimation of Urinary Water as an Index of Renal Function. Experimental Polyurla. Albarran's Method The healthy kidney possesses the power in a high degree to adapt itself to those tendencies, such as addi- tion or subtraction of water to or from the circulating Tests of Kidney Function 73 blood, which would tend to alter the molecular concen- tration of the fluid. It quickly re-establishes both mo- lecular and water equilibrium, thus maintaining an equable osmotic tension in the blood and lymph. Super- fluous water is, normally, quickly eliminated through the glomerulus and reabsorption in the canaliculus is in- hibited. When the supply of water to the organism is deficient, the resorptive function of the canaliculus is raised and a more highly concentrated urine is elimi- nated. This function of the kidney may be properly termed its diluting-concentrating power. Since water secretion is a function of the glomerulus, the diluting power of the kidney is a glomerular func- tion. The functionally weak kidney is not only unable to produce a highly concentrated urine but also unable to elaborate a much diluted one. The concentrating power of the kidney is a function of the epithelium of the uriniferous tubes (canaliculus). The diluting-concentrating power of the kidney suf- fers in diffuse kidney disease in proportion to the amount of parenchyma involved. In parenchymatous nephritis the water secreting power of the kidney is lowered ; in contracted kidney it is more or less retained. The increased urine following an experimental pro- vocative polyuria test differs from the increased secre- tion following a heavy meal, since in the former case the freezing point ( A ), molecular concentration, chloride, phosphate and urea content (specific gravity) are all diminished, while in the latter they are all increased. With respect to continuity of function the diseased organ possesses a greater constancy and invariability in proportion to the amount of disease. The normal kidney function tends to vary, that is, to adapt itself to the constantly changing conditions in the organism. The diseased organ has no such power. 74 Manual of Vital Function Testing Methods The healthy kidney always functions below its maxi- mum strength; always possesses, in other words, a cer- tain reserve power which can be used under extraor- dinary circumstances, such as great increase in water and solid molecular intake. The insufficient kidney is unable to meet these requisitions for added energy, and responds but little if at all to extra stimulation. It has lost its reserve. As a corollary to the above it may be added that if one kidney is diseased and the excretion from the two organs be compared, the facts as above stated will apply. The affected kidney is less able to respond to adaptation requirements than the normal and the degree of its failure to do so may properly be taken as the measure of its incapacity. For these reasons polyuria tests may be employed with the view of conducting examinations upon the total excretion or whenever necessary upon the excretion ob- tained by ureteral catheterization from each organ separately. The Water Tests. The provocative polyuria tests are usually carried out with water. The tests should be applied in the morning on an empty stomach. The morning urine prior to the test should be measured and examined for quantity and specific gravity, total sodi- um chloride and urea elimination and perhaps cryo- scopically. The patient is then given 500-700 c.c. of mineral water or ordinary water. The urine should be collected every half hour by voiding or catheter if the total amount is to be examined (general renal function) or by ureteral catheterization if the separate kidney functions arc to be compared. Under normal circumstances the polyuria appears within the first half hour, reaching its maximum at this time, and quickly sinking. The content of solids sinks. Tests of Kidney Function 75 The freezing point (A) diminishes. If the functional power of the organ is below normal, the polyuria is delayed or does not occur and the amount of variation from the normal may be taken as a fair measure of the incapacity. Straus-Grunwald Method. The patient takes noth- ing after 7 P. M. into the stomach. At 6:30 A. M. the following morning a pint of water is ingested. The night urine is collected; also that voided at 7, 8, 9, 10, and 11 A. M. The amount and specific gravity of each portion are recorded. The patient remains in a reclin- ing position during the time of the test. In normal cases an amount of urine is passed in the first 3 hours equal to that which was drank. That is by 10 A. M. at least a pint is voided. At 8 A. M. the sp. gr. is lowest. Variations in the amount voided, time required, and specific gravity will indicate abnormal renal function. The Diuretic Tests {pharmacological}. Caffein, diuretin, theophyllin (theocin), euphyllin and other diuretic substances have been employed, but none of these drugs has been found to possess much advantage over the simple water test. The diuretic drugs appear to increase the solid constituents of the urine as well as the fluids. Blum, who introduced euphyllin, does not recommend it because he has found a fall of blood pres- sure follow its hypodermic or intravenous administra- tion. None of these tests have acquired a sufficient promi- nence to justify their description. While the so-called water or polyuria tests have proven of some value in estimating relative function in the separate kidney secretions, it is generally agreed 76 Manual of Vital Function Testing Methods that they arc of much less importance in estimating total kidney function. The absolute quantity of urine voided varies very greatly under normal and abnormal circumstances. According to Rowntree and Fitz there is no constant relation between the existence of polyuria or oliguria and the condition of kidney function as shown by other well-recognized tests. This applies to nephritic and cardiac cases and to combinations of these. The specific gravity of the urine in advanced nephri- tis according to these authors is usually low. 2. Estimation of Sodium Chloride as an Index of Renal Function Ten to fifteen grams of sodium chloride are excreted by a normal adult in 24 hours. The rapidity with which the kidneys can excrete a considerable amount of sodium chloride has been suggested and employed as a test for renal function. With regard to the method of so-called forced elim- ination of sodium chloride, it must be freely granted that the problem is a very difficult and complicated one. If diminished secretion of sodium chloride always indicated renal impermeability the problem would be solved, but this is by no means the case. The tissue fluids themselves, everywhere in the body perhaps, have varying affinities for sodium chloride and a diminution of chloride elimination may not signify a diminished permeability of the kidney for salt but only an increased retention of salt in the body. Nevertheless there is, under normal circumstances, a very close relation between the intake of sodium chloride in the food and its elimination by the kidneys. So that if an individual is placed for a period of time upon a Tests of Kidney Function 77 regimen containing a low percentage of salt, the excre- tion of that substance will become reduced to a lower equilibrium. If now the quantity of sodium chloride ingested be suddenly increased there will be an immediate and proportionate increase in the amount secreted. If there is no increase, it will be difficult to determine whether there is chloride retention or defective excre- tion so that the chloride test is seldom used alone as a measure of renal function. The exact situation in the glomerulo-tubular mechan- ism where sodium chloride is excreted, is not known with certainty. . According to the rather recent investi- gations of Schlaycr and Takayasu and Von Monakow, sodium chloride is excreted by the tubular epithelium, or more exactly the excretion of salt following its ad- ministration in amounts in excess of the usual daily intake is accomplished by the tubules. When large amounts of salt are ingested the excre- tion, according to Schlayer, takes place in one of two ways, depending upon the amount of water simultane- ously absorbed. If the salt is given without extra water it is almost entirely secreted within 24? hours without diuresis, by an increased concentration of the urine. If, however, it is given with an excess of water it is secreted partially through increased concentration and partially through diuresis. If the vascular structure of the kidney is injured, the ingestion of salt may be followed by marked diure- sis, the salt all escaping in the urine in 21 hours with- out the percentage content being increased. The spe- cific gravity may be low and tends to remain at a fixed point. To tin's combination of phenomena, Schlayer gave the name vascular hyposthenuria, and, according to his idea, the inability of the kidney to eliminate a urine concentrated in salt is not due to tubular defect 78 Manual of Vital Function Testing Methods but to a hypersensitive condition of the blood vessels which allows the secretion of the salt in relatively large amounts of water; in other words, the sensitive vessels respond to the salt administration by the diuresis. When the vascular injury is more marked the vessels do not so react but respond with oliguria. In severe tubular epithelial disease, however, the quantity of salt eliminated is not raised by the adminis- tration of salt. Here a urine of fixed low specific grav- ity of moderate quantity is obtained. The salt is re- tained. There is, according to Schlayer, a tubular hyposthenuria. These interesting findings reported by Schlayer and his school have not been completely corroborated and it does not appear to be agreed at the present time that any absolute distinction between vascular and tubular hyposthenuria can be founded upon the response of the kidney to tests with sodium chloride. In fact the number of extrarenal factors concerned in the salt output are so many and so illy understood that the salt test alone is considered of no value. But when considered in conjunction with other functional tests and with clinical findings in cardiac and renal cases it may be of some diagnostic and prognostic value. In advanced nephritis there seems no doubt that salt elimination is lessened to a certain extent but if the patient has been on a salt-poor diet for a time previous to the test, the tissues will retain salt when administered regardless of the cardiorenal condition. (Edema is the symptom in chronic nephritis which is usually associated with the idea of chloride reten- tion. It is not positively known just what factors are concerned in this, or whether they are chiefly renal or extrarenal. Tests of Kidney Function 79 It is on the basis of the supposed connection be- tween oedema and chloride retention that the now well- known method of salt reduction in the treatment of oedema in Bright's disease was introduced. Technic of Sodium Chloride Test. Test of Alimen- tary Chloruria. The patient is placed on a diet con- taining about 5 gms. of salt per day. After several days or when the salt output is approximately con- stant, 5-10 gms. of sodium chloride are given, dis- solved in 125 c.c. of water. The quantity is taken in three portions during the day. This is kept up for four consecutive days. The daily output of chloride is determined by the method described below. If the patient is kept on a salt-poor diet, say 2.5 gms. daily for some time previous to the test, it will be found that excretion will always be lessened from a normal ten- dency of the tissues to retain chlorides. For this reason the best method consists in merely establishing chloride equilibrium before the test is started. Estimation of Sodium Chloride in the Urine. The principle of the test is that chlorides are precipitated by solutions of nitrate of silver. Volhardt's method with its various modifications is regarded as the most accurate quantitative method of chloride estimation, and is used when the exact amount must be known as in metabolic experiments. For all practical purposes Mohr's method will suf- fice. 2 The strength of silver solution used in the test is such that 1 c.c. corresponds to .01 gm. of sodium chloride. Such a solution contains 29.06 gms. of pure fused silver nitrate to the liter. The technic of the estimation is as follows : 2 The Lutke-Martius method is often recommended, see Sahli's Diagnostic Methods, 1911, p. 455. 80 Manual of Vital Function Testing Methods 10 c.c. of urine previously freed from albumen are put in an Erlenmeyer flask or porcelain capsule and 100 c.c. of water added and several drops of potassium chromate solution, enough to produce a distinct yel- low color. The standard silver solution is added from a burette, stirring until the reddish orange color which appears first where the drop falls is distributed. The first permanent orange color trace is the end of the reac- tion. The operation should be repeated if necessary, to make certain of results. The number of c.c. used multiplied by .01 gives the amount of sodium chloride. The results are a little high but near enough for prac- tical purposes. A rough estimate of the amount of chloride in the urine may be made as follows : To a test tube of clear urine non-albuminous, add 10 drops of pure HNO 3 and one drop of AgNO 3 (1 to 8). If chlorides are normal or increased, the precipitate is a compact ball which sinks to the bottom. If dimin- ished, the ball is less compact. If much diminished, only a cloud is produced without solid flakes. This last represents a chloride content of \% or less. 3. Estimation of Urinary Nitrogen as an Index of Hcnal Function When functional tests of the liver were being dis- cussed (z'.s.) considerable emphasis was laid upon in- terpreting ureagenetic disturbance of that organ by taking into consideration the different phases of nitro- gen metabolism which arc so closely connected with liver activity. It was then shown that a knowledge of total nitrogen elimination in the urine and particu- larly of the different forms in which the nitrogen is eliminated, will shed light upon the condition of liver Tests of Kidney Function '81 functional power. The reasons for these assumptions were given in their proper place, but it may be serviceable to refer again to the fact that the liver is regarded as a very important locus for the synthesis of urea in the body. For this reason it is quite reasonable to suppose that functional depreciation of the liver cells would be re- flected to an appreciable extent by the quantitative relative variations of the nitrogen constituents of the urine. When now we come to the significance of nitrogen elimination in the urine to disorders of kidney activity, it will be necessary to remember that the kidney has nothing to do whatever in a specific way with the nitro- gen metabolism. Its only function is to excrete the nitrogenous waste products which are brought to it by the blood. That urea is not manufactured by the kidney is proved by the fact that if blood is perfused thro an isolated kidney, no urea is formed even tho substances such as ammonium carbonate, from which urea is readily produced, are added to the blood. It is well known that if the kidneys are removed in ani- mals or their function paralyzed, urea will continue to accumulate in the blood as long as the animal sur- vives. No physiological fact is bettor known than that the relative amount of urea nitrogen in the urine varies directly with the amount of protein food ingested. Other nitrogenous constituents of the urine, the purin bodies and creatinin, are unaffected by the intake. This suggested to Folin that most of the urea in the urine may come directly from food proteins which, having been hydrolyzed in the intestine into amino acids, are absorbed and further hydrolyzed and oxidized and the nitrogen constituent immediately eliminated as 82 Manual of Vital Function Testing Methods urea. The liver has most to do with this process though the urea forming function of this organ is known to be shared by some other tissues since even after the removal of the liver some urea is formed. These physiological principles being agreed upon, it is easy to appreciate that the question of the estima- tion of urinary nitrogen as an index of renal function will be practically confined to the estimation of the amount of urea eliminated under normal circumstances, the patient being on a fixed diet, with an estimation of the power of the kidney to eliminate more urea when the proteid intake is increased, or when urea itself is ingested. The question of nitrogen accumulation in the blood as an index of renal insufficiency will be dis- cussed below, when the study of the blood as an index of renal function is considered. It may be admitted here that the study of the partition of nitrogen and particularly quantitative estimations of urea and in- coagulable nitrogen in the blood serum are of much greater significance in the estimation of kidney function than the same or related studies applied to the urine. Diminished and Delayed Excretion of Urea. This is an old criterion of functional renal power. The physiological principles upon which it is based have just been given. In order that this test of renal func- tion shall be really conclusive, the patient must be put for some days upon a fixed regimen in which the amount of protein is definitely known. The feces and urine must be examined to determine what part of the nitrogen has escaped in both. In carrying out such procedure, the experiment rises to the dignity of a metabolic investigation and requires great care and patience besides considerable technical skill. For this reason such a procedure cannot be re- garded as adapted to clinical use. Tests of Kidney Function 83 Without the above precautions the value of ordinary routine urea estimation in the urine as a criterion of renal function is extremely doubtful. If the dietary conditions can be reasonably con- trolled and a perfectly and persistently normal output of urea results, the renal function is at least equal to the ordinary demand of that person. If the proteid intake is increased and the urea excretion undergoes a corresponding and immediate rise, it may be con- cluded that the reserve force of the kidney is not materially damaged. But negative results need not necessarily be taken to indicate a defect of renal function because of the fact that digestive disturbance and diminished liver function will cause the same thing to occur. If these extrarenal factors of error can be eliminated, then a diminished output of urea may become a valuable and reliable index of renal inadequacy. Under a later chapter, concerning study of the blood as an index of renal function, it will be shown that the quantitative estimation of nitrogen partition in that fluid is a much more reliable test of renal functional power than urea estimations in the urine. Repeating what has been given under general con- siderations it may be stated that a normal adult se- cretes from 30 to 34 grams of urea in the urine every 24 hours. The simplest quantitative test for urea in urine is that of Marshall, which has been described (v. s.}. This method is so simple and so accurate that every clinician should familiarize himself with it. Forced Urea Elimination. Provocative Urea Test of McKasky. Technic- Thirty grams of urea dissolved in four to six ounces of water are given with a small 84 Manual of Vital Function Testing Methods breakfast, such as a cup of gruel. Follow this with four or five ounces of water to assist diuresis. The urine is collected every two hours for twenty-four hours, beginning two hours before the urea is given, so that a standard for comparison may be had, to determine the amount of increase. Quantitative determinations of urea are made in the different specimens at the end of the time. The maximum excretion, its time, incidence, and the curve for the 24 hours, is thus determined. Under normal conditions there is a sharp rise in urea excretion in the second two-hour period. When the kidney function is deficient the sharp rise is absent or is much delayed. The only factor liable to disturb the interpretation of this test is gastric stasis. Theoretically, the provocative urea test should be useful. There does not appear to be any reason why the urea could not be injected in smaller amounts, di- rectly into the circulation, in which event the liability of misinterpretation through retention in the stomach would be avoided. The test has not been subjected to any clinical examination, so far as I know. 4- Estimation of Urinary Coloring Matter as Test of Renal Function Thudichum's Test. This test is now of only histor- ical interest. It was proposed by its author on the clinical ground that in many chronic kidney diseases the urine becomes distinctly paler in color. Therefore careful quantitative estimations of color excretion might be of value as an early sign of renal imper- meability. But unfortunately for the value of the test, the quantity of coloring matter excreted in the urine Tests of Kidney Function 85 depends upon a great variety of factors (liver, intes- tine, food, etc.), of which the most insignificant of all is perhaps renal permeability. 5. Estimation of Urinary Diastase as an Index of Renal Function Wohlgemuth's Test? Te clinic. After neutraliza- tion urine is placed by means of an accurately grad- uated pipette in a series of twelve test tubes, the amount decreasing from .6 c.c., .5 c.c., A c.c., to .1 to .09 c.c., .08 c.c., to .04 c.c. A sufficient quantity of 1% sodium chloride solution is then added, to bring the amount of fluid in each tube up to 1 c.c. In order to more readily obtain the fractional quantity of urine required, 1 c.c. of the urine may be diluted up to 10 c.c. and from this diluted urine the required measures may be taken. To each tube is added 2 c.c. of a 1-1000 solution of freshly prepared soluble starch. The tubes are im- mersed in a water bath at 38 C. for 30 minutes, after which they are placed in cold water for 3 minutes. To each tube is then added sufficient 1/50 normal iodin solution to elicit a permanent color, violet or blue occurring where digestion is not complete. The tube in the series immediately preceding incom- plete digestion of the starch indicates the diastase content of that particular urine. From this is calcula- ted the diastasic activity represented by 8. 8 is expressed as the number of c.c. of 1/10% starch solution which can be digested by 1 c.c. of urine. This test can be applied to the whole urine or to the samples obtained unilaterally by ureteral catheterization. The diastase test is particularly adapted to unilateral estimations of kidney function applied to urine obtained by ureteral 3 Lancet Clinic, 1913, CX, p. 164. 86 Manual of Vital Function Testing Methods catheterization. But altho it is capable of indicating in the majority of cases which is the diseased or more diseased kidney, in the opinion of most genitourinary surgeons it is not necessary and adds nothing to the information obtained from the phthalein test or urea estimations, which latter are operations somewhat more easily performed. The diastase test has never been used to any extent in estimating total functional capacity of the kidneys. B. The Study of Physical and Biological Character- istics of the Urine as Criteria of Renal Function. There are three tests which come under this category : 1. Cryoscopy or determination of the freezing point of the urine. 2. Electrical conductivity of the urine. 3. Determination of urinary toxicity. 1. Cryoscopy of the Urine. Significance for Estimat- mg Renal Function. Von KoranyVs Test The theoretical bases upon which this test is founded are of extreme interest both from physical and physio- logical points of view but naturally cannot be com- pletely considered here. The freezing point of distilled water is zero. The freezing point of any solution is below zero, and the depression of freezing point below zero is proportionate to the molecular concentration of the solution. Conse- quently the freezing point of a solution is a measure of its molecular concentration. It is also a measure of its osmotic pressure. The freezing point of a solution is independent of the nature, size, and molecular weight of the dissolved Tests of Kidney Function 87 molecules and is only dependent on their number. The specific gravity of a solution is on the contrary de- pendent upon the nature and molecular weight of the dissolved molecules. Solutions of similar molecular con- centration have the same freezing point and the same osmotic pressure but not necessarily the same specific gravity. By cryoscopy the molecular concentration of urine and blood can be estimated and in this way a certain insight into the functional power of the kidney can be obtained, since the global function of this organ is to regulate the osmotic pressure, or, what is the same, the molecular concentration of the blood. One can obtain from an estimation of the lowering of the freez- ing point of the urine below the zero of distilled water a somewhat more adequate idea of the functional ca- pacity of the kidney than from the specific gravity. 4 Of two urines of equal specific gravity the one with the lower freezing point comes from the better func- tioning kidney. Investigations have shown that the freezing point of the urine ( A ) in health varies between rather wide limits (A= .90 to 2.30) and that it is to some extent affected by miscellaneous factors of extra- renal nature. Altho much was expected originally from the de- termination of the freezing point (cryoscopy) of urine in estimating the integrity of renal function, it is not depended upon to any great extent at present. The molecular concentration of the blood hence its freezing point ( 8 ) is much more constant than that of the urine. It is supposed to remain somewhere 4 Some authors do not agree that cryoscopy is superior to sp. gr. estimation in determining renal permeability, v. Sahli, Diag- nostic Methods, 1905, p. 551. 88 Manual of Vital Function Testing Methods near .56. It was thought that insufficiency of renal function by allowing the accumulation of molecules in the blood which should be excreted would raise the molecular concentration therein or, in other words, lower the freezing point. Unfortunately, experience has not confirmed the hopes of those who thought that cryoscopic examinations of the urine and blood would solve the great problem of estimating the renal func- tions and the method is not extensively used. The relation between the lowering of freezing point of blood (& ) and urine (A) was proposed by Dreser as a measure of the work done by the kidney and by Bernard as the basis of a sort of mathematical con- ception of the eliminatory power of the kidney. The g formula X V = R will represent the molecular A elimination of the kidney according to this conception. A represents the freezing point of urine, 8 that of the blood, V the quantity of urine in 24 hours. In normal cases R varies from 3000 to 5000, whereas in renal insufficiency these numbers are considerably re- duced. Unfortunately all attempts to reduce our con- ceptions of organic function to mathematical terms have not been eminently successful. Technic of Cryoscopy.- The technic of cr} r oscopy is not especially complex but requires a certain ap- paratus for its performance. It is usually carried out in the laboratory and has never become a routine clinical procedure. Only the necessary outlines of the method need here be given since those who desire to master it can easily refer to numerous texts in which the technique is minutely described. 5 The technic of cryoscopy is carried out with Beck- B Consult in this connection Wood's Chemical and Microscopical Diagnosis, 1909, p. 61; Sahli's Diagnostic Methods, p. 546. Tests of Kidney Function 89 mann's freezing apparatus carrying a special ther- mometer. The freezing mixture is made of ice, water and salt. In the freezing mixture is plunged an ordi- nary thermometer and a mixer, thus enabling the tem- perature to be kept at a fixed point ( 3 to 5). Thro an opening a tube containing the special ther- mometer immersed in the liquid to be frozen can be immersed in the freezing mixture. The estimation of the exact points of freezing is not difficult and usually the operation can be performed in its entirety in 15 or 20 minutes. 2. Electrical Conductivity of the Urine The electrical conductivity of the urine in health and disease was first studied by Turner. The electrical conductivity is estimated in ohms of resistance and depends upon the number of ions of salts dissolved in the urine. The method measures, in other words, the amount of salts or mineral content of the fluid. The Kohlrausch method of performing the test, which is the one usually employed, requires a whetstone bridge, a resistance box, telephone and cells, besides other paraphernalia, and partly on account of this complexity and also the fact that the practical results obtained are meagre, the test has never come into gen- eral use. 3. Estimation of Urinary Toxicity as a Test of Renal Function Bouchard's Test.- It was for a long time believed that the toxicity of the urine was proportional to the functional power of the kidney. Urine of human be- ings produces symptoms of poisoning and death when 90 Manual of Vital Function Testing Methods injected intravenously into lower animals. Bouchard developed from this fact a method of testing renal function 6 by determining the quantity of a 24-hour specimen of urine, required to kill a kilogram of lower animal. Bouchard established a so-called urotoxic co- efficient which was that quantity of poison elaborated by every kilogram of body weight of the person whose urine was tested. The same objections exist with respect to the theo- retical basis of this test, as in the case of chemical urinalysis previously discussed, namely that so many factors besides renal sufficiency or insufficiency enter into the production of results that the test becomes devoid of scientific value. It simply shows the toxicity of a given urine injected intravenously into a given animal and by no means reflects the actual functional power of the kidney thro which it was derived. The test is quite complex and has been abandoned. II. STUDIES OF THE BLOOD AS CRITERIA OF RENAL FUNCTION. ESTIMATION OF BLOOD UREA AND OF IN- COAGULABLE (RESIDUAL) NITROGEN IN BLOOD When it is considered that a major portion of the nitrogenous waste of the body makes its escape thro the kidney by way of the urine, it becomes evident that a diminution of the functional capacity of these organs must often result in an accumulation of nitrogenous products of metabolism in the blood. Such an idea is very old and as long ago as 1821 Prcvost and Dumas " reported an increase of urea in the blood after extirpation of the kidneys in animals. 8 Also toxopexic liver function (y. .?.). 1 Quoted by Schondorff in Pfliiger's Archiv f. de ges. Physiol., 1899, LXXIV, p. 307. Tests of Kidney Function 91 The clinical importance of their experiments was rec- ognized by Bright in his observations upon nephritis in 1836. 8 After B right's time it became well recognized that in chronic nephritis there may be a tendency toward accumulation of nitrogenous matters in the blood, but the quantitative study of nitrogen retention was forced to await the development of accurate chemical methods of investigation. In this place we can speak only of modern technic. The development of accurate technic in nitrogen estimation of the blood and its application to clinical medicine is a subject which has been perfected only since the beginning of the century. Ascoli, 9 Strauss 10 and others showed that in many cases of chronic Bright's disease nitrogenous matter accumulates in the blood and the increase is more marked as death ap- proaches. Muller 1: showed that in outspoken uremia the accumulation becomes proportionately more marked. Obermayer and Popper 12 first laid stress upon the increase of incoagulable nitrogen in the blood serum in uremic states and found that in this incoagulable nitrogen increase, urea plays the most important part. Hohlweg 13 substantiated these facts and altho he showed that an increase of urea in the blood is not necessarily pathognomonic of uremia, nevertheless its accumulation therein may be regarded as an evidence and to some extent at least as an index of the function s Guy's Hosp. Rep., 1836, I, p. 358. u Pfluger's Arch. f. de ges. Physiol., 1901, LXXXVII, p. 103. 10 Chronisch Nierenenzundung; ihrer Eimvirkung auf die Blut- flussigkeit, etc., Berlin, 1902. "Verb. d. Deutsch. path. Gesell., 1904-5, VII to IX, Erg. 80. "Zeit. f. klin. Med., 1909, LXVII, p. 332. M Deut. Archiv f. klin. Med., 1912, CIV, p. 216. 92 Manijuil of Vital Function Testing Methods of the kidneys. Widal, 14 however, believes that the amount of urea retention is an actual quantitative index of renal func- tion and that the severity and prognosis of a given case may be predicated upon the basis of such findings. Widal's clinical method of estimating urea in the blood is not commonly used in this country as it gives, ac- cording to Rowntree and Fitz, an error of 10 to 60% and is therefore useless as a quantitative method. The estimation of urea in blood serum has remained until recently a rather difficult chemical operation, but of late one or two practical clinical methods have been devised which render the test much more practical. Marshall's method appears to be the simplest and most practical of these and will he described in full (.*.). The normal figures for the elimination of urea are between .300 to .500 gm. per liter of serum. The simplest methods by which the total incoagulable nitrogen in the blood serum may be determined still remain even more difficult and complicated than those of urea estimation, especially since they involve in the end a nitrogen determination by Kjeldahl's method. A fair laboratory equipment is therefore necessary. The most practical method of determining the in- coagulable nitrogen seems to be that of Hohlweg and Meyer which has been modified by Morris ]r> in this country. This method will be described below. The normal figures for total incoagulable nitrogen in the blood serum are .500 to .600 gm. per liter. The recent work of Folin and Denis indicates that a urea concentration in the blood of .5 gm. and total in- 14 Bull, et Mem. Soc. Med. d. hop. de Paris, 3, 1011, XXXTI, p. 627. 15 Archiv. of Int. Med., 1911, VIII, p. 457. Tests of Kidney Function 93 coagulable nitrogen content of .6 gm. per liter, which was formerly considered normal, is too high an esti- mate. They found the normal urea concentration .13 gm. and incoagulable nitrogen .26 gm. per liter. In their experience no great prognostic significance is to be attached to urea concentration of less than .55 gm. per liter and incoagulable nitrogen less than .50 gm. per liter. Greater concentration than this, especially if the freezing point of the serum drops lower than 60 are of considerable prognostic significance. In pure passive congestion Rowntree states that he has never seen the rest nitrogen in the blood serum higher than .63 gm. per liter. Marked accumulation of incoagulable nitrogen or of urea in the blood is now regarded as a valuable evidence of renal insufficiency and in cases of nephritis it is an unfavorable prognostic sign. The relation between non-protein (incoagulable) ni- trogen retention in the blood and the excretion of phe- nolsulphonphthalein has been studied in experimental uranium nephritis by Frothingham, Fitz, Folin and Denis. 16 These investigators found that the results of the two tests paralleled very closely. For this reason and because in clinical studies the same parallelism has been found to obtain, these two tests have come to be considered as among the best for conjoint use. It will now be necessary to give the simplest, most practical and accurate methods by which the clinical investigator may determine the two important phases of the blood which have been discussed, namely, the amount of urea in the blood and the amount of in- coagulable or rest nitrogen contained in the same fluid. The chemical operations which are used at present for these purposes are simple enough to bring about their "Arch, of Int. Med., 1913, XIII, p. 2L5. 94 Manual of Vital Function Testing Methods frequent use in the clinic. The best method of quickly and accurately determining the amount of urea in the blood is that of Marshall. The incoagulable or rest nitrogen in blood serum is usually determined by either of two general methods, that of Hohlweg-Meyer and that of Folin and Denis. The details of these three important methods will now be given. Marshall's Method for the Determination of Urea in the Blood. 17 The blood is drawn in the usual man- ner and allowed to stand on ice until clotting is com- plete. As shown below, the urea content of the serum does not change after standing even for three or four days ; the blood can, therefore, be kept on ice over night, if desired. Two equal portions of the serum are measured into ordinary test tubes, 1 c.c. of the soy bean extract lf added to one tube, and about 0.5-1.0 c.c. of toluene to each. If sufficient serum is available, 10 c.c. portions should be used; however, perfectly satisfactory results can be obtained by using 5 c.c. or even 3 c.c. portions of the serum. The tubes are tightly stoppered and allowed to remain at room temperature until the con- version of the urea into ammonium carbonate is com- plete. Generally, they are allowed to stand over night, altho four to five hours is usually amply sufficient for the completion of the reaction. The contents of the tube containing the serum and extract arc transferred to cylinder A (see illustration), and washed in with a "Jour, of Biol. Chem., 1913, XV, No. 3. 18 The preparation of the soy bean extract is as follows: Ten grams of finely ground soy beans are treated with 100 c.c. of water and allowed to stand with occasional agitation for one hour. 10 c.c. of ^ hydrochloric acid are added and the mixture allowed to stand about fifteen minutes longer. It is now filtered and pre- served with toluene. Such a solution is perfectly satisfactory for use at least five or six days after its preparation. Tests of Kidney Function 95 very small amount of water (not more than 5 c.c.). Two grams of sodium chloride, an equal volume of al- cohol and a layer of kerosene oil are added to the cylin- der. The contents of the other tube are transferred to cylinder B, and treated in exactly the same manner. 25 c.c. of - hydrochloric acid and about 25 c.c. of water are placed in each of the 200 c.c. Erlenmeyer flasks used for the absorption of the ammonia. The different parts of the apparatus are now connected and about 0.5 gram of sodium carbonate added to each cylinder. A rapid air current is passed through the apparatus until all the ammonia has been removed from the cylinders. With a good suction pump, one hour suffices. The excess of acid in the absorption flasks is titrated with OU sodium hydroxide and alizarin sodium sulphonate. The amount of acid neutralized in the flask attached to 96 Manual of Vital Function Testing Methods cylinder B corresponds, of course, to the ammonia 10 present in the serum, while the amount used in the other two flasks represents the urea plus the ammonia. The difference corresponds to the urea in terms of r-r OU hydrochloric acid, and multiplied by 0.0006 gives the urea in grams present in the amount of serum taken for the determination. Details in Connection With the Apparatus and De- termination. 1. On account of the large quantity of protein in serum, it is advisable to use both alcohol and kerosene to prevent foaming. 20 2. The tubes C and C' are ordinary calcium chlor- ide drying tubes packed loosely with cotton. These in conjunction with the bulbs prevent any splashing or mechanical transmission of the alkali into the ab- sorption flasks. While the bulbs are probably not absolutely necessary, they are convenient in keeping the cotton filters dry. 3. For the better absorption of the ammonia, the tubes in the Erlenmeyer flasks are closed at one end, and pierced with six or seven small holes, as suggested by Folin.' 1 Even with this device one absorption flask is not always sufficient to completely absorb the am- monia. Two flasks are always used for safety in con- nection with the urea determination; however, since from the serum alone only a very small amount of am- monia (corresponding to 0.10-0.70 c.c. of -^ HC1) oU 19 We can, however, place no value on this as a determination of the true ammonia content of the blood, for on standing even a few hours tin 1 blood develops much more ammonia than the original amount (Folin). !0 This has been pointed out by Folin, in connection with the use of the air current method for determining ammonia in blood. (Zeitsch. f. physiol. Chem., XXXVII, p. !(>.>, l!)OJ-0.'i.) -'Jour. Biol. Chem., XI, p. 493, 19U. Tests of Kidney Function 97 is ordinarily obtained, one absorption flask is here sufficient. 4. A layer of toluene is placed on the liquid in the absorption flasks, for. due probably to the alcohol car- ried over by the air current, considerable foaming some- times occurs. If not prevented, this results in a loss of a portion of the contents of the flask. 5. The bottle contains dilute sulphuric acid to free the air from any traces of ammonia before passing it through the apparatus. 6. No correction is necessary for the ammonia de- rived from the 1 c.c. of soy bean extract used, as the amount obtained from this source is unappreciable. Another method also suggested by Dr. Marshall is to draw 5 c.c. of blood from a vein with a hypodermic needle, into a 5 c.c. pipette, and immediately transfer the specimen to a test tube, containing 1 to 2 c.c. of 1% sodium oxalate solution. To this is added 25 milligrams (one tablet) of Urease-Dunning, the tablet having been previously crushed and dissolved in 5 c.c. of water. This mixture is allowed to stand until the urea of the blood is decomposed; at ordinary room temperature, one-half an hour is usually sufficient; it is better, however, to place the test tube in a beaker of water at 30 to 40 C. for one-half hour. After the urea has been changed, the contents and sufficient wash- ings of the tube are transferred to a cylinder. The ammonia is then removed by a current of air collected in the - hydrochloric acid and titrated with 50 50 sodium hydroxide. Calculating Urea Content. As the purpose in using Urease-Dunning is to convert the urea present in a specimen into an easily estimated substance am- monium carbonate and as the amount of this salt 98 Manual of Vital Function Testing Methods produced from this source, by the enzyme, is indicated by the increased alkalinity of the specimen to methyl orange, it is obvious that the quantity of standard hydrochloric acid required to exactly neutralize the contents of the flask containing urease, less the quan- tity required for the control specimen, corresponds to the ammonium carbonate formed by the conversion of the urea originally present in the specimen. By the following equation: NH 2 ONH 4 CO +2H 2 0=CO NH 2 ONH 4 it may be calculated that 60 grams of urea would be converted, by urease, into 96 grams of ammonium car- bonate, which amount would require 72 grams of hydro- chloric acid to neutralize it. As this quantity (72 grams) of hydrochloric acid is contained in 20,000 c.c. of decinormal (N/10) hydro- chloric acid solution and is equivalent to 60 grams of urea, as represented by 96 grams of ammonium car- bonate, it follows that one twenty-thousandth of this quantity or 1 c.c. of decinormal hydrochloric acid would be equivalent to one twenty-thousandth of 60 grams = 3 milligrams (60 -f- 20,000 = .003), there- fore each c.c. of decinormal hydrochloric acid required to neutralize an enzyme-treated specimen, that is in excess of the number of cubic centimeters required to neutralize the control specimen, represents three milli- grams of urea, and, as the 5 c.c. specimen is the one two-hundredth part of a liter, it will be only necessary to multiply the number of c.c. of the decinormal hydro- chloric acid solution, in excess of the control's require- ments, by the factor .6 (.003 X 200 = .6) to ascer- tain the urea per liter, when estimating the daily output. Tests of Kidney Function 99 1. Technic of Estimating Total Incoagulable or So- catted Rest or Residual Nitrogen in the Blood Serum Two methods are in common use. These are Morris' modification of the Hohlweg-Meyer method, and the method of Folin and Denis. Morris' Modification of Hohlweg-Meyer Method. To 10 c.c. of blood serum obtained by venipuncture or otherwise, in a 300 c.c. Erlenmeyer flask is added a reagent consisting of equal parts of 1% acetic acid and a 5% solution of monocalcium phosphate, until the reaction is acid to litmus but neutral to Congo red. The volume is brought up with distilled water to 80 c.c. and 80 c.c. of saturated solution of sodium chloride are poured into the flask. The mixture is boiled to precipitate the coagulable proteins, and the filtrate, from which the proteins have been shown to be completely removed, subjected to a nitrogen determination by Kjeldahl's method. For a description of the technic of Kjeldahl's method see page 28. Folin and Denis Method. This method is considered at the present time as the most practical way of quan- titatively determining the amount of rest nitrogen in the blood. It will be given in the words of its authors from their communication published in 1912. (Jour. Biol. Chem. 1912, XI, 527, Ibid. 1913, XIV, 29.) Method for Drawing Blood. "Before going into de- tails of the chemical work it would seem worth while to describe our method of drawing blood because sc far as we have been able to learn it is somewhat different from the procedures employed by physiologists and because we believe it to be expeditious, neat and exact and therefore particularly suitable for quantitative work. 100 Manual of Vital Function Testing Methods "We use neither cannulae nor syringes but simply hypodermic needles and pipettes. The needles are about 1 mm. in diameter, and about 25 mm. long. They are immersed in a dilute solution of vaseline in ether and then allowed to drain and dry on a clean paper for a few minutes before being used. (This does not apply of course to the drawing of human blood when the needles must be thoroughly sterilized.) An adequate supply of these needles is kept on hand so that we do not need to use any needle more than once in any one experiment. The needle is attached to the tip of a 2 or 5 c.c. pipette by means of a short piece of narrow pure gum tubing. A small pinch of powdered potas- sium oxalate is introduced into the upper end of the pipette (which must be clean and perfectly dry) and is allowed to run down into the tip and the needle. The other end of the pipette is connected with a rubber tube which in turn connects with a mouthpiece con- sisting of a short tapering glass tube. Close to the pipette the rubber tube carries a pinchcock. "To draw the blood insert the needle in the vein or artery and regulate the flow of the blood by means of the pinchcock and by suction. The exact quantity of blood desired is thus obtained without any waste and without clotting." Isolation of Non-Protein Nitrogenous Constituents. "To separate the non-protein nitrogenous constitu- ents from the protein materials we make use of pure (acetone-free) methyl alcohol and an alcoholic solu- tion of zinc chloride. Ordinary methyl alcohol cannot be used because the impurities in it, particularly the acetone, combine Avith more or less of the urea so that it escapes decomposition in the subsequent treatment and is not quantitatively recovered. We have satisfied ourselves by means of determinations on pure urea Tests of Kidney Function 101 solutions that the presence of acetone results in a loss of urea. "As soon as the blood is drawn it is transferred into measuring flasks half filled with methyl alcohol and the flasks are then filled up to the mark with methyl alcohol and vigorously shaken. Two cubic centimeters of blood are diluted to 25, while for 5 c.c. of blood use 50 c.c. flasks. At the end of two hours, or as soon after that as is convenient, the contents of the flasks are filtered through dry filters. To the filtrate are then added two or three drops of a saturated alcoholic solution of zinc chloride and after standing for a few minutes the mix- ture is again filtered thro a dry paper. The zinc chlor- ide brings down an appreciable precipitate and the last traces of coloring matters so that when the second filtration is made, a perfectly colorless filtrate is ob- tained. 5 c.c. of these filtrates, corresponding to 0.4 or to 0.5 c.c. of blood, depending on whether 2 or 5 c.c. of blood were drawn, are taken for each determina- tion. "The precipitation procedure described above is the one which we ordinarily use. There are objections to it. We are not certain that protein-like materials may not escape precipitation by this as by every other method and we do know that the filtrate does not con- tain all of the non-protein materials. When relatively large quantities (equivalent to 100 mgm. of nitrogen per 100 c.c. of blood) of creatine or asparagine are added to blood and treated as described above there is invariably an appreciable loss of material. To over- come this loss we have tried to triturate and wash the first alcoholic precipitate with methyl alcohol, and with some substance as, for example, with glycocoll, urea and acetamide, we are thus able to get practically quantitative results, while with others, such as creatine, Manual of Vital Function Testing Methods asparagine and tyrosine, we still do not get quite all. Moreover, such trituration and washing docs leach out a small amount of the coloring matters of the blood so that except for special experiments with less soluble substances we consider the simpler procedure rather more satisfactory. Determination of the Total Non-Protein Nitrogen. "To determine the non-protein nitrogen of the blood 5 c.c. of the alcoholic filtrate is transferred to a large Jena test tube. One drop of sulphuric acid, one of kerosene and a pebble are added and the methyl alcohol is driven off by immersing the test tube in a beaker of boiling water for five to ten minutes. When the alcohol is removed 1 c.c. of concentrated sulphuric acid, a gram of potassium sulphate, and a drop of copper sulphate solution are added and the mixture is boiled, cooled and diluted. "From this digestion mixture the ammonia is removed in the usual manner. It is, however, not collected di- rectly in a measuring flask (as in urine analysis) but in a second large test tube previously charged with 1 c.c. of acid added to 3 c.c. of water. The reason for this variation is that 0.4 to 0.5 c.c. of blood con- tains only 0.1 to 0.2 mgm. of non-protein nitrogen. The final Nesslerizcd solution cannot be diluted to 100 c.c. and smaller volumetric flasks cannot be used as re- ceivers during the air current treatment because of spattering. Large test tubes are therefore used as receivers and the ammonia is Nesslcrized in these before the liquids are transferred to measuring flasks. "Ordinarily the colored solutions when obtained from cat's blood are transferred to 25 c.c. flasks and are then found to have a depth of color which permits of a sure and accurate reading in the colorimeter. In some Tests of Kidney Function 103 of our absorption experiments the total non-protein nitrogen runs up to very high figures and then the solu- tions are diluted to 50, sometimes even to 100 c.c., be- fore being read in the colorimeter. "Human blood contains scarcely more than one-half as much non-protein nitrogen as cat's blood. In the case of human blood we therefore never draw less than 5 c.c. and we take 10 c.c. of the filtrate for each de- termination. In all other respects we use the same procedure for human blood as for cat's blood. "In all ordinary cases 7 to 8 c.c. of diluted Nessler's reagent (dilution 1:5) are added for the production of the color. If much ammonia is present so that the resulting colored solution must be diluted to 50 or 100 c.c. correspondingly larger amounts of Nessler's reagent are added. "The calculation of the analytical results to milli- grams of nitrogen per 100 c.c. of blood is not difficult, but the formulae given below may prove useful. In these formulae, the standard solution contains 1 mgm. of nitrogen (as ammonium sulphate) Nesslerized in a 100 c.c. flask and the colorimeter prism of the standard 50 is set at 20 millimeters. ^- X D, in which R stands XV for the reading of the unknown and D represents the volume to which its ammonia has been diluted, gives the desired figure. The reason for the figures is that we are working with 4 c.c. of blood. "When 5 c.c. of blood is taken and it is diluted to 50, 40 the formula becomes -=? X D- xv "When working with human blood and taking 10 c.c. of the filtrate obtained from 5 c.c. of blood diluted to 20 50 the formula is X D- xv 104 Manual of Vital Function Testing Methods "It may be thought that we are using unnecessarily small amounts of blood in these analyses. We are, however, by no means sure that working with larger amounts would yield more accurate results and we have satisfied ourselves by scores of duplicate analyses that the method as outlined gives trustworthy figures. Further, the smaller the quantity of blood which can be made to give reliable results the greater becomes the usefulness of the method. The work which we have already done on cats could not have been done on such a small animal except by means of these micro- chemical methods. Finally, small amounts of blood must be used for the urea determinations because of the disturbing effects of the sugar present." 2. Estimation of Blood Coagulation Time as Test of Renal Function Bachrach-Tittinger Test. In cases of renal insuf- ficiency, such cases as those in which it is claimed that the freezing point of the blood is lowered (high fig- ures for 8 ) the coagulation time has been found de- layed. This delay is supposed to be connected with salt retention in the plasma. A rather large amount of blood is required according to the original technic of the originators (20 c.c.). The test is not credited with much value. For method of estimating blood coagulation time see page 41. 3. Cryoscopy of Blood as Test of Renal Function Cryoscopy of the Blood. This method has been employed by some investigators on the principle that under conditions of renal impermeability the waste Tests of Kidney Function 105 products which fail to be eliminated in the urine will accumulate in the blood, thereby increasing the molecu- lar concentration. This means, of course, a lowering of its freezing point. The technic of estimating cryoscopy of the blood is not different from that applied to the urine, which has been already described (v. page 86). This method has not come into general use, however, and for this reason it will not be further considered here. III. STUDY OF THE ELIMINATION OF FOREIGN SUB- STANCES BY THE KIDNEY, AS CRITERION OF KIDNEY FUNCTION This category of tests may be divided into two parts : A. Miscellaneous Chemical Substances : 1. Potassium iodide. 2. Phloridzin. 3. Hippuric acid. 4. Lactose. B. Elimination of Dyes by the Kidney. 1. Methylcne blue. 2. Indigo carmin. 3. Phenolsulphonephthalein. CHEMICAL TESTS A. Tests with miscellaneous chemical substances. 1. The Potassium Iodide Test This was one of the first chemical substances applied to the estimation of renal function since it was sug- gested by Duckworth as long ago as 1867. 22 22 St. Barthol. Hosp. Rep., 1867, III, 216. 106 Manual of Vital Function Testing Methods Potassium iodide is rapidly absorbed from all the mucous membranes. 23 It is absorbed unchanged and appears quickly in the excretions. Only a few min- utes normally elapse before it can be demonstrated in the urine. According to the investigations of Quetsch, 24 Roux 25 and Studeni 26 it appears at any time from 9 to 18 minutes after doses of 1 to 3 grams have been swallowed. The greater part of the iodide ingested is excreted in the urine. Some, however, es- capes in the saliva and other secretions. Iodide is rapidly excreted, since 65-80% of the amount ingested is eliminated in 24 hours. Several investigators have reported the exact time required for complete elimination to take place. According to Antem, 27 .5 gram requires 40 hours to be excreted, and Schlayer and Takayasu, 28 and Monakow 29 state that they found the same amount required 48 hours to eliminate. Schlayer concluded from his studies that the demonstration of iodide in the urine beyond 60 hours after its administration may be considered de- layed, therefore a pathological excretion. Schlayer and his followers endeavored to fix as they did with sodium chloride the exact locus of elimination for iodide in the kidney. They believed that iodides are excreted by the tubular epithelium. They also con- tended that the elimination of iodide is not delayed in passive congestion (cardiac) while it is delayed in chronic tubular nephritis. These suppositions have neither of them been substantiated by subsequent in- 23 Gushing Pharmacology, 5 Ed., 1910, 510. M Berl. klin. Wchnschr.,' 1884, XXI, 353. 28 Thfcse de Paris, 1890, no. 248. 20 Inaugural Dissertat., Zurich, 1897. 27 Arch. f. Pathol. u. Pharmacol., 1902, XL VIII. '"Deutsch. Arch. f. klin. Med., 1911, CI, 354. ""Deutsch. Arch. f. klin. Med., CII, 248. Tests of Kidney Function 107 vestigations. Rowntree and Fitz in their experience with the iodide test have found it to vary markedly and they believe that the observation of excretion time of potassium iodide as a test of renal function is unreliable. Technic of Iodide Test. .5 gm. (7^ grains) of potassium iodide is given in solution by mouth in the morning on arising. The urine is collected at the end of 48 hours and every four hours thereafter and tested for iodide until a negative result is obtained. One of the simplest and best qualitative tests for iodide in the urine is that of Sandow. The test is made by taking 30 c.c. of urine, 2 c.c. of 2% solution of sodium nitrate and 2 c.c. of dilute sulphuric acid, adding chloroform and shaking. A purplish or violet color appears in the chloroform if iodide is present. 2. The Phloridzin Test Von Mering 30 discovered the fact that the injection of the glucoside phloridzin into animals, produces a glycosuria without a hyperglycemia, thus proving that the conversion is a vital act of the renal parenchyma. Achard and Delamare 31 built upon this fact a method of testing the functional capacity of the kidney. Technic of Phloridzin Test. .005 gm. of phloridzin in fresh aqueous solution is injected hypodermically. At 15-minute intervals the urine collected by catheter or voided spontaneously is examined for sugar. The maximum excretion takes place normally in an hour and disappears in 2 or 3 hours. 00 Centralbl. f. med. Wissensch., 1885, 531. 31 Bull, et Mem. Soc. Med. d. Hop. de Paris, 1899, 379. 108 Manual of Vital Function Testing Methods 3. The Hippuric Acid Test It has been long known that bcnzoic acid or benzoates are eliminated by the kidney as hippuric acid which is synthesized in the kidney from benzoic acid and glycocoll. Altho the fact has been used as a basis for testing kidney function the results have been disap- pointing and the method lias been abandoned. 4- The Lactose Test Voit 32 was the first to demonstrate that lactose is eliminated by the healthy kidney following its sub- cutaneous or intravenous injection. De Bonis 33 claimed that the elimination takes place exclusively in the glomerulus. Lactose was suggested as a means of estimating renal functions in 1911 by Schlayer and Takayasu, 34 who with their co-workers studied the question of renal func- tion in experimental and clinical nephritis. These workers studied the elimination of lactose, potassium iodide, salt and water, dividing the nephritides into vascular and tubular varieties with subdivisions. Schlayer believed that the elimination of lactose be- ing exclusively, as he thought, a glomerular function could be taken as an index of the vascular functioning power of the kidney. Lactose being a foreign sub- stance that is not found in the organism would not be influenced in its elimination by extrarenal factors, and should therefore be an ideal criterion of glomerular function, any delay in its passage through the kidney indicating glomerular insufficiency. M Dcut. Arch. f. klin. Med., 1897, LVII, 545. "Giorn. intern, cl. scien. Med., 1907, XXIX, 4iG. : " Deutsch. Arch. f. klin. Med., 1911. Tests of Kidney Function 109 Following Nussbaum's 35 technic of obtaining in the frog an exclusively tubular secretion from the kidney by artificially excluding the glomerular secretion, Rowntree and Fitz 36 concluded that the tubular epithe- lium can secrete a certain amount of lactose, hence its elimination is not exclusively a function of the glomeru- lus. But they concluded also from their clinical experi- ments with lactose in the study of renal function in practice and also from some studies they have made in experimental passive congestion, that the mechanism of lactose excretion differs essentially from that of phthalein, salt, indigo carmin, etc., and that estima- tions of lactose excretion may be looked upon as a satisfactory index of the vascular, if not exclusively the glomerular function of the kidney. Technic of Lactose Test. Two and five-tenths (2.5) gms. of chemically pure lactose are dissolved in 25 c.c. of freshly distilled water, placed in small cotton stop- pered Erlenmeyer flasks and pasteurized for four hours for four successive days at 75 to 80 C. By this method the dose injected amounts to a little over 2 gms. lactose in 20 c.c. of water. A fresh solution is used for each injection and a careful technic for in- travenous injection carried out. Following the injection there are usually no consti- tutional disturbances altho occasionally there may be some headache malaise or even chill followed by fever. The urine is collected four hours after the injec- tion and every hour or two hours after for twelve hours. Each specimen is tested for sugar by Nylander's reagent, using the same amount of urine, solution and length of time for boiling. Polarimetric readings may be made. ^Pfliiger's Arch. f. d. ges. Physiol., 1878, XVI, 179; XVII, 580. 36 Archives of Int. Med., 1913, XI, 258. 110 Manual of Vital Function Testing Methods The normal excretion time for this amount of lac- tose is four to six hours. The time required for secre- tion is the main point. Over six hours is delayed ex- cretion. (Nylander's reagent consists of Rochelle salts, 4 gins, dissolved in 100 c.c. of 10% NaOH (sp. gr. 1015); warm and saturate with bismuth subnitrate (about two grams are necessary). When cool, filter and keep in a dark bottle. The solution remains perma- nent for years.) B. ELIMINATION OF DIFFERENT COLORING MATTERS OR DYES AS A MEASURE OF RENAL FUNCTION. URINARY CHROMOSCOPY There are three of these tests used at the present time, namely: 1. The Methylene Blue Test. 2. The Indigo Carmin Test. 3. The Phenolsulphonphthalein Test. Other coloring matters such as rosanilin, fuchsin, etc., have been suggested and employed at different times for estimating renal function, but with the ex- ception of the three named they appear to have fallen into disuse at the present time. Numbers 2 and 3 are most extensively employed, namely, indigo carmin which is used particularly in Europe and phenolsulphonphthalein which is by far the most popular colorimetric test in this country. Rosanilin (sodium trisulphate) was introduced by Lepine. 37 One c.c. of a \ c /o solution is injected hypo- dcrmically. The dye appears normally in the urine in less than half an hour, total elimination requiring twenty-four hours. The test has never attained wide 37 Lyon Medical, 1898. Tests of Kidney Function 111 use, probably because of the greater success attending the use of phenolsulphonphthalein according to the method of Rowntree and Geraghty (q. v.). 1. The Meihylene Blue Test The introduction of methylene blue as a test for renal function is credited to Achard and Castaigne. 38 These authors gave the drug intramuscularly, using 1 c.c. of a 5 % solution. Later Czyhlarz and Donoth 39 recommended it by mouth in 1/4 grain dose. The drug is rapidly eliminated in the urine, appearing therein in about fifteen minutes as a colorless chromogen, as dem- onstrated by Voisin, 40 which may be shown by boiling the urine after addition of acetic acid. Normally the color itself appears in the urine in half an hour. The excretion of both forms continues for 36-48 hours, but even in health the time may be very much prolonged. The authors of the test recommended that the time of first appearance, time of maximum intensity of excretion and time required for total excretion should be noted. Diminished renal permeability was thought to delay or prolong all three. Various observers confirmed these suppositions. In some cases of chronic inter- stitial nephritis the elimination was found to be pro- longed for two weeks. Various modifications of the type of elimination under pathological conditions were described, consisting of remittances or intermittances of excretion. Later observers noted that elimination is not delayed in all forms of kidney disease and that the elimination 38 Bull, et Mem. Soc. Med. d. hop. de Paris, April, 1897, 63T. 39 Wien. klin. Wchnschr., XXIV, 1899, 649. 40 Gaz. Hebd. Med., 1897, 493. Manual of Vital Function Testing Methods under certain circumstances might be normal or even accelerated. Attempts to measure quantitatively the elimination of methylene blue were made, one of which described by Rowntree and Geraghty will be mentioned under technic. The methylene blue test was later applied to diag- nosis in surgical diseases of the genitourinary tract and was at one time considered the best test available for estimating the functional capacity of one or both kidneys. Walker showed that the elimination is re- tarded in lower urinary tract obstructions as in cer- tain types of prostatic hypertrophy. Casper made similar observations. Methylene blue produces some pain when given sub- cutaneously and occasionally when given intramuscu- larly. This of course is a drawback though perhaps not a serious one. Another and greater drawback is its prolonged elimination necessitating a large number of urine examinations. A third objection is the difficulty of accurate colorimetric estimations. Finally, the methylene blue test is imperfect in that part of the substance is converted into a colorless chromogen in the body secreted as a leucobase and therefore does not contribute to the color results in the urine. Only 50% of the drug is normally passed out in the urine. It has not been demonstrated with certainty in what part of the kidney the elimination of methylene blue takes place. In disease of the tubular epithelium that is in the chronic parenchymatous nephritis, methylene blue is quickly and completely eliminated ; in interstitial neph- ritis, the elimination is delayed. The duration of elimination is diminished in paren- Tests of Kidney Function 113 chymatous and increased in interstitial nephritis. A cyclic, polycyclic or intermittent elimination of the coloring matter has been said to indicate several dif- ferent conditions : disturbance of kidney innervation, hepatic insufficiency, interstitial nephritis and pyo- hydronephrosis. Up to the time of the advent of the phenolsul- phonphthalein test, the methylene blue test was the most extensively used method of determining renal per- meability. At the present time, certainly in America, the Rowntree-Geraghty test has quite superseded it. Teclinic of Methylene Blue Test. After urination 1 c.c. of a 5% solution of methylene blue is injected intramuscularly. A sterile catheter may be introduced into the bladder or the patient may empty the bladder in 15 minutes if possible to determine the presence of the leucobase or chromogen. This is done by boiling the specimen and adding a few drops of acetic acid. A greenish color denotes the presence of the chromogen. At the end of half an hour the bladder should be emptied spontaneously or by catheter and the urine examined for color. A greenish blue color denotes the presence of the dye. As above mentioned, the time of appearance, time of maximum intensity and time required for total elimina- tion (disappearance of color) should be noted. Quantitative Estimation. Before administration of the drug the urine is collected for some time and kept. The methylene blue is given in the usual manner, the urine collected for as long a time as necessary, all chromogen being converted into dye. An equal quan- tity of urine previously collected is taken, to which is added from a burette, drop by drop, a sufficient quantity of a solution of methylene blue of known strength until the colors are alike. Compare against 114 Manual of Vital Function Testing Methods a white background. From the quantity of methylenc blue used, the amount of coloring matter may be esti- mated. 2. The Indigo Carmm Test Volcker- Joseph Test 41 The dye was first used by Heidenhain in his famous investigations into the physiology of the kidney. He believed that this substance is eliminated exclusively by the epithelial cells of the convoluted tubules. Indigo carmin possesses the advantages over methy- lene blue that the quantity required for the test is completely eliminated thro the kidney and that no leucoderivative is formed in the tissues. After the intramuscular injection of .08 gm. to .16 gm. of indigo carmin, elimination begins in 6 to 8 min- utes if the kidney is normal. The intensity of the color will give some idea of the concentrating power of the kidney, that is to say, the water resorbing power of the tubules, and consequently, its capacity to produce a urine of high molecular concentration. A polycyclic or intermittent elimination is said by Blum to indicate intermittent hydronephrosis. In strongly alkaline urine the dye may be discolorized. In parenchymatous nephritis, the elimination of indigo carmin may be normal. In interstitial nephritis the elimination begins later than normal, is diminished in quantity and much prolonged. The delayed elimination indicates a diminished reaction power of the kidney, the diminished elimination a loss of water resorbing power, a hyposthenuria, a loss of the concentrating function of the organ, while the long duration of elimination indi- cates a general loss of secreting power as always accom- panies the sclerotic kidney. "Munch, med. Wchnschr., 1903, 2081. Tests of Kidney Function 115 The indigo carmin test has been quite extensively used, especially in Europe, in testing the functional capacity of the single kidney by ureteral catheteriza- tion and in general functional testing of the kidney. It is considered superior to methylene blue because of more rapid elimination. But in this respect, as in others, it is inferior to phenolsulphonephthalein, which substance, since its introduction for this purpose by Rowntree and Geraghty, has become the most exten- sively used chromoscopic test, at least in this country. Rowntree and Geraghty consider the indigo carmin test of more value than the methylene blue test because of its more rapid appearance in the urine, but that it is less adapted to functional work than phenolsul- phonephthalein. This opinion is now shared by most other workers in this field. Technic of Indigo Carmin Test. A 4% solution is made. Twenty c.c. of this solution are injected into the muscles usually of the gluteal region. There is some pain produced by the injection. In 10 to 15 minutes the urine is collected. In normal persons it is tinged greenish blue in this time. Excretion is usually com- plete in 24 hours, but practically the greater portion escapes in 12 hours. The color of the dye in the urine does not lend itself well to colorimetric estimation. In this respect it resembles methylene blue. Purulent urine decolorizes indigo carmin. It is estimated that not more than 25% of the amount injected finds its way out thro the kidneys. The fate of the balance is unknown. 3. The Phenolsulphonephthalein Test. Rowntree- Ger eighty Test (The Red Test} The phenolsulphonephthalein test of kidney function had its origin in the pharmacological researches of 116 Manual of Vital Function Testing Methods Abel and Rowntree, upon the phthaleins generally. Of all the phthaleins studied by these investigators, phe- nolsulphonephthalein stood out in striking contrast with all others because of the fact that it is almost ex- clusively eliminated by the kidney. This fact suggested its use as a test of renal func- tion to Rowntree and Geraghty and their first com- munication upon this subject appeared in July, 1910. 42 Phenolsulphonephthalein has the following formula: 'C fi ILOH C G H 4 OH C 6 H 4 It was first made by Remsen. 43 The substance is a red crystalline powder, partly soluble in water, the solution when alkaline being red, becoming more purple as the alkalinity is increased. Abel and Rowntree in their pharmacological inves- tigations of the phthalein group showed that the sub- stance appears in the urine after administration by mouth in one to one and a half hours and after sub- cutaneous injection in about 10 minutes. They found that after fair-sized doses (1 gm.) the drug appears in the bile, is passed into the intestine, there reabsorbed, and, except for a mere trace, is excreted wholly by the urine. Phenolsulphonephthalein is practically non-toxic. 43 Jour. Pharmacol. and Exp. Therap., I, 1910, 579. "Amer. Chem. Jour., VI, 280. Tests of Kidney Function 117 When a dose of .006 gm. is injected subcutaneously, 40-60% of this quantity is recovered in the urine dur- ing the first hour after injection. From 15-25% more is recovered in the second hour, making a total excretion for the first two hours following the injection of 60-85%. Normally when the urinary flow is free, the dye ap- pears in the urine in 5 to 10 minutes after injection. The maximum excretion appears in 15 to 20 minutes. This density of excretion continues an hour to an hour and a half. The elimination then begins to diminish. At the end of the first hour the pink color on addition of alkali is slight, and after the expiration of two hours excretion is practically complete. In acute nephritis, Rowntree and Geraghty 44 found a diminished excretion of phthalein in two out of three cases. In parenchymatous nephritis there was a marked diminution of excretion in seven out of eight cases. In chronic interstitial nephritis a low output was encoun- tered in all the cases experimented upon, 10 in number. These results concerning the lowered excretion of phenolsulphonephthalein in the nephritides have as a general thing been entirely corroborated in subsequent investigations by many different observers. In their first researches Rowntree and Geraghty made over two hundred functional tests in one hundred and fifty persons. To them the phenolsulphonephtha- lein seemed to possess advantages over all other func- tional tests, these advantages consisting chiefly in the following points: 1. The early appearance of the dye in the urine and its rapid and complete elimination by the kidney. 2. The accuracy and simplicity of quantitative esti- mation of the drug in the urine. "Jour. Phar. and Exp. Ther., I, 1910, 656. 118 Manual of Vital Function Testing Methods They showed by these researches that the permeabil- ity of the kidney for phenolsulphonephthalein is de- creased in both parenchymatous and interstitial nephri- tis, the decrease being most marked in the latter form. Further than this they showed that the test is of value to the surgeon in determining the true condition of the kidney, in cases with prostatic obstruction. In such cases the authors believed the phenolsulphoneph- thalein test to be of greater service than urinalysis or nitrogen estimations and that the use of the test in cases of obstruction in the lower urinary tract prior to operations would disclose the necessity of prelimi- nary treatment. Finally they pointed out that the test lends itself and is well adapted to unilateral esti- mations of the functions of the separate organs in con- junction with ureteral catheterization. In surgical cases with urinary obstruction, the authors contended that when the phthalein excretion is delayed beyond twenty-five minutes and the output for the first hour is below 20% the operation may prof- itably be postponed until treatment by drainage has improved conditions, such improvement being shown by an increase in the elimination of phthalein at a subse- quent time. Technic of Phenolsulphonephthalein Test. Twenty minutes to half an hour before giving the test the patient is given 200 to 400 c.c. of water to insure diuresis. The bladder is cathetcrized or completely emptied. The time being noted, 1 c.c. of a solution of the drug is injected into the lumbar muscles. The solu- tion is prepared as follows : .6 gm. phenolsulphoneph- 2 thalein and .84 c.c. - NaOH are added to .75% NaCl n 2 solution. Add two or three drops of NaOH. The n Tests of Kidney Function 119 color becomes Bordeaux red and the solution is non- irritant. The urine is passed into a test tube containing a drop of 25 % NaOH and the time of appearance of the first pinkish color noted. If there is no urinary obstruction the catheter is not necessary after the appearance of the color, and the patient may then retain the urine and urinate at the end of one hour in one receptacle and again at the end of the second hour in another. A rough estimate of the time of the appearance of the drug in the urine may be gained by having the patient urinate, frequently, a small amount without the catheter. In prostate cases it seems better to keep a catheter in situ. If this is done the catheter may be corked and this is removed at the end of the first and second hours. Each sample of urine is measured. Twenty-five per cent. sol. NaOH is added to make the color maximum. The urine is usually yellow or orange and becomes deep purple on addition of the alkali. The solution is put in a liter flask and diluted with distilled water to make a quart. This is thoroughly mixed and a portion is fil- tered and compared with a standard in a colorimeter. 45 The standard solution consists of .003 gm. phenolsul- phonephthalein (^ c.c. of solution used for injection) diluted to 1 liter and made alkaline with a few drops of 25% NaOH. The test solution retains its fine purplish color for a week or more. The colorimeter contains a wedge-shaped cup which is filled with the standard solution. The rectangular 45 The colorimeter used by Rowntree and Geraghty is a modi- fication of the Autenrieth-Konigsberger instrument. This can be obtained from Hynson and Westcott, Balto., Md., who also sup- ply convenient ampoules containing .006 gm. in each c.c. of phe- nolsulphonphthalein. 120 Manual of Vital Function Testing Methods cup is filled with the solution to be tested. The wedge- shaped cup is manipulated by a screw until the color fields are identical. The percentage is read off on the indicator scale. Technic of the Phenolsulphonephthalein Test as Ap- plied to Estimation of the Function of the Individual Kidney. Twenty minutes previous to the application of the test the patient is given 600 to 800 c.c. of water to provide a free flow of urine. The ureters are catheterized, a special catheter being recommended, namely, the flute end catheter of Albarran No. 6 or No. 7. The catheters are passed four inches into the ureters. The cystoscope is withdrawn, leaving the catheters in situ. A small urethral catheter is passed into the bladder to empty that organ and detect later leakage. The other details of the test are similar to those of the ordinary technic (q. v. ). In September, 1911, Geraghty and Rowntree 46 made a report of their previous experience with the sul- phonephthalein test and reiterated their first opinion that the test devised by them appeared to possess dis- tinct advantages over all other methods of examining renal function. The reasons upon which this opinion was based have been given above. They concluded that the sulphonephthalein test will enable the clinician to determine quantitatively the amount of functional derangement of the kidneys in his nephritis cases, whether of the acute or chronic type. That in cardiorenal cases the test will show exactly to what extent the kidney is involved. That the test is of special value in the diagnosis of uremia from other conditions which may simulate it and also to foretell in many cases an impending uremia before the appearance of indubitable clinical signs. 46 Jour. Arner. Med. Assn., 1911, LVII, 815. Tests of Kidney Function The authors reiterated their confidence in the value of this test in cases of urinary obstruction, it being in their judgment superior under these circumstances to measuring the urinary quantity, and to urea or total nitrogen estimations. In many surgical cases studied by them in the genito- urinary clinic of Young at Johns Hopkins, they found that separate studies of unilateral kidney function re- vealed more accurate and dependable information than any other method of examination. In a third and very complete report of their phenol- sulphonephthalein test of kidney function published in 1912, Rowntree and Geraghty studied carefully the in- fluence on the rate of excretion, of the various methods of administration, subcutaneous, intramuscular and in- travenous. They concluded from their researches that intramuscular injection into the lumbar region is the method of choice. Studying the influence of various diuretics upon the excretion of sulphonephthalein the authors found that while under the conditions of animal experimentation, some slight increased activity was caused by certain stimulating diuretics like caffein, yet clinically these substances do not affect the phthalein output. The route of phthalein through the kidney was in- vestigated and it was demonstrated that the drug is excreted chiefly by the uriniferous tubules and the smaller remainder by the glomerulus. In nephritis of all types the output of phthalein was found diminished, the diminution of excretion be- ing apparently in proportion to the amount of damage to the kidney structure. So that the test is of consider- able value from a diagnostic and prognostic standpoint since the amount of functional incapacity is revealed. In those cases in which the heart and kidney are Manual of Vital Function Testing Methods both affected (the so-called cardiorenal cases) the test was found useful in determining just what proportion of the trouble could be referred to the heart disease and what to the renal lesions. In uremia the test proved in the hands of its authors of value in differentiating uremia from conditions sim- ulating it. In certain cases, when no clinical evidence of the imminence of uremia was present, a very low phthalein output frequently enabled the authors to fore- see the danger. From a surgical standpoint the earlier opinions held by Rowntree and Geraghty as to the utility of the test in cases of urinary obstruction were completely cor- roborated by their subsequent work. According to them the test is more dependable than estimation of urinary output, total solids, urea or total nitrogen, in indicating to the surgeon the propriety of operation or the institution of preliminary treatment, in contemplated nephrectomy and prostatectomy. Finally their studies tended to show that no other test is so adaptable to the examination of unilateral kidney function to determine the relative amount of work performed by each organ separately. The medical and surgical aspects of the phthalein test will be further developed later on under the general summary of renal function tests in their medical and surgical aspects, (v.i. ) Since the introduction of the phenolsulphonephtha- lein test for kidney function in July, 1910, a very con- siderable literature upon the subject has appeared. It is very striking how few are the criticisms and how numerous are the encomiums which have been passed upon the test of Rowntree and Geraghty. It might al- most be said that, in this country at least, the opinion of those who have used it is unanimously favorable and Tests of Kidney Function 123 tends to corroborate in every particular the claims which were advanced for it by its authors. Clinical and experimental corroboration of the sul- phonphthalein test have been given by the publications of Austin and Eisenbrey, 47 Boyd, 48 Cooke, 49 Sehrt, 50 Lance, 51 Sanford, 52 Behrenroth, 53 Frank, 53 Bonn, 54 Erne, 55 Mouriquand, 56 Lohnstein, 57 Frothingham, Fitz, Folin, Denis, 58 Christian, Janeway, Cabot, Dock, 59 Snowden, Thayer, 59a and many others. While many of these reports are extremely illuminat- ing and important, it cannot be said that they have added anything noteworthy to the test itself, or to its indications, which fact is a strong testimony of the thor- oughness and care with which the work had originally been performed by Rowntree and Geraghty before its publication. From the standpoint of pure experimental corrobora- tion, the work of Austin and Eisenbrey should be noted. These authors in 1911 studied the elimination of phe- nolsulphonephthalein as compared with the elimination of nitrogen and chlorides, in experimental nephritis in dogs set up by administering uranium, cantharidin, and potassium bichromate. They concluded, among other "Jour. Exper. Med., 1911, XIV, 367; 4,62. 48 Jour. Amer. Med. Assn., 1912, LVIII, 620. 49 Providence Med. Jour., 1912, XIII, 118. 80 Centralbl. f. Ch., 1912, XXXIX, 2, 1121. 51 Gaz. d. hop. de Par., 1912, LXXXV, 32. 62 Cleveland Med. Jour., 1912, XI, 763. 53 Ztsch. f. Exp. Pathol. u. Therap., 1913, XIII, 72. 54 Jour. Ind. State Med. Assn., 1913, VI, 154. "Munch, med. Wchnschr., 1913, LX, 510. 5S Lyon Medicale, 1913, CXXL, 299. "Allg. Med. Centr. Gtz., 1913, LXXXII, 591. 58 Arch. Int. Med., 1913, vols. XI-XII; also Jour. Exper. Med., 1911, XIV, 366. 59 Tr. Cong. Amer. Phys. & Surg., 1913, IX, 45. 69 aAmer. Journ. Med. Sc., 1914, CXLVIII, 781. Manual of Vital Function Testing Methods things, as a result of their researches, that a marked and early decrease in the elimination of phenolsulpho- nephthalein takes place in the experimental nephritides and that the phthalein test is the better indicator of renal function under the circumstances than total ni- trogen or chloride elimination, which latter are more irregular and inconstant. Other investigators have experimented along the same line, studying the phthalein elimination in experimental nephritis and their results have tended to corroborate the earlier researches. Perhaps the most recent con- tribution to this phase of the subject is that of Potter and Bell. 60 These authors have studied the phthalein elimination, also that of lactose and potassium iodide in experimental tartrate nephritis in rabbits. It may be recalled that Underbill, Wells and Gold- schmidt G1 discovered that the injection of racemic tar- taric acid into rabbits produces a type of acute nephri- tis in which the great majority of the convoluting tu- bules become necrotic and the rest are fatty and gran- ular. The glomeruli may be anatomically intact. In kidneys of this type it has been found that the excretion of phenolsulphonephthalein, likewise indigo carmin and methylene blue, is completely suppressed. The excre- tion time of lactose is over twice as long as normal, while that of potassium iodide is four times as long as normal, but both lactose and potassium iodide are ex- creted by this type of kidney. Potter and Bell suggest that their results appear to show that phthalein, indigo carmin and methylene blue are excreted exclusively by the tubules, while potassium iodide and lactose are at least partly excreted by the glomeruli. In experimental chronic passive congestion of the Amer. Jour. Med. Sci., CXLIX, 1915, 236. 91 Jour. Exper. Mcd., 1913, XVIII, 322. Tests of Kidney Function 125 kidney in animals, produced by compression of the vena cava and renal veins, Rowntree, Fitz, and Geraghty 62 found that the functional capacity of the kidney as judged by the phthalein output is reduced. The reduc- tion, however, only occurs as the degree of passive congestion becomes marked. Goldsborough and Ainley in 1910 63 studied the renal function in pregnancy and the puerperium by means of sulphonephthalein, and concluded that even normal fe- males in pregnancy eliminate less phthalein than non- pregnant. In the ninth month the power of elimination may be very low. The exact meaning of these facts is not known. This result was confirmed by Roth, 64 who also claimed that women with diseases of the genital tract were unsuitable for the test. These investigations of Goldsborough, Ainley and Roth have not been subse- quently developed. In this connection it may be men- tioned that Pepper and Austin 65 reported in 1913 that occasionally cases of parenchymatous nephritis will show a quite prompt and fairly normal elimination of phthalein. In one of their cases there was a phthalein output of 67% for one hour, strongly suggesting hyperpermeability. Baetjer has also encountered sim- ilar cases. Such cases do not appear to represent the rule, however, and just what meaning is to be attached to these facts is at present unexplained. A few suggestions for slight variations of technic in the test have been published but none of them appears to have been generally adopted. They are few in num- ber and the most important may be given. Fromme and Rubncr GG in 1912 suggested that the 82 Arch, of Int. Med., 1913, XI, 121. ""Journ. Amer. Med. Assn., 1910, LV, 2058. 64 Berl. klin. Wchnschr., 1913, L, 1609. 65 Amer. Journ. Med. Sc., 1913 n. s. CXLV, 254. 66 Berl. klin. Wchnschr., 1912, XLIX, 1889. 126 Manual of Vital Function Testing Methods phthalein should always be given hypodermically and that the observation period should be extended to three hours. Keyes and Stevens 67 also recommend hypoder- mic injection when the ureters are to be catheterized. Bonn, 68 as a result of his experience, thought that the time of appearance of the dye in the urine is not of much importance except to determine the time for per- centage estimation. He recommended the intravenous injection of the phthalein when the ureters are to be catheterized. He does not believe that the test will inform the surgeon when the patient can be operated on safely. He, however, states that, in his belief, the test is the best one yet devised for studying the renal func- tion. Fanz 69 suggests to add a quantity of the patient's urine obtained just before injecting the indicator, equal in amount to the first hour's urinary output after the injection. The standard solution now will have ap- proximately the same amount of urinary salts as the specimen solution, and the standard and urinary solu- tion will equal each other in opaqueness and yellowish tint, making color comparison easy. To make up the standard solution, he uses 1 c.c. of the contents of an ampoule * of the phcnolsulphonephthalein, adds the pa- tient's urine (obtained before injecting the indicator) in amount to equal the first hour's urinary output after appearance of the drug. This mixture is alkalinizcd with 25 c.c. of 10 % solution of potassium hydroxide, filtered, and sufficient distilled water is added to make 1000 c.c. This is the standard and contains 100% of the indicator in 1000 c.c. 67 N. Y. Meet Jour., 1912, XCV, 1134. 88 Indiana State Med. Assn. Jour., 1913, VI, 154. 68 N. Y. Med. Jour., 1915, C, 1193. * Put on market by Hynson and Westcott. Tests of Kidney Function 127 The first hour's urinary output, after injection of 2 c.c. phenolsulphonephthalein, is now alkalinized with 25 c.c. of 10% potassium hydroxide and filtered. To this is added distilled water to make 1000 c.c. This is the test specimen. By diluting a unit of the standard, say 50-100 or 200 c.c., with distilled water until it matches the 1000 c.c. solution of the first hour's urinary output, the direct percentage of the indicator in the first hour's specimen can easily be estimated. Say 100 c.c. of the standard had to be diluted up to 500 c.c. before it matched the first hour's specimen dilution, then the standard would be five times as strong as the speci- men, or the specimen would contain 20% of phenolsul- phonephthalein. The second hour's output of phenol- sulphonephthalein is estimated precisely like the first. As a matter of fact it does not appear that any modifications of the original test method as described by Rowntree and Geraghty has contributed materially to its simplification or improvement, and for this reason the original method is usually followed. Thayer and Snowden 70 have recently attempted to compare their results obtained by the test with the anatomical changes found in the kidneys at autopsy. They conclude, as the result of their quite extensive investigations, as follows : In severe chronic nephritis there is always a low phthalein output. This rule, in their experience, has absolutely no exception. The phthalein output in cases of chronic nephritis diminishes steadily, according to them, until the terminal uremia, when it approaches zero, just prior to death. In the passive renal congestion of heart disease, there is often a reduction in the elimination of phthalein, especially when the amount of decompensation of the 70 Amer. Jour. Med. Sci., 1914, CXLVIII, 781. 128 Manual of Vital Function Testing Methods heart is considerable. When these symptoms become ameliorated, the phthalein output increases. If passive congestion of the kidney from heart disease is accompanied by concomitant chronic nephritis the output of phthalein is lower than in cases of uncom- plicated congestion. In one case of acute nephritis and one of amylosis the phthalein output was reduced. In acute infectious diseases, during which cloudy swelling of the renal parenchyma occurred, there was found a considerable reduction of phthalein output. From this study Thayer and Snowden concluded that the phenolsulphonephthalein test of Rowntree and Geraghty is a procedure of great diagnostic and prog- nostic value, especially in the study of chronic neph- ritis. General Summary of the Application of Renal Func- tion Tests This subject may be divided into two parts : 1. Renal Function Tests and Their Medical Application. 2. Renal Function Tests and Their Use in Surgery. 1. Medical Application. A decade ago the burning question in renal pathology was whether the varying clinical findings in chronic nephritis could be divided into classes and each class correlated with certain defi- nite histopathological findings in a kidney post mortem. Although no absolute answer to the question was reached, there is no doubt some progress was made. During the past decade, in line with the general depar- ture of interest somewhat away from the anatomical toward the functional view in pathology, a serious at- tempt was made to divide the clinical symptomatology of nephritis into groups ; to correlate these groups with Tests of Kidney Function 129 certain definite types of renal involvement. Although this worthy attempt has not been entirely successful any more than its anatomical prototype which preceded it, some important facts have been learned which are of value from a practical as well as a theoretical stand- point. The method by which these advances have been gained is none other than the application of functional tests to a study of the different phases of renal activity. Different portions of the glomerulo-tubular structure of the kidney have been supposed to possess selective secretory activities. It cannot be said that these selec- tive activities are yet understood, so that at the present time there is no such thing as an exact topical diagnosis of the kidney functions. Nor can it be said in any case that the results of the most complete and comprehensive functional examina- tion will reveal with any certainty the anatomical changes which are present in the kidney. Certainly they will not reveal the extent to which the vascular, glomerular or tubular structures are involved in a given case, and will shed but little light upon the relative proportions of the changes. Our chief clinical terms in renal pathology still re- main as they were in the older tradition ; we still speak currently of acute nephritis, chronic parenchymatous nephritis and chronic interstitial nephritis, but we are not surprised when the autopsy shows the extreme rar- ity of these arbitrary types and presents us with patho- logical pictures of such great complexity that it is no wonder that the intricate problem is not to be solved by the most skillful and painstaking ante-mortem exami- nation. Great hopes were aroused some years ago in this direction when it was discovered that experimental in- flammation of the kidney may be set up in animals by 130 Manual of Vital Function Testing Methods the use of certain poisons 71 which make it possible to submit some of these difficult problems in renal pathol- ogy to experimental investigation. Although little has come of this work so far, it appears to be founded upon rational and scientific principles and the work must be enthusiastically encouraged to go on. Schlayer, Hedinger, and Takayasu, in the Romberg Clinic at Tubingen, have applied themselves assiduously to working out the problems of pathological kidney function from an experimental standpoint. In this country the purely experimental method has yielded interesting results in the hands of Rowntree, Fitz, Geraghty, Christian, O'Hare, Folin, Karsner, Denis, Frothingham, Austin, Eisenbrey, Potter, Bell, and others. For an interesting contribution dealing with the relation of functional tests to pathological diag- nosis, the reader is referred to an article by Christian, 72 to which an excellent bibliography is appended. The author of this review very justly concludes that tests of renal function are quite capable of demonstrating the bare fact (the important fact indeed) that the kid- neys are diseased but are quite unable to disclose the exact type of pathological lesion of the kidney, which exists in any given case. While no functional test is capable of disclosing the nature or location of the pathological lesion, it may be perfectly capable of disclosing the inability of the kid- ney to perform a given function or set of functions, such, for example, as the elimination of water, salt, urea or some foreign substance such as lactose or phthalein. Such an inability on the part of the kidney will indicate a depreciation of its functional power if 71 Uranium, ohromates, mercuric chloride, cantharidin, tartrates, etc. 72 Trans. Cong. Amer. Phys. and Surg., 1913, IX, 1. Tests of Kidney Function 131 the test is properly carried out, and this particular information may be important in prognosis and treat- ment although we shall be unable with certainty to point to the seat of the disturbance in the kidney or identify with exactitude the nature of the underlying patho- logical lesion. From a medical standpoint renal function tests are most important in defining the state of kidney activity in the acute and chronic nephritides, orthostatic and other albuminurias, arteriosclerosis, uremia, and myo- cardial insufficiencies. In some medical clinics renal function tests are ap- plied as a matter of routine to all these classes of cases. While they do not in themselves make the diagnosis or settle the prognosis it is contended, very properly per- haps, that they will occasionally reveal an unsuspected latent deterioration of kidney function, just as routine blood examinations may reveal an unsuspected leukemia. Renal functional tests will be of prognostic value be- cause they will often serve to show whether the disease is stationary, progressing or recovering. Of course it hardly needs to be said that renal function tests will always be carried out in the medical clinic in conjunc- tion with the clinical study of the patient. A progres- sive lowering of the kidney function in chronic nephri- tis may indicate impending uremia. The question of the prognostic value of renal func- tional tests has been made the theme of a very complete and excellent article by Rowntree. 73 According to this investigator the prognostic value of renal functional studies is as great in medical as it is in surgical cases. In acute nephritis, the prognosis depends upon the eti- ology more than the result of functional tests. In mild chronic nephritis with slight albuminuria and cylindru- 73 Trans. Cong. Amer. Physic, and Surg., 1913, IX, 23. Manual of Vital Function Testing Methods ria, slight hyperpiesis, moderate arteriosclerosis and hypertrophy, the tests may show how great the func- tional deterioration may be, and the regular repetition of the tests will show whether the disease is progressing or not. In advanced nephritis also, the proper func- tional tests will disclose the severity of the disease and the imminence of uremia. Cases of clinically well- marked nephritis, even with some uremic signs and a less marked functional derangement, will be more diffi- cult to prognose. Head and brain complications cannot be foreseen by means of renal functional tests. If the renal function remains fair, say 30% of phthalein out- put (Rowntree) and the blood tests do not show marked cumulative phenomena the prognosis is favorable and vice versa. In cardiorenal cases it is always difficult to deter- mine clinically just how much the heart and kidney are separately responsible for the conditions. Renal func- tional tests are of considerable service in showing just how far the kidney is affected. A low phthalein output with cumulative signs in the blood indicate a severe degree of renal involvement. In mere passive conges- tion these signs are not apt to be found. Moderately advanced nephritis with slight heart fail- ure may show a fairly good renal function, and, if so, the prognosis depends more upon the response of the heart to treatment than on the kidney. If the phthalein output increases in such cases it is a favorable sign, and if the phthalein output is fairly good in an appar- ently severe cardiorenal case, the heart may be judged the principal offender in the symptom complex. On the contrary, if the phthalein output is very low and there are signs of cumulation in the blood tests (-{-urea, -{-rest nitrogen, low 8*) both kidneys and heart may be * 5, symbol for freezing point of the blood. Tests of Kidney Function 133 regarded as failing and the prognosis is grave. As a rule in cases of pure myocardial insufficiency the renal function tests give surprisingly good results. When the kidney is passively congested for long periods, however, as a result of heart failure, the functional tests give low results just as they do in severe types of nephritis. The prognosis becomes proportionately lugubrious. 2. Surgical Application. The surgeon is extremely interested in the problems of renal function. With him the question often becomes a very vital one in connec- tion with important and serious operations upon the genitourinary tract. The proper selection of tests is a matter of great importance to the surgeon. Geraghty rightly insists that it is only through familiarity with the reliability, limitations, and significance of findings of individual tests of renal function, in their relation to various types of disease and various kinds of problems to be solved, that a proper selection can be made. Certain of the tests or combinations of tests are more suitable to surgical investigations, while other tests or combinations of tests may be of greater value to the internist. In discussing the utility of renal function tests to the surgeon, we shall draw freely upon Geraghty, since he more than any other investigator has identified himself in his researches with the question of their value and limitations in surgical practice. Some of the tests of renal function are adapted to estimating the total func- tional capacity of the kidneys at any given time. Other tests are not of great use in this respect but are of considerable importance in determining the relative functional capacity of the organs when applied to the excretion obtained by ureteral catheterization. Finally there are a few tests which are apparently useful in 134 Manual of Vital Function Testing Methods both cases. As an illustration, it is known that the estimation of urea output in a 24-hour specimen is of no great value by itself in judging the total func- tional capacity of the kidneys, while comparisons of urea elimination in catheterized specimens from both sides may give very important information. From a purely practical standpoint, as was men- tioned under preliminary considerations, all tests for kidney function come under two general heads: 1. Tests to determine how much of a given substance is excreted by the kidney or kidneys, comparing the amount with what is known to be normal. The sub- stance excreted may be one which is normally found in the urine or it may be a substance artificially intro- duced into the circulation to determine the capacity of the kidneys to eliminate it. 2. The blood may be exam- ined for substances which are normally passed through the kidneys, with a view of detecting an accumulation of such substances in the body, such accumulation being regarded as an evidence of defective kidney function. Renal functional tests arc especially valuable to the genitourinary surgeon in two types of cases : 1st, Sur- gical diseases of the kidney secondary to obstructions in the lower urinary tract. 2nd, Unilateral and bilateral surgical diseases of the kidney not associated with ob- struction. Rowntrce and Geraghty, in many of their publica- tions concerning the phenolsulphonephthalein test, have called attention to the fact that inasmuch as many cases with obstruction in the lower tract also have hydroneph- rosis, pyoncphrosis, pyelonephritis, or pressure at- rophy, an examination of the total function by means of the phthalein test will often show diminished functional activity. In these cases the urinalysis may be mislead- ing, for urea output and total solids secretion may be Tests of Kidney Function 135 normal, and yet the kidney function may be so unstable that the shock of a surgical operation, such, for exam- ple, as prostatectomy, may be sufficient to inhibit func- tion altogether, and death will result. In just such cir- cumstances they believe a total examination by phtha- lein will serve to differentiate these cases with severe from those with slight renal involvement. Experience has shown that cases of obstruction in which the phthalein output is deficient may be so greatly improved by preliminary treatment with proper drain- age that the subsequent radical operation may be per- formed with greatly diminished risk. According to Geraghty the phthalein test affords the truest index of functional capacity of the kidney for surgical work. The diastase test and urea estimation are, he thinks, of equal value, but are unreliable indices of functional capacity ; but when persistently low values are given by them they may be important from a prognostic point of view. Estimations of blood urea and incoagulable blood nitrogen are extremely impor- tant when they are associated with the phthalein test. When all three are positive they are extremely sig- nificant. The functional tests, one and all, can never be said to arbitrarily answer the important question, when to operate and when not to operate, because there are other factors to consider in a surgical case besides renal function. If the phthalein test is very low operation should be postponed until efforts are made by improved drainage (either by suprapubic cystotomy or by cathe- ter) to bring about improvement in the functional capacity of the kidneys. In the second group of surgical cases above men- tioned, namely, unilateral surgical kidney diseases, the relative functional power of the kidneys is a question 136 Manual of Vital Function Testing Methods of vital importance. It is here that ureteral catheter- ization is of supreme importance, for by this means the excretion from each kidney can be separately obtained. Many functional tests, such as urea estimations, cer- tain chromoscopic tests, as indigo carmin and methylene blue, cryoscopy, diastase estimation, phloridzin reac- tion, and experimental polyuria, give information only with respect to the relative functional value of the two kidneys. This is insufficient, since one kidney may be doing a great deal more work than the other, and yet be incapable of doing the work of both, and this is the question of vital importance to the surgeon, requiring a definite answer in each individual case. The phenol- sulphonephthalein test has proven of special value here because by it not only is an idea of the total functional capacity, but also a quantitative estimate of the work done by each kidney obtained. There are two difficulties, however, which belong to all tests of renal function when carried out with ureteral catheterization ; one is inhibition of function produced by the presence of the catheter in the ureter ; the other is leakage around the catheter. Special catheters are now used to prevent the latter difficulty. As to the first, the best method of procedure is to test the total func- tional capacity with phthalein before ureteral catheter- ization is performed. If the total phthalein output is nearly normal one kidney at least is satisfactorily nor- mal. If, after ureteral catheterization, one kidney is found with an exceedingly low output, which, together with the clinical findings, indicates that this is the dis- eased organ, it may be safely removed even if the output in the sound side is low, for here the natural inference is that inhibition has produced the deficiency. Inas- much, however, as inhibition is not necessarily equal on the two sides it will always be necessary to combine the Tests of Kidney Function 137 phthalein test with comparison of diastase and particu- larly urea percentages, on the two sides. The actual procedure recommended by Geraghty is, first, the esti- mation of total output by phthalein and the estimation of relative function by ureteral catheterization com- bined with pigment, diastase and urea estimation on the two sides. If the phthalein output is low, cryoscopy of the blood serum and estimation of blood urea may profitably be done. Geraghty says : "Most of the criticism of functional tests has come from those who have not used them, and are unfamiliar with the nature of the information sup- plied. It is true that in the vast majority of cases a successful nephrectomy on the diseased side from the standpoint of renal function, can be performed when the urine from the opposite kidney is found apparently normal on analysis. Unfortunately, however, the prob- lems are not always so simple. Cases of bilateral dis- ease are encountered in which a knowledge of the renal function becomes of absolutely vital importance, and in which every source of information must be called upon before the proper line of procedure can be employed. Again, in certain cases, particularly those of tubercu- losis, it may be possible to introduce a catheter only on one side. In such cases one must depend to a great extent upon the information derived from function tests as to the condition of the opposite kidney since the cys- toscopic appearance of the ureteral orifice is frequently deceptive. The recognition of hypoplastic and infantile kidney is practically impossible without functional esti- mation. The infantile kidney is a particularly danger- ous type because the urine which is secreted by the kid- ney is apparently normal in every respect except that of quantity. Functional estimation has proved also of great value in the differentiation between pyelitis and 138 Manual of Vital Function Testing Methods pyelonephritis. In pyelitis the renal function is prac- tically normal, while in pyelonephritis there is dimin- ished function." Selection and Practicability of Renal Function Tests In this regard we can do no better than quote the words of Geraghty, spoken at the conference on Renal Function Tests at the ninth triennial meeting of the Congress of American Physicians and Surgeons, 1913 74 : "The number of functional tests has become so great that it is impracticable to employ all of them in any individual case ; and, even if not impracticable, nothing would be gained by employing all of these tests. The information furnished by many is of the same char- acter, but more accurately furnished by one test than by others. For example, there is a parallelism between the excretion of the different dye substances; but as phthalein furnishes more accurately all the information obtainable from this group of substances, no advantage attaches to the employment of all. "For chromocystoscopy alone indigo carmin is un- questionably the test of choice. The estimation of rest nitrogen and blood urea bear about the same signifi- cance. Lately we have discarded estimations of resid- ual nitrogen in the blood and are depending entirely upon the blood urea determined by Marshall's method or upon cryoscopy for evidence of cumulative phe- nomena. "From a practical standpoint certain tests can be entirely discarded without loss, such as cryoscopy of the urine and electrical conductivity of the urine. Total urea estimations in urine are of doubtful value and dias- 74 See Trans. Cong. Amer. Phys. and Surg., 1913, IX, 45. Tests of Kidney Function 139 tase determination furnishes only information that is obtainable more accurately and quickly by other means. Certain other tests, such as potassium iodide elimina- tion, can be discarded as furnishing at times unreliable information. We have seen potassium iodide excretion delayed in cases with normal function (proven by sub- sequent history) and excreted within normal limits in cases of the most severe nephritis. The tests which we consider of the greatest value in the excretory group, based upon actual experience, are: Phthalein, lactose, and chlorides ; and of the tests of retention, blood urea, rest nitrogen and cryoscopy. The indications for the specific employments of the individual tests are as fol- lows: "Chloride estimation in the urine is useful in all forms of nephritis and cardiorenal disease, especially if oedema is present. "Lactose is indicated for the detection of slight in- jury to the kidneys and also in severe nephritis, since its suppression indicates a bad prognosis. It is not par- ticularly helpful in surgical diseases. "Of the retention tests either blood urea, rest nitro- gen or cryoscopy is indicated wherever there is a severe lesion of the kidneys. "We consider that one of these tests should be used as a routine in conjunction with phthalein wherever functional tests are desirable, particularly if the phtha- lein function is low. "Tests in conjunction with ureteral catheterization : in this connection, phthalein, urea in urine, and urinary diastase are most serviceable. The diastase and urea give practically the same information, but only give relative functional values, while phenolsulphonphthalein p-ives relative and absolute values. The total function O should always be estimated by means of phthalein with- 140 Manual of Vital Function Testing Methods out ureteral catheterization, in order to detect the amount of catheter inhibition, should this exist. Where severe bilateral lesions exist, one of the retention tests should be used. "As to practicability, the simplest and easiest test is undoubtedly the phthalein test, as it requires the least amount of time and apparatus. "The lactose test, if quantitative determination is re- quired, necessitates the employment of an expensive po- lariscope. Furthermore the preparation of the lactose for injection requires attention and consumes time. Its use also requires familiarity with the technic of in- travenous injection. "Diastase requires the daily quantitative preparation of soluble starch, accurately graduated pipettes, a large series of test tubes, a water bath, and one-fiftieth nor- mal iodine solution. For total estimation it requires 24-hour specimens of urine with preservatives. The time necessary for a simple determination is scarcely warranted by the information obtained. Urea estima- tions of the urine can be accurately and rapidly done by the Marshall method ; and from the standpoint of practicability it leaves little to be desired. It is useful only in conjunction with ureteral catheterization. "Chloride estimation requires standardized solutions and carefully graduated apparatus when accurately done. It consumes considerable time, and, besides, re- quires daily collections of the urine with the knowledge of the daily chloride intake. "All retention tests require, of course, the withdrawal of blood ; and cryoscopy of the blood is undoubtedly the simplest, provided that proper apparatus is at hand. It requires careful attention to the details and con- sumes considerable time. "Blood urea can be done by either the Folin or the Tests of Kidney Function 141 Marshall method, and the total rest nitrogen, preferably by Folin's method ; but any of these methods is imprac- ticable for the general practitioner. "Where only one test can be employed the most value is unquestionably to be obtained from the use of phtha- lein ; and this is particularly so from the standpoint of the surgeon." CHAPTER III TESTS OF PANCREATIC FUNCTION GENERAL CONSIDERATIONS As Stadmiillcr 1 rightly says, "The recognition in the living subject of pathological conditions of the pan- creas belongs, without doubt, to the more difficult prob- lems of the diagnostic technic of the present day." Aser 2 also said years ago that there is no organ in the body in which such a disparity exists between its known physiological importance and our capacity to clinically estimate its functioning power. In other words, the enormous importance of the pan- creas as a digestive organ and as a gland of internal secretion is granted, thanks to the work of Von Mering, Minkowski, Pawlow, Boldyreff, and many others. Our ability to estimate the functioning power, the physio- logical capacity, the anatomical condition of the organ in a given case is limited. The importance of the pancreas as a digestive gland is very great. It is the only gland which furnishes an enzyme for each class of foodstuffs. The activated proteolytic ferment trypsin breaks up protein into simpler structures (amino acids) than the gastric juice. Amylopsin or pancreatic diastase con- verts starch into sugar. Steapsin or pancreatic lipasc 'Archiv. of Diag., 1911, IV, 20. "Nothnagel's Spec. Path, und Therap., Wien, 1898, I. 142 Tests of Pancreatic Function 143 splits neutral fats into fatty acids and glycerine. Glaessner has estimated that the pancreas pours into the duodenum through the ampoule of Vater perhaps a pint or more of its mixed secretion per day. But beyond this great act, which a priori would ap- pear to be more than sufficient for a single organ, the pancreas elaborates a mysterious secretion which pre- sides in an equally mysterious way over the sugar metabolism of the body. Von Mering and Minkowski in 1889 discovered that typical diabetes follows total extirpation of the pan- creas in dogs. This fact, which has received an immense amount of experimental corroboration, still stands out as an epoch-making discovery in the history of pan- creatic physiology amidst much that is obscure and un- certain. It is pretty generally agreed that the internal secre- tion of the pancreas is elaborated in the islands of Langerhans, closely crowded groups of polygonal cells without excretory duct of any kind scattered in the stroma of the gland between the external secreting acini and surrounded by a profusely developed net of blood vessels. Besides controlling the carbohydrate intermediate metabolism of the body the internal secretion of the pan- creas is claimed by Loewi and some others to exert an inhibitory effect upon the sympathetic. From the standpoint of the clinician it is an exceed- ingly difficult matter to determine the existence of pancreatic disease, and to ascertain its nature. All the customary methods of examination are, of course, employed in such a search. Inspection, palpation, blood examination, and a careful study of the semiology are made use of. The subjects of pancreatic semiology, pain, tenderness, tumor, pressure, etc., and pancre- 144 Manual of Vital Function Testing Methods atic exploration arc extremely important aids and in- dispensable adjuncts in pancreatic diagnosis. They do not come, however, under the functional investiga- tion of pancreatic activity, and will, therefore, be necessarily left undiscussed in this place. 3 In considering the question of studying the functional capacity of the pancreas, two possibilities become im- mediately apparent. (A) Functional investigation may be made of the pancreas regarded as an organ of external or digestive secretion, and (B) the func- tional examination may relate to an inquiry concerning the internal secretory activity of the organ, its power, in other words, of adequately performing its endocrin- ous or metabolic function. A. TESTS FOR PANCREATIC FUNCTION, WHICH CONCERN THE EXTERNAL OR DIGESTIVE ACTIVITY OF THE ORGAN These may be divided into four subdivisions, as fol- lows: 1. Proteid digestion tests. 2. Fat digestion tests. 3. Starch digestion tests. 4. Demonstration of pancreatic ferments in secre- tions and excretions. The protcid digestion tests are: Demonstration of waste muscle fibre in the stools, the so-called azotor- rhoea or creatorrhoea. Schmidt's test for digestion of nuclei. Sahli's glutoid capsule test. The fat digestion tests are: Demonstration of ex- cess fat in the stools, so-called steatorrhoea. Identifica- tion of split fat in the stools. Winternitz Diagnosti- cum. 8 Consult Opie's Text Book, Diseases of the Pancreas. Tests of Pancreatic Function 145 The starch digestion test consists of identification of undigested starch in the stools. Besides the above tests there have been devised special qualitative and quantitative methods of identifying the various pancreatic ferments themselves (trypsin, amyl- ase and lipase) in the stools, urine and gastric contents. It is somewhat difficult, therefore, to separate the operations of routine urinalysis and coproanalysis in- tended to show the presence or absence of pancreatic enzymes, from certain special tests which have been de- vised with the express purpose of testing pancreatic function. For example, Schmidt's test for nuclear di- gestion, and Sahli's glutoid capsule test might, upon superficial consideration, be considered as special tests for pancreatic function, but in reality they are both merely special methods of demonstrating the presence or absence in the intestine of pancreatic enzymes. However, the fact that in the performance of these tests something particular is done in the way of special preparation with a certain definite end in view, would, in all likelihood, insure their inclusion in any scheme or list of functional pancreatic tests. All the other tests mentioned in the above synopsis are, in reality, only special methods of urinalysis or fecal examination. The same difficulty of classification becomes apparent when we come to consider the tests of internal secre- tion. The Cammidge test, as it is called, is but a specially technical urinalysis. The Loewi test for pupillary reaction to adrenalin and the test for alimentary glycosuria might be said to answer all the requirements of special functional tests, but if our knowledge of pancreatic function were lim- ited to these experimental inquiries alone, it would be 146 Manual of Vital Function Testmg Methods meagre indeed. From all these considerations it will be apparent that any attempt to give a description of methods of study- ing pancreatic function must include all of the above- mentioned methods, although a very strict interpreta- tion might relegate some or many of them to the domain which is usually covered by books dealing with chemical or microscopical laboratory methods. B. TESTS FOR PANCREATIC FUNCTION WHICH CONCERN THE INTERNAL OR METABOLIC FUNCTION OF THE ORGAN There are three tests which belong to this class. 1. The Cammidge reaction. 2. Loewi's pupillary reaction. 3. Provocative alimentary glycosuria. 1. Proteid Digestion Tests. Estimation of Undigested Protein in the Stools as a Means of Determin- ing Pancreatic Hypofunction The patient is placed for three days on Schmidt's test diet. This diet is as follows: 1.5 liters of milk, 100 gins, of zwieback, 2 eggs, 50 gms. of butter, 125 gms. of beef, 190 gms. of potatoes, and gruel made from 80 gms. of oatmeal. In this diet are contained 102 gms. of albumen, 111 gms. of fat, 191 gms. of carbohydrates; making a total of 2234 calories of energy. According to Schmidt the diet is distributed thro the day as follows: Morning, .5 liter of milk (if milk does not agree .5 liter of cocoa made from 20 gms. of cocoa powder, 10 gms. sugar, 400 gms. water and 100 gms. milk are substituted), 50 gms. zwieback; noon, 125 Tests of Pancreatic Function 147 gms. chopped beef (raw weight) broiled rare, with 20 gms. of butter (the interior of the meat must be raw), 250 gms. of potato broth made of 190 gms. of mashed potatoes mixed with 100 c.c. of milk and 10 gms. of but- ter ; afternoon, same as morning ; evening, same as fore- noon. It would be well if investigators should agree to use this well-known standard diet so that comparisons of results could be made. Pratt, 4 while using the diet, recommends that the whole quantity be given in three meals instead of five, conforming thus to American custom. The Schmidt diet, containing as it does a good amount of all three varieties of foodstuffs, is adapted to all tests which involve an examination of the feces. The presence of azotorrhoea or defective proteid digestion due to deficiency of trypsin zymase, is deter- mined by examining the feces microscopically after the test diet. Meat consists of connective tissue and muscle fiber. Connective tissue is digested promptly by the gastric juice, the muscle fiber by trypsin. Large quantities of striated muscle fiber in the feces may indicate defective pancreatic activity. The bulkiness of the stools should also be noted. Oser, Musser and others have claimed that there is no single symptom of pancreatic disease of greater significance than bulkiness of the stools. The dried weight of the stools may be rather readily ascertained. A ventilating hood and scales are all that are necessary. If the pancreatic juice is not appearing in the bowel the dried stools will weigh more and the stools themselves will be more than ordinarily voluminous. In six healthy individuals on Schmidt's diet Pratt 4 Amer. Jour. Med. Sc., 1913, CXLIII, 313. 148 Manual of Vital Function Testing Methods found the average weight of the dried feces to be 54.3 gins. The maximum was 62 and the minimum 45 gms. Schmidt's Cell Nuclei Test for Pancreatic Suf- ficiency. 5 According to Schmidt, the nuclei of animal cells is digested only by the pancreatic enzymes. Con- siderable has been written pro and con concerning this test and, although it does not appear to be extensively used, there is no doubt that it is not devoid of a certain value. Technic. Raw beef somewhat fibrous is cut into cubes of .5 c.cm. These are hardened in alcohol and placed in small bags of coarse silk gauze. Before using they must be soaked several hours in water. The bags are recovered in the stools, hardened, paraffined, cut and stained, and the sections mounted and examined micro- scopically for nuclei. If the nuclei are not digested, they will take the stain and become visible. Thymus gland has been substituted for the meat by Einhorn. Kashiwado, one of Schmidt's pupils, has altered the original test by giving a powder consisting of equal parts of stained thymus nuclei and lycopodium. The mixture is given in two capsules, each containing .25 gms, at the evening meal. The stool is examined for stained nuclei. In performing the Schmidt test the sac must be recovered in the feces before the expiration of 30 hours at the latest, otherwise a longer sojourn in the bowel will enable the bacterial enzymes to dissolve the nuclei. Sahli's G Glutoid Capsule Test of Pancreatic Func- tion. The glutoid capsules used in this test are made of gelatin and hardened in formalin. This is intended Verhandl. d. Kong. f. inn. Med., 1904, XXI, 335. 6 Deutsch. med. Wchnschr., 1897, XXIII, 6; also Deutsch. Ar- chiv f. klin. Med., 1898, LXI, 475. Tests of Pancreatic Function 149 to make them resist digestion by the gastric juice, to permit of their entering the bowel, there to be softened and disintegrated by the pancreatic juice if it be pres- ent in sufficient amount. It is the trypsin zymase which affects this solution. The capsules may be filled with sodium iodide, iodo- form or salol. The last is usually employed. Salol is split up by the pancreatic juice into salicylic acid and phenol. The former is eliminated by the kidney and escapes in the urine as salicyluric acid, which is easily recognized in the excretion by the violet color produced by adding to the urine a few drops of a solution of ferric chloride. Normally the reaction is obtained in five hours. Sahli did not disclose the precise manner of preparing the capsules and this, according to Pratt, has hindered the generalization of the test which is so extremely simple. The capsules are made and sold by Hansmann of St. Gallen, Switzerland. Sailer 7 says that a satisfactory capsule may be prepared by placing ordinary gelatin capsules in pure formalin for three minutes. This test is practically the same as that introduced by Sahli for estimating gastric motility. Its extreme facility of execution would make it an excellent test if there were unanimity in the findings. Unfortunately this is not the case and at the present time no one would pretend to depend upon its results alone, though it seems undoubtedly true that in the absence of pyloric spasm or stenosis, the capsule test of Sahli is worth considering from a corroborative point of view. 7 Amer. Jour. Med. Sc., 1910, CXL, 330. 150 Manual of Vital Function Testing Methods 2. Fat Digestion Tests. Demonstration of Excess of Fat in the Stools (Steatorrhaea) and Diminu- tion of Split Fats in the Stools as a Means of Estimating Pancreatic Insufficiency Here the insufficiency of pancreatic function, if it exists, is reflected upon the fat-splitting activity of the external secretion and shows itself by phenomena which are due to diminution or absence of the lipasic enzyme of the juice. Steatorrhoea, or increased fat in the stools, is known to be common in pancreatic disease. The light color of the stools is an important macroscopic feature. They may be almost white. The stools are often rancid in smell and bulky. Since the stools in icterus may also be fatty it may be necessary to test for bile in order to make sure that the pale color is not due to hydrobiliru- bin. When fat crystals are present in excess, the stools have a metallic lustre like aluminum. A white stool, fluid when passed and becoming solid on cooling, is fatty and is said to be quite characteristic of defective pan- creatic secretion. The most important function of pan- creatic lipase is to split up the neutral fats into fatty acids and glycerine. The free fatty acids combine with the alkalies of the pancreatic juice to form readily assimilable soaps. If the functional activity of the pancreas is deficient, one important consequence will be a considerable dim- inution in the amount of split fats, and hence of soaps. The fat will remain unsplit, therefore unassirnil- able and consequently unabsorbed. Normally from 7 to 11 % of the fat ingested in the food escapes action by the pancreatic juice and is passed in the stools. If bile is absent from the intestine because of occlusion of the duct, there will be incomplete Tests of Pancreatic Function 151 emulsification of the fats and consequent reduction of pancreatic effects. Under such circumstances as much as 45% of the fats ingested may escape in the stools. If, in a case of icterus, the loss of fat in the stools is less than 60% the pancreas is probably not implicated. If the fat loss exceeds 60% it points to pancreatic disease. Under the microscope, fat appears in the stools as droplets, needle crystals, or as structureless plates or flakes. Unfortunately the quantitative estimation of fat in the stools is not a simple matter. The stools must first be dried on a water bath; the neutral fats and fatty acids are extracted in a Soxlet apparatus with ether, from a known quantity of dried stool ; the residue is treated with diluted HC1 to convert soaps into fatty acids and these are extracted in the same manner. The amount of free acid in the first extract is calculated with titration with alkali. Like the Cammidge reaction or test (v.i.) the quanti- tative estimation of fat in the feces is not to be regarded as a clinical procedure likely to be carried out by the physician. The method requires some apparatus and more chemical experience than the clinician is likely to possess and, besides, is time consuming. Of course, in many hospitals, the examination can readily be made. It is not considered advisable to give the details of the method for fat extraction and quantitative estimation, the skeleton of which is above outlined, especially as the details may readily be obtained from texts on chemical diagnosis. For convenience two references may be given Sahli 8 and Wood. 9 Winternitz's Test of Pancreatic Fat-splitting Func- tion. Winternitz Diagnostikum. The Sajodin Test. This test is founded upon the fat-splitting power of 8 Diagnostic Methods, 190,5, p. 446. 9 Chemical and Microscopical Diagnosis, 1909, p. 334. 152 Manual of Vital Function Testmg Methods steapsin to set free iodine from iodine-containing fats. Winternitz was the first to apply the artificially iodized fats to the investigation of the fat-splitting power of the pancreatic juice. The first substance used was iodi- pin, but it was found that the iodine present in this sub- stance is so firmly bound that even normal pancreatic juice may not split it. He finally selected monoiodo- behenate of calcium or sajodin as the most suitable sub- stance. Sajodin is a thin, oily liquid containing 25 c /o of iodine. If 3 c.c. of this substance are administered by mouth to a fasting individual and the urine examined for iodine 3 to 5 hours later the reaction will be nega- tive. If the same substance in the same amount is given with a meal, the iodine reaction will be present. In the first instance no pancreatic secretion has been stimulated to appear in the duodenum and consequently the sajodin is not split. In the second instance the opposite prevails. It was further found that in cases of icterus no splitting of sajodin takes place because of the absence of bile salts in the intestine, which are necessary to actuate the fat-splitting ferment and to stimulate ab- sorption. Several investigators have investigated the Winter- nitz test and the question has been made the subject of a thesis by Stegman. 10 He concludes that the failure to find iodine in the urine 3-5 hours after the ingcstion of 3-5 c.c. of sajodin with a meal, is indicative of lipo- lytic pancreatic insufficiency in most cases, and that, in combination with other well-known tests, the method of Winternitz may be regarded as of considerable cor- roborative value. 10 Uebcr eine neue Methode tier Pankreasfunktions prufung Winternitz Diagnostikum. Dissertation Otto Stegman, 1911. Tests of Pancreatic Function 153 In a recent inaugural dissertation upon the Winter- nitz sajodin test by Syring, 11 this author finds that without exception in normal cases iodine appears in the urine in 3-5 hours after the ingestion, with a meal, of 5 c.c. of calcium monoiodobehenate (sajodin). The question as to its real value in determining the existence of pancreatic insufficiency can only be settled by further investigations. 3. Starch Digestion Test. Identification of Undi- gested Starches in the Stools as an Evidence of Pancreatic Insufficiency The presence of starch in the stools is generally un- derstood to be of little value in this connection. Nor- mally scattered granules of undigested starch are to be found and can be easily identified under the microscope, when stained with iodine solution (Lugol's), which colors them blue. Any great excess, however, may fairly awaken suspicion, especially if there is persistent diarrhoea and the condition tends to be permanent or long continued. Under these circumstances it is legiti- mate to conclude that there is pancreatic insufficiency. 4- Identification of Various Ferments. Tests Where Examination Is Made for the Pancreatic Fer- ments Themselves in the Excreta. The Meaning and Interpretation of These Results in Relation to Estimating the Pancreatic Function The different pancreatic ferments can be demon- strated by proper methods, in the stomach (after an oil meal), in the urine, and feces. Einhorn, Gross, and others have devised special ap- 11 Ueber die Funktionsprufung des Pankreas. Leipzig, 1913. 154 Manual of Vital Function Testing Methods paratus for obtaining the pancreatic juice directly from the duodenum. Einhorn's duodenal tube is used consid- erably in this country, but more particularly for thera- peutic purposes. One objection which urges against all tests for tryp- sin in the stools is that erepsin, a ferment coming from the mucous membrane of the small intestine, may digest albumen, etc., even in the absence of trypsin. The ob- jection cannot be urged against the gastric estimations, and, in fact, seems to be exaggerated even when applied to fecal analysis methods. Demonstration of Trypsin in the Stools. Two methods are chiefly used ; the Serum Plate method and the Casein method. The Serum Plate Method of Mutter and Schlecht. 12 Trypsin acts upon the surface of serum agar plates. Method. One drop of the stool obtained by a laxa- tive (calomel or phenolphthalein) is placed upon a Loffler serum agar plate and kept at a temperature of 55 to 60 C. for 6-12 hours in an incubator. If trypsin is found in the stool there will appear a depression or hole in the serum due to digestion by the enzyme. Ordinary diphtheria culture tubes have been sug- gested by Stadmiiller as being sufficient for the purpose of the test. This test is simple and seems to be rather highly re- garded (Brugsch, Hirshberg, etc.). Other practical methods of demonstrating trypsin in the feces have been devised. Arthur and Hubert add a 2% solution of sodium fluoride to the stools, also fibrin, and incubate at 40 for 24 hours. Crystals of tyrosin are formed if trypsin be present. Abderhalden's technique has also been employed, using glycyl-tyrosin. The Miiller- 12 Munch, med. Wchnschr., 1908, LV, 225. Tests of Pancreatic Function 155 Schlecht method is sufficient, however, and much more simple. For a quantitative variation of the serum agar plate method of stool examination for trypsin, put in one part of the plate undiluted feces and in orderly sequence in other portions of the plate use dilutions of the feces 1:10, 1 : 20, 1 : 100, 1 : 200, and note which still forms indentation. If the stools are fatty the fat should be extracted with ether. The Casein Method of Demonstrating Trypsin in the Stools. Casein in alkaline solution is precipitated by dilute acetic acid. If casein is digested by trypsin it will no longer give the precipitation reaction with dilute acetic acid. This is the foundation of a test devised by Gross. 13 In Gross' method the feces are mixed with an alkaline solution of casein .1% casein, .1% sodium carbonate. Various quantities of the filtered feces are added to the casein solution, incubated for an hour and tested with dilute acetic acid. (v.i. ) Mette's tubes are also sometimes used in testing for the presence of trypsin. They consist of glass tubes about 1 or 2 mm. in diameter, containing coagulated egg albumen. These are suspended in the dilute feces or other solution to be tested for a fixed time and the amount of albumen digested off, measured. The strength of the ferment will be proportional to the square of the length digested. Technic of Gross' Quantitative Test for Trypsin. Prepare a .19c solution of casein by adding 1 gm. of pure casein (Merck) and 1 gm. of sodium carbonate to 1000 c.c. of chloroform water. Place in a flask and 13 Arch. f. Exper. Path, und Pharm., 1907, LVIII, 157; also Deutsch. med. Wchnschr., 1909, XXXV, 1706. 156 Manual of Vital Function Testing Methods allow to stand for 24 hours, after which the solution is shaken vigorously. Five gms. of feces are placed in a mortar. Add 45 c.c. of \ c /o solution of sodium carbonate. Titrate thoroughly and filter. The first cloudy portion of filtrate is discarded. The second is used. To each of six reagent glasses marked for identifica- tion add 10 c.c. of the casein solution. With graduated pipette add respectively 1 gm., .5 gm., .25 gm., .2 gm. and .1 gm. of filtered feces to specimen and mix thor- oughly. Place all in incubator, adding to each 3 drops of 1% acetic acid. Specimens in which the casein are digested (presence of trypsin) will remain clear; others are cloudy. In normal stools, glasses 1 to 3 are clear, 4 to 6 cloudy. A trypsin unit equals the amount of feces which digests 10 c.c. of starch casein solution. If .33 gm. feces which is diluted tenfold digests 10 c.c. of casein solution, there are 30 trypsin units, which is nor- mal. In clinical work, disease of the pancreas may be suspected when no trypsin or, at most, 10 units are found in examination of the feces. Demonstration of Trypsin in the Stomach Contents. Method of Boldyreff-Volhard. 14 Boldyreff noted in 1904 in Pawlow's Institute that feeding olive oil to dogs caused regurgitation of duodenal contents into the stomach. Volhard 15 applied the principle to the clinic. A breakfast of 200 c.c. of olive oil is given by stom- ach tube or 250 c.c. of cream may be substituted, the latter being swallowed. Half a tcaspoonful of magnesia usta are given just prior to the meal and is repeated "Ccntrbl. f. Physiol., 1904, XVIII. 457; also Zentbl. f. Phys. und Path. d. Stoffw., 1909, III, 209. 15 Munch, med. Wchnschr., 1907, LIV, 403. Tests of Pancreatic Function 157 twenty minutes afterward. This is to prevent acidifica- tion. At the end of 45 to 60 minutes, the stomach con- tents are removed by tube. Usually a liquid is obtained which tends to separate into two layers, the lower one containing the duodenal juice. The presence of trypsin can be demonstrated by the Gross casein test described above or by that of Arthur Hubert, 16 previously mentioned, the details of which follow : Fresh fibrin obtained from horse blood by whipping and washing the coagulum is covered with 2% solution of sodium fluoride and kept for 24 hours at 40 C., then filtered. The fluid to be examined is diluted with equal volume of 2% sodium fluoride solution, and one volume of this dilution is added to two or three volumes of the fibrin solution and digested at 40 for some hours. Crystals or crusts of tyrosin form on the wall of the vessel. According to Sahli trypsin can qualitatively be most easily demonstrated by digesting in alkaline fluid at incubator temperature a flake of fibrin stained with magenta red. The fibrin becomes digested and dissolved and the fluid is colored red. Stadmiiller mentions a simple qualitative test devised by Von Oefele. A few drops of Fehling's alkali solution with a few drops of a 1-1000 solution of casein are added to a .07% copper sulphate solution and .1% sodium carbonate. The mixture is incubated at 55. To 5 c.c. of this, in a warm test tube, are added five drops of the fluid (intestinal juice) to be tested, the whole being shaken. If trypsin is present the solution which at first is blue or green if bile is present becomes red-violet or rose color. "Archiv. de Physiol., 1894, 622. 158 Manual of Vital Function Testing Methods 5. Estimation of Diastatic and Lipolytic Ferment in the Feces as a Measure of Pancreatic Function The results obtained by a study of the diastase con- tent of the stool should, provided all controllable fac- tors in performing the test are standardized, give val- uable information concerning the functional integrity of the pancreas. The physiological basis upon which the test is founded is, that practically the whole amount of diastatic ferment found in the feces is of pancreatic origin, i.e., provided certain possible sources of error are understood and obviated (Wohlgemuth). Several satisfactory methods for estimating diastase in the feces have been devised, chief of which are those of Wohlgemuth 17 chiefly used in Germany ; that of Durand, 18 chiefly used in France and England, and that of Brown, 19 chiefly used in the United States. The methods of Durand and Brown will be described. Durand's Method of Estimating Diastase in the Feces, One c.c. of the total diluted feces is added to 50 c.c. of starch solution (1 c /c starch and 2% decinor- mal HC1). The tubes are incubated for half an hour at 39.5 C. and digestion is then stopped with three drops of strong soda solution. The sugar formed is estimated quantitatively with Fehling's solution. Ten c.c. Fehling's solution is reduced by .0124 gm. of sugar. If x be the number of c.c. of the incubated mixture used to reduce the Fehling's solution, the amount of sugar present in the 51 c.c. of the mixture . , .0124 X 51 . will be or, in the whole amount or the x "Biochem. Zeitsch., 1908, IX, I; also ibid., 1909, XXX, 432. "Archiv. des Mai. d. Appnr. Digest., 1911, V, 76. 19 Johns Hopkins Hosp. Bull., 1914, XXXV, 200. Tests of Pancreatic Function 159 .0124 X 51 X 20,000. ,. teces (obtained as below), ims x figure is multiplied by two to give units of grams of sugar formed in the hour. The normal limits are 1500- 2000 units. The feces for the test are obtained as follows: The patient is well purged 12 hours after his last meal. He is then given % liter of milk and 45 minutes later 50 grams sodium sulphate in water, and one-half hour after this a glass of vichy. The feces of the next 3^/2 hours are passed into a vessel containing ice. They are then diluted with water to 20 liters and tested as above. Technic of Brown's Test. The patient is given a high enema the night before. The evening meal should be very light. At 7 A. M. the next morning 750 c.c. of milk are given. At 7:30 and again at 8:30 A. M. 1/2 ounce of Epsom salts are taken. At 8 :30 a glass of water containing 1/4 teaspoonful of sodium bicarbonate is swallowed. All stools up to 2 P. M. are saved in a vessel con- taining 2 ounces of toluol which is kept in a cool room or on ice. If less than 400 c.c. of stool are obtained an enema of a pint of water is given. The average quantity collected up to 2 P. M. will be from 400 to 1100 c.c. usually. The stool should be examined as soon as possible after 2 P. M. Dilute the amount up to 3000 c.c. with normal salt solution, stir the whole amount until abso- lutely homogeneous. Centrifugalize a portion for 5 minutes and use the supernatant fairly clear fluid for testing. Diminishing amounts of the fluid are put into a series of tubes, 1.8 c.c. in the first, 1.6 c.c. in the second, 1.4 c.c. in the third, 1.2 c.c. in the fourth, 1 c.c. in the 160 Manual of Vital Function Testing Methods fifth, .8 c.c. in the sixth, .6 c.c. in the seventh, .4 c.c. in the eighth, .2 c.c. in the ninth, .1 c.c. in the tenth, .05 c.c. in the eleventh and .025 c.c. in the twelfth. Bring the fluid in each of the tubes up to 2 c.c. with normal salt solution. If the test shows a negative reading in the first tube or if a low reading is expected, a supplemen- tary set of tubes is prepared containing respectively, 2 c.c, 3 c.c., 4 c.c., and 5 c.c. centrifugalized mixture. In each of the tubes is added 2 c.c. of 1% solution of soluble starch (Kahlbaum) and the tubes are incu- bated at 38 C. in water bath i/o hour, then cooled by adding a little tap water and by holding them under the cool tap. They are then quickly tested with a few drops of one tenth normal iodine solution. The limit is held to be that tube before the one in which the first definite blue color appears. Demonstration of Lipolytic Ferment. Two simple tests for the presence of lipolytic ferment may be men- tioned. These are the Grutzner-Gamgee 20 method and the von Oefele 21 method. The first is as follows : An emulsion of ten parts of oil, five parts of gum and thirty-five parts of water is prepared. A neutral solution of litmus is made up which in test tubes of 12 mm. diameter appears violet against white paper. Ten c.c. of litmus solution and 5 drops of the emulsion are placed in several of these tubes and increasing quantities, 2, 4, 8, 16, 32 drops, of the fluid to be tested are added to the successive test tubes. These are put in a water bath at 37 C., and af- ter a short time the tubes are compared. If any fat- splitting ferment is present the color of the fluid will have turned redder the larger the amount of solution added. 20 Quoted by Stadmiiller, loc. cit. 21 From Sahli's Diagnostic Methods, 421. Tests of Pancreatic Function 161 Von Oefelc's method of demonstrating steapsin is as follows : sweet butter is melted and the resulting clear fat mixed with an equal proportion of a \ c fo aqueous solution of potassium carbonate and some phenolphtha- lein, and then titrated with a soda solution until there is a red tint. This liquid is heated in the incubator to 55 C. and 5 c.c. of it well shaken in a warm test tube with 5 drops of intestinal juice. In the presence of a normal amount of steapsin the red tint will disappear in from 2 to 5 minutes. According to the rapidity of the discoloration the quantity of actual steapsin can be estimated. C. TESTS FOR PANCREATIC FUNCTION WHICH CONCERN THE INTERNAL, OR METABOLIC FUNCTION OF THE ORGAN All the functional tests of pancreatic activity which have been described have related to the external secre- tion with its enzymes, which is poured out thro the pancreatic duct and the ampoule of Vater into the small intestine. There is, however, another phase to the question of functional deficiency of the pancreas and this relates to its internal secretion, which in some mysterious or quite unknown manner presides over the mobilization and destruction of sugar in the body. When the in- ternal secreting function of the pancreas is lowered, the power of assimilation of carbohydrates is reduced and when a certain limit is reached a hyperglycemia results which tends at a certain point to manifest itself by the elimination of sugar in the urine, glycosuria, diabetes. There are three functional tests which concern par- ticularly the internal function of the pancreas. These are: 162 Manual of Vital Function Testing Methods 1. The Cammidge Reaction. 2. Loewi's Pupillary Reaction. 3. Provocative Alimentary Glycosuria. 1. The Cammidge Pancreatic Reaction There is still much dispute as to just what position the reaction holds in the clinical diagnosis of pancreatic function. The original Cammidge reaction consisted of two parts or analyses which were known as A and B tests. It was held that the presence of a pancreatic lesion and even its nature could be determined by these tests. The original theory upon which the test was based was about as follows. If there is a real pancreatic lesion, the pancreatic juice will escape into the parenchyma of the organ and lead to fat necrosis with splitting of neutral fat into fatty acids and glycerine. The fatty acids will remain in the necrotic areas and the glycerine will be absorbed into the blood and excreted by the urine. The Cammidge test was de- vised to demonstrate the presence of glycerine in the urine by the presence of glycerosazone crystals. The original theory was subsequently modified and the two original tests were abandoned and replaced by one process known as the C test. The present theory of the Cammidge test is this. The crystals produced in the test when positive, result from the presence in the urine of a sugar complex which upon hydrolysis with HC1 yields a substance giving a pentose reaction. The crystals of a positive reaction are be- lieved to be pentosazone. The pancreas contains four or five times as much pentose as any other organ in the body and conse- quently when any disintegration of pancreatic tissue Tests of Pancreatic Function 163 takes place as a result of disturbance or disease of the organ, crystals of pentosazone, the Cammidge crystals will be demonstrated in the urine. Neither a mere blocking of the pancreatic secretions nor a pure fibrosis of the organ will produce a positive reaction. It is usually held as Cammidge himself believes that a posi- tive reaction is evidence of active degeneration such as occurs in acute or chronic pancreatitis. A negative reaction contraindicates active degeneration but does not exclude old pancreatitis nor malignant disease of the pancreas. In fact, in 75% of malignant cases the reaction is negative. But the test is not always posi- tive even in pancreatitis and a negative test does not definitely exclude pancreatitis. The results must al- ways be taken in conjunction with clinical, urinary and fecal findings. The test is not considered generally by pathologists or clinicians as having great practical value in diagnosis. But when positive it constitutes an interesting abnormality which seems to be connected in some rather cryptic or obscure manner with dis- turbances of the pancreatic function. Teclinic of the Cammidge Test. Filter a portion of a 24-hour specimen of urine. Test for Albumen. If albumen is present in amount more than a trace, measure out 50 c.c. of filtrate and add a few drops of acetic acid, boil, cool, filter and make up to 50 c.c. Test for Sugar. Either Fehling's or Nylander's test is performed. The result must be absolutely nega- tive. If there is any reduction on standing about 50 c.c. of the albumen free urine must be mixed with yeast fermented for 12 to 2-1 hours and filtered. Stage I. Measure 20 c.c. of the clear albumen and sugar free filtrate into a small flask with an in- verted filter funnel placed in its mouth as a condenser. 164 Manual of Vital Function Testing Methods Add 1 c.c. of strong HC1. Boil on sand bath for 10 minutes from commencement of ebullition. The boil- ing should not be too vigorous and the flame should be turned low for the greater part of the time. Stage II. Cool under the tap. Make up contents to 20 c.c. with distilled water. Slowly add 4 gms. of lead carbonate ; shake gently at first and more thor- oughly later. Stand, and shake occasionally until no more gas comes off. Filter through a paper moistened with distilled water. Stage III. Add 4 gms. of powdered tribasic acetate. Shake thoroughly for some minutes and allow to stand. Filter thro a moistened filter paper. Stage IV. To the clear and almost colorless filtrate add 2 gms. of powdered sodium sulphate, shake thor- oughly for several minutes. Bring slowly up to the boiling point on a sand bath, shaking from time to time. The excess of lead is removed at this stage and it is important that the shaking and heating should be done carefully. Stage V. Cool under the tap and filter. Measure 10 c.c. of clear filtrate. Make up to 18 c.c. with dis- tilled water. Add 8 gms. of phenyl-hydrazine hydro- chlorate, 2 gms. powdered sodium acetate and 1 c.c. of 50^? acetic acid. Boil in a flask with a funnel condenser on the sand bath for 10 minutes from the commencement of ebulli- tion. Do not boil too vigorously. Filter hot through a filter paper moistened with boiling distilled water into a 15 c.c. measure. Should the filtrate fail to reach the 15 c.c. mark make up to 15 c.c. with hot distilled water. Stand for from 4 to 5 hours or longer at room temperature or in ice chest. Examine the filtrate for the appearance solubility and amount of crystal formation. Tests of Pancreatic Function 165 The typical crystals examined under the microscope are of the osazone type and more circular and tuft-like than glucosazone crystals. Run under the cover slip 33% H 2 S0 4 ; the crystals should dissolve in 10-15 sec- onds. The crystals have to be distinguished from the coarse yellow needles which may be deposited if the ex- cess of lead was not removed in Stage IV. In a strongly positive reaction the deposit of crystals may occupy half the bulk of the filtrate. In a completely negative reaction the filtrate remains clear. *2. Loewi's Pupillary Symptom or Test of Pancreatic Insufficiency In 1908 Loewi 22 made the observation that after re- moval of the pancreas in certain animals, the instilla- tion of adrenalin into the eye will cause dilatation of the pupil. Ordinarily the instillation of adrenalin into the eye does not cause dilatation altho intravenous injec- tion will do so. Loewi attributed the mydriasis fol- lowing instillation to increased excitability of the sym- pathetic system brought about by the removal of the inhibitory effect of the pancreatic internal secretion. From this fact it was thought that a mydriasis in man following local instillation of adrenalin would indicate pancreatic internal insufficiency, provided hyperthy- roidism or Graves disease did not exist. According to Sladden 23 this test has given interest- ing and encouraging results and should not be dis- missed with the comparatively scant attention it has received lately. The technic of the test is extremely simple since it consists merely of dropping into the conjunctional sac 22 Archiv. of Exper. Path, and Pharm., 1908, LIX, 83. "Quart. Jour, of Med., 1913-14, VII, 455. 166 Manual of Vital Function Testing Methods a few drops of a 1 : 1000 solution of adrenalin and ob- serving the effects upon the pupil. 3. Spontaneous and Provocative Alimentary Glyco- suria in Their Relation to Pancreatic Function The intimacy of relationship between the pancreas and control of the carbohydrate metabolism is close and undisputed, but what particular cells of the pan- creas are concerned or just what the mechanism of the control may be is very imperfectly understood. Glyco- suria is usually present in many of the more serious pancreatic diseases, but glycosuria is by no means an infallible index of either the extent or the nature of the pathological processes. In many cases of pan- creatic disease, however, glycosuria does not appear (e.g. many cases of chronic pancreatitis, carcinoma, etc.). According to Cammidge the presence of glycosu- ria means only a one to three chance that the pancreas is diseased, and in case of pancreatic disease about one in fourteen shows sugar in the urine. The truth is well expressed by Sladden when he says, "viewed arith- metically, glycosuria is not a sign of great diagnostic value." If, however, glycosuria either spontaneous or provocative be present together with other confirmatory evidence of pancreatic disease the symptom then ac- quires a greater value. It must be remembered that the liver is also concerned to a very important and intimate extent with carbo- hydrate metabolism, and tests for alimentary glycosuria have long been employed with a view of estimating the functional integrity of that organ. Under a previous chapter these tests have been given and discussed. It must be perforce admitted that the question of applying the so-called carbohydrate tests for provoca- Tests of Pancreatic Function 167 tive glycosuria to the elucidation of pancreatic func- tion is one which for the present must be left open. It would appear rational to assume that the pres- ence of glycosuria after the provocative tests indicates either hepatic or pancreatic insufficiency or both, and such tests are never in themselves sufficient to elucidate the problems involved, but where they are corroborated by other more specific evidences of insufficiency, they assume an importance in diagnosis of no mean value, an importance which is entirely lacking to them when interpreted alone. General Conclusions Concerning the Tests for Pancreatic Insufficiency It is certainly true that no one functional test of pancreatic activity so far devised constitutes an abso- lute or pathognomonic sign of disease of this organ. It is quite natural that this should be so in view of the manifold functions of the pancreas. In other words it is often a very difficult question to determine in a given case whether the pancreas is diseased or insufficient at all, much less to make out by means of the most complete and comprehensive semeiological study the precise nature and extent of the pathological processes. The whole subject of pancreatic clinical pathology and diagnosis and that of tests for functional activity of the organ is in its infancy. Nevertheless the value of those functional tests so far devised is considerable and it is the duty of clini- cians to apply them in practice to such an extent that their individual and collective worth or lack of worth may be definitely determined. CHAPTER IV TESTS OF HEART FUNCTION GENERAL, CONSIDERATIONS IN recent times a considerable transformation has occurred in the viewpoint of clinicians toward the cardiopathies and cardiac pathology. A few years ago all cardiopathology was discussed in terms of anatomical lesion. The chief interest lay in the exact localization and delimitation of the lesion. The ten- dency at the present is to bring more and more into the foreground the idea of functional capacity of the organ. Recent studies in cardiac physiology and pathology have shown that the fundamental factor is the muscle itself rather than its innervation as was be- fore believed. It has been likewise shown that the func- tion of the cardiac muscle is a complex one and that at least five subdivisions of function may be made (Englemann). These may be enumerated. The heart muscle possesses the power of originating contractile impulses (impulse formation, chronotropic function) ; it possesses the faculty of susceptibility to the receipt of these functions (excitability, irritability, bathmo- tropic function) ; it is endowed by means of the conduct- ing system of fibers including a histological differenti- ated tissue, the bundle of His with power to transmit these impulses from the point of their formation, the 168 Tests of Heart Function 169 sino-auricular node to the cardiac muscular fibers (the conducting function, conduction, dromotropic func- tion) ; it possesses the fundamental power of contrac- tion (contractility, inotropic function), and finally, the muscle possesses that vital function by which it nor- mally refuses to dilate beyond a certain point (tonic- ity). A perfect method of estimating cardiac function would be one in which all these five functions of the cardiac muscle could be separately measured. The function of the entire organ would be then their arith- metical sum, if all possessed some degree of integrity or their algebraic sum if certain of them were below a normal point which might be diagrammatically repre- sented by zero. But as a matter of fact we are far from being able to accomplish such an estimate of cardiac function at the present time. There are many instances, however, in which a care- ful physical examination of the organ together with the use of special methods of cardiac investigation which have come into use in recent years (sphygmo- graph, electrocardiograph, sphygmodynamometer, the X-ray) will enable the physician to conclude that one or more of the five functions of the cardiac muscle are deficient. When some or all of these functions have become so insufficient and incompetent that the occurrence of heart failure (asystole) is imminent, the symptoms of the condition (cyanosis, decompensation, edema, signs of venous congestion) become so patent and evi- dent that the presence of cardiac insufficiency is simple to recognize from a study of the physical signs and the symptoms. This stage of true insufficiency may be called a ter- minal stage of the cardiopathies no matter what the 170 Manual of Vital Function Testing Methods anatomical lesion may be in a given case. This stage is known, however, to be preceded by a long period of latency in which cardiac insufficiency, if it is present, cannot be so easily discovered and has to be looked for in order to be recognized. It is the desire of the clinician to increase his powers of observation, to so lengthen as it were his cardiac vista, that he may be enabled to recognize the earliest signs of cardiac incompetency. It is for this very evi- dent reason that tests for estimating the integrity of heart functions have been devised. As yet no one of them has succeeded in providing an entirely adequate means of obtaining this much to be desired end, never- theless several interesting and valuable methods have been developed. The work of the past gives promise of future developments and improvements in this extreme- ly important domain of functional diagnosis. To Rosenbach is generally given the credit of in- sisting upon the necessity of devising proper tests by which the functional integrity of this most im- portant organ, the heart, might be measured. The various methods of testing the cardiac func- tion may be divided into a few classes. The first and largest group includes those tests which depend upon the reaction of the heart muscle to various types of exertion active or passive. There is a second and much smaller group based upon the behavior of the heart to reflex stimulation. A third and extremely insignificant group includes but one test, based upon the supposition that sodium chloride elimination is effected by cardiac insufficiency. A fourth group in- cludes modern clinical and instrumental methods of investigating cardiovascular conditions so far as they are concerned with the question of elucidating heart functional power. Tests of Heart Function 171 The following synopsis shows how the various tests are to be placed in the four categories above men- tioned. I. Reaction to muscular exertion active or passive as a basis for estimating cardiac function. 1. The staircase test. 2. Graupner's test. 3. Mendelsohn's test. 4. Katzenstein's test. 5. Herz's self-checking test. 6. Gymnastic resistance test. 7. The Russian test "Holding the breath" test. 8. The Venous pressure test. II. Application of cardiac reflex estimations in de- termining heart function. Merklen's test. III. Estimation of sodium chloride elimination as a test of cardiac sufficiency. Vaquez-Digne test. IV. Modern clinical and instrumental methods of in- vestigating cardiovascular conditions : their applicability to estimating cardiac function. 1. The sphygmomanometer as an index of cardiac function. 2. Rontgenoscopy and Rontgenography as indices of cardiac function. 3. Sphygmocardiography and electrocardiogra- phy ; their relation to cardiac functional capacity. I. REACTION TO MUSCULAR EXERTION ACTIVE OR PAS- SIVE AS A BASIS FOR ESTIMATING CARDIAC FUNCTION The majority of the methods so far suggested for estimating heart functional power have consisted in 172 Manual of Vital Function Testing Methods the subjection of the patient to a certain measured degree of physical exertion followed by the systematic observation of the phenomena produced by the exer- tion as compared with conditions carefully ascertained prior to the beginning of the test. All these methods, however, have the common objec- tion that the same work prescribed to different indi- viduals, will under normal circumstances produce quite different results, according to certain circumstances, among which are the size and general muscular strength of the individual, the usual mode of life with respect to physical exertion, the condition of the nerv- ous system, etc. The result, therefore, of exertion tests may not always be comparable even in healthy persons. If these factors can be properly estimated and provided for the exertion tests are rendered more certain and hence more useful. Herz has emphasized the fact that the cardiac phe- nomena produced by exertion tests are varied by the type of effort attempted: whether, for example, the movements are rhythmical and gymnastic, whether they are resisted or not, and especially whether the muscular groups called into play are weaker or stronger. A much higher rise of blood pressure is produced by the effort attempted by a weaker set of muscles than the same operation performed by a stronger set. Grebner and Grunbaum have contended that the increase in blood pressure produced by muscular contractions is inversely proportional to what may be termed the specific energy of the muscles employed in the effort. The influence of psychic factors in varying the re- sults of muscular tests has always been recognized (Kornfelds). The same may be said of the nerve factors. All these facts tend of course to render un- certain the results obtained by the exertion tests (pulse Tests of Heart Function 173 rate, blood pressure), but even with these defects this type of method of estimating cardiac function is of practical value. The chief points taken into consideration in this type of test are the rate of the pulse, the blood pressure (systolic and diastolic) and the area of cardiac dull- ness or size of the heart (percussion, rontgenography). As long ago as 1833 Donnell showed that the pulse rate is normally slower in the recumbent than in the semi-erect and erect positions. Christ in 1894 l pro- posed to register the exact pulse rate after exertion with the sphygmograph provided with a time marker. He likewise invented an apparatus by which the patient could undertake a measured amount of exertion on a steppage machine. Rosenbach in the same year em- phasized the importance of noting the condition of the skin after the performance of the exertion. It was his belief that the skin remains dry if the heart is competent but becomes quickly moistened with perspir- ation if there is cardiac insufficiency. He explained the increased sudation on the ground that the excretory function of the skin is called from the list of reserve forces to compensate as far as possible for the cardiac inadequacy. This is probably not the correct interpre- tation of the phenomenon since sweating itself re- quires the expenditure of force. It is probably due to vasomotor causes. 1. The Staircase Test. Selig's Test 2 Technique. Count the pulse and take the systolic pressure. Have the patient ascend a flight of twenty steps, rapidly. Count the pulse and take the systolic pressure after the ascension. 'Archiv. f. klin. Med., 1894, LIU, 1902. 2 Prag. med. Wchnschr., 1905, XXX, 418, 432. 174 Manual of Vital Function Testing Methods Under normal circumstances, there is an increase in the pulse rate of 20 beats per minute on an average and a rise of blood pressure of 8 millimeters of Hg. If the myocardium is insufficient there will be an increase in the pulse rate of 30 beats per minute or more. The blood pressure rise will be slower and average about 6 mm. of Hg. The rise may be fol- lowed rather suddenly by a fall below normal or the preliminary rise may be absent. The length of time required for recovery to the normal systolic pressure may be taken as a measure of the amount of cardiac insufficiency present. The staircase test on account of its simplicity is often employed by clinicians. The "hopping test" is a modification of the Selig test, which has been used for years as a routine method of eliciting a latent cardiac insufficiency. The patient is instructed to hop 20 paces on one foot and a com- parison is then made between the pulse rate before and after the exertion. One serious objection to the "hopping test" is that the actual amount of work performed by the indi- vidual to be tested cannot be computed. In the method of climbing stairs, the amount of energy expended can be approximately known. The amount of work done in foot pounds will be equal to the product of the weight of the individual in pounds into the number of feet ascended. The advantage of this simple test is that it can be performed without any special apparatus, which is the chief objection from a practical standpoint to some of the functional cardiac tests which have been sug- gested. Tests of Heart Function 175 2. Graupner's Test 3 Graupner found at Nauheim in observing the reac- tion of patients after the exercises carried out as a part of the treatment of cardiopathies, that persons with weakened hearts showed a different type of reac- tion from those with normal or nearly normal myo- cardia. Under normal circumstances, as is well known, the pulse rate and the systolic blood pressure rise after exertion, returning to normal after a fairly short in- terval. If the exertion is sufficiently prolonged and arduous they may fall below the normal. Graupner discovered that after the pulse rate has risen and again fallen to normal after an exertion, the systolic pres- sure rises gradually to a maximum, which is reached in a few minutes, usually about six, declining to normal in about 18 to 20 minutes. The rise of blood pressure following the pulse rise is called the normal erholung. In weakened hearts, even if the weakness is slight, Graupner found that the erholung occurs but it is less in amount than normal and is delayed beyond the normal interval, usually to about 12 minutes. If the heart is seriously weakened the erholung may be ab- sent altogether, the blood pressure declining from the start then gradually rising to normal. In normal cases the pulse reaches its normal in 5 to 10 minutes. Technique of Graupner's Test. A Zuntz Ergom- eter 4 of the bicycle or weight and pulley type is used in conducting the test. The patient turns a wheel which is supplied with a brake and adjustments for measuring the amount of work expended. Tests are 3 Bcrl. klin. Wchnschr., 1902, 174; Deutsch. med. Wchnschr., 1906, XXXII, 1029. * Centrbl. f. Physiol., 1898, 502. 176 Manual of Vital Function Testing Methods made on successive days at the same hour. The work is therefore done by the same muscle groups. It is im- portant not to carry the work to the point of exhaus- tion or strain. Mental excitement must be absent. The pulse rate, blood pressure and size of the heart are noted before and after the test. Arm muscle work may be substituted for thigh muscle work on the same machine and this was done by Graup- ner in his later researches. Cabot 5 and Bruce have recommended using a meas- ured amount of stair climbing, which of course is a more practicable and generally useful method. They estimate the amount of work in foot pounds which is readily computed by multiplying the number of pounds the individual weighs by the number of feet ascended. Graupner came to the following conclusions as a result of his rather extensive investigations : If the blood pressure remains constant after the exercise the heart muscle is sufficient. If the blood pressure falls after the exercise there is cardiac insufficiency. If the blood pressure rises but returns to normal there is com- pensatory sufficiency. If the blood pressure rises then rapidly falls without a tendency to subsequent rise the heart muscle is fatigued. Graupner stated as his belief that if the pulse is accelerated and the patient becomes short of breath after the performance of work equivalent to 45 to 300 kilograms the heart is manifestly insufficient. Several authors have corroborated Graupner's view that a persistent tendency toward a fall of blood pres- sure after the exertion denotes cardiac insufficiency. According to Graupner's later observation persons with normal hearts can perform arm muscle work on the ergometcr equivalent to from 3,000 to 20,000 kilo- Amer. Jour. Med. Sc., CXXXIV, 1907, 491. Tests of Heart Function 177 grams per hour. If the figures fall below 1000 kilo- grams per hour there is cardiac insufficiency. If meas- urements are made every half minute after exercise it was found that the amount of variability in the blood pressure corresponds with the insufficiency of the heart, in other words, that the greater the heart weakness the greater are the variations in the pressure and the longer 'the time required for the status quo to be restored. This does not wholly occur for 30 to 35 minutes. Some observers have found that a lowering of the blood pressure after exertion may be found in trained athletes and according to the terms of the test this should denote cardiac insufficiency. But as Hirsch- felder 6 states, "the heart of the trained athlete is habitually throwing out an amount of blood suited not to the needs of the moment but to the needs of the periods of exercise to which he has accustomed himself. The systolic output is above normal when the exercise (and hence the increased production of C0 2 ) is slight. The heart is then able to take care of the excess of C0 2 production in exercise without in- creasing the output and hence the vasodilatation in the muscles is the only factor influencing the blood pressure. When the exercise becomes severe the other mechanisms begin to play a role. Also in certain patients with diseased hearts the blood pressure has been found to rise, it is claimed, because of high pressure stasis. This rise, however, comes later than in normal cases. Cabot and Bruce as a result of their trial of Graup- ner's test in seventy-five experiments believe that it is reliable. They say "the main outlines of Graupner's contention can be easily verified by anyone. Run 6 Hirschfelder, Diseases of the Heart and Aorta, p. 286. 178 Manual of Vital Function Testing Methods quickly up two flights of stairs and then stop and count your pulse. After the immediate acceleration is passed or during the slowing of the pulse following it you will note that the heart beat and the strength of the pulse become markedly exaggerated. One feels the thump thump of the heart against the ribs much more strongly after the pulse has almost or quite reached its normal rate than during the period when the pulse is most accelerated. . . . As regards the phenomenon designated by Graupner as the normal erholung we can verify his findings and we likewise agree with him in the results of our experiments upon seriously weakened hearts. In some of the cases be- lieved by us from ordinary examination of the heart to be normal there was considerable variation from the ordinary curve of blood-pressure after exertion. Cases of valvular disease with good compensation showed, as might have been expected, a normal curve." Tests like that of Graupner in which blood pres- sure estimations are used as a criterion of cardiac function are founded upon observations of Masing 7 and others that the normal blood pressure rises during exercise and falls immediately afterward. When a normal individual exercises with chest weights, for example, the blood pressure may rise 10 to 30 mm. of Hg. If the individual is arteriosclerotic the rise of blood pressure may go to 40 to 60 mm. of Hg. and outlast the exercise a variable length of time. Bauer in employing this test used a stationary bicycle and this is a good method because the blood pressure estimations can be easily taken in the arm during the performance of the exercise. According to Bauer the bicycle test gives for normal individuals a rise of 5 to 10 mm. of Hg., while in those with cardiac insufficiency ' Dcutsch. Arch. f. klin. Mcd., Lcipz., 1901, LXXI, 253. Tests of Heart Function 179 there may be a fall of equal degree (5 to 10 mm. of Hg.)- The great difficulty with this test is that it has been found that in trained athletes the blood pressure may fall instead of rising at the commencement of mild ex- ercise and the fall may last for a considerable period, thus making the reaction of the strong man some- what similar to that of the weak. The proper interpre- tation of this fact has be.en given. As Hirschfelder states all functional tests of cardiac efficiency if based upon mathematical changes in pulse and blood pressure may lead to ambiguous results. This is no objection, however, to the application of the tests but only to their too strict interpretation. The appearance of the patient after the performance of the physical tests is of course extremely important. Accelerated or labored breathing, holding the breath, dilatation of the nostrils, drawing in of the corners of the mouth, darkness or pallor of the cheeks, sweating, palpitation and so forth, all these are signs of cardiac insufficiency more important perhaps than the mathe- matical results of individual tests. According to Hirschfelder the most reliable nu- merical criterion of cardiac efficiency is whether a given strain causes the heart to diminish in size (increase in tonicity) or to dilate (decrease in tonicity over- strain). 8 3. Mendelsohns Test Q Teclinic. -The pulse is carefully counted in the standing, sitting and recumbent postures and the fig- ures noted. This may be repeated several times and 8 Hirschfelder, loc. cit., 199. 8 XIX Kongr. f. Inn. Med., 1901. 180 Manual of Vital Function Testing Methods an average taken. The person to be tested then per- forms muscular work upon a Gaertner ergostat by means of which the amount of work may be measured. 10 The Gaertner ergostat is an instrument not easily se- cured and for this reason the original test is not much employed. By the simple method, however, of Cabot and Bruce above mentioned of having the patient per- form a given amount of work in stair climbing which can be easily calculated, the reaction of the pulse rate and the return of the latter to normal, which is the basis of the Mendelsohn tests, can be readily estimated. After the performance of varying amounts of work the patient assumes the recumbent posture immediately and the time is noted which is required for the pulse to return to the normal figure previously ascertained for that posture. The first criterion of the Mendelsohn test is based on the principle that when the heart is healthy or well compensated, a transition from vertical to horizontal position is accompanied by a slowing of the pulse of 10 or 12 beats per minute. If the heart is insufficient or decompensatcd an opposite condition may prevail, namely, the pulse becomes quicker in the recumbent posture or tends to remain constant. Mendelsohn contended that if there is not a well marked difference in the pulse rate between the erect and recumbent postures the heart is incompetent. The second criterion suggested by Mendelsohn de- pends upon the principle that the competent heart is able to return immediately to a normal when rest- ing after a strain. He suggested, therefore, that an estimate of the functional capacity of the heart can be obtained by noting the degree of facility displayed by the organ to return to normal conditions after meas- 10 Allg. AVien. Med. Zeit., 188T, Nos. 49 and 50. Tests of Heart Function 181 ured exertion in which extra cardiac energy is called into play. Mendelsohn found that a normal heart after performing work equivalent to 100-200 kilograms returns immediately to the normal with rest in recum- bent posture. After 500 kilograms of work the normal heart is accelerated somewhat for a varying period of time. If, however, there is cardiac insufficiency very much smaller amounts of muscular exertion prove exces- sive and disturb the pulse rate. A disturbance of rate with failure to return immediately to normal following the expenditure of 25-50 kilograms of work denotes cardiac insufficiency. A Variant of Mendelsohn's Test. When a normal individual rises from the reclining to the standing posi- tion the heart rate is accelerated, but it is usually stated that the increase ought never to be more than 20. Beyond 20 one has the right to assume that the myocardium is insufficient. This test from its extreme simplicity has been much used and is capable of giving some valuable informa- tion. But nevertheless, under some circumstances it fails to do so ; for example, under conditions where the psychic role may play a part. Here the increase of pulse rate in a normal individual, that is normal so far as the myocardium is concerned, may be inordinate and out of proportion. Hirschf elder X1 says that per- sons with enteroptosis may give a false increase. 4- The Katzenstein Method 12 Katzenstein found from animal experiments that ligature of peripheral arteries produces an increase in 11 International Clinics, Vol. IV, 1910, p. 39. 12 Dcutsch. med. Wchnschr., 1904, Xo. 30, p. 807; also, ibid., 1907, XLIV, No. 16. 182 Manual of Vital Function Testing Methods the general blood pressure without change in the pulse rate. In animals with weakened hearts he demonstrated that ligature of peripheral arteries produced other re- sults, namely increased pulse frequency and irregulari- ties in the blood pressure. He therefore proposed ap- plying the principle involved to clinical medicine as an aid to determining the functional capacity of the heart muscle. His method consists essentially in making compres- sion upon peripheral arteries (the femorals) so as to shut off the circulation in the lower limbs, and observ- ing the effects upon the pulse and blood pressure. The author of the test found in cases of cardiac insufficiency a lowering of the blood pressure and a simultaneous increase in the pulse rate, both of which deviations from the normal appeared to maintain a proportionate relation to the incompetency of the heart muscle. It has been proposed to substitute an Esmarch band- age for digital compression of the arteries, thereby doing away with the necessity of an assistant. Technic of Kcitzenstein Test. Sometimes called also the Marey-Katzenstein-Shapiro Test. The patient is put in a reclining posture and the pulse rate and blood pressure taken. Pressure is then made for two and one-half to five minutes over both femoral arteries in the groins by means of the fingers of an assistant or by clastic Esmarch bandage, or according to Morelli by inflatable rubber stockings. The pulse rate and blood pressure are again recorded. In normal individuals with sufficiency of the myocar- dium the pulse diminishes in number. The blood pres- sure rises 5 to 15 mm. of Hg. With sufficient but hypertrophic hearts the pulse diminishes or remains the same. An increase of 15 to 40 mm. of Hg. takes place in the blood pressure. Tests of Heart Function 183 In cases of moderate latent cardiac insufficiency the blood pressure remains unchanged. The pulse rate is unchanged or increased. In higher grades of cardiac insufficiency the blood pressure sinks and the pulse rate increases. For practical purposes it may be said that with sufficiency of the heart muscle the pulse remains un- changed or diminishes in number and the blood pres- sure rises. If the pulse increases and the blood pres- sure remains the same or falls after the Katzenstein test the heart is insufficient. Norris 13 made an investigation in 1907 with a view of determining the adaptability of Katzenstein's test to clinical use. He found that generally speaking the results were accurate and confirmatory of its author's findings but many exceptions were noted. Some of the cases, many in fact, of cardiac weakness which re- sponded positively to the test did so in an extremely equivocal manner, leaving practically the final deter- mination a matter of personal equation on the part of the investigator. As a corroborative test the method of Katzenstein appears to possess some value, but as an independent test of cardiac sufficiency or insufficiency no great de- pendence can be placed upon it. The method should be used with caution in cases of severe cardiac weak- ness where it may prove to be actually dangerous. 5. Herz's Self -Che eking Test. 14 Selbst-Hemmungs Probe Teclinic, The patient is placed in a sitting posture and remains so until the pulse rate has become con- 13 Blood Pressure and Clinical Applications, Phila., 1914, p. 145; International Clinics, 1907, I, 17s, p. 66. 14 Deutsch. med. Wchnschr., 1905, 31, XXXI, 215. 184 Manual of Vital Function Testing Methods stant. He is then directed to contract all the muscles of hand and forearm with all his force and to flex and extend the forearm with all possible force, performing the motions slowly, paying strict attention to the per- formance and endeavoring to antagonize his movements as forcefully as possible. In healthy persons the pulse rate is unaffected by this maneuver, whereas in persons with a weak heart the rate increases 5 to 20 beats per minute. This test has been found to possess a certain degree of reliability but it does not possess so absolute a value as was originally ascribed to it by its author. Some- times healthy persons give a positive reaction. Hirsch- felder believes that the vagus plays a part in it and that the results are not altogether indicative of cardiac output and vigor. 6. Gymnastic Resistance Test. Herz-Harancliipy Test This consists in having the patient execute three types of resisted movements. First a movement of flexion-extension of the forearm, the patient being seated. Second, a movement of separation-approxi- mation of the thighs in sitting posture. Third, a move- ment of abduction-adduction of the extended lower limbs, the patient seated. All of these motions are re- sisted equally. A slight rest is given between each series. The whole test lasts 25 to 30 seconds. Prior to the movements the systolic blood pressure is taken. While the movements arc being executed and during repose the blood pressure is retaken. In a normal in- dividual there should be a variation in the blood pres- Tests of Heart Function 185 sure as a result of the exercises of not more than 10 to 15 mm. of Hg. In cases of cardiac insufficiency it reaches 20 to 30 mm. of Hg. 7. The Russian Test. 15 "Holding the Breath" Test This simple test for estimating the integrity of the cardiac muscle has been in long use empirically. We have called it the Russian test because Herz has men- tioned the fact that he could not find any specific men- tion of it in the literature but knew that it was com- monly employed by certain Russian physicians. The test is well called "Holding the Breath" test since it consists simply in directing the patient to stop breathing for as long a time as possible. This ma- neuver puts a considerably added strain upon the myocardium, particularly the right ventricle. Great variations of the length of time in which the breath can be held by different persons are found, but any marked limitation of the time during which a person can inhibit the act of respiration indicates cardiac insufficiency. If the period of voluntary apnoea is less than 15 seconds the myocardium may be considered insufficient. 8. The Venous Pressure Test. Schott's 16 Test The principle of the Schott test depends upon the fact that in health if the arm is elevated to an angle of 60 degrees with the patient in a recumbent posture and making no other exertion the venous pressure in- creases only .5 cm. of H 2 O or sometimes may remain 35 Die Herz Krankheiten, \Vien, 1912, p. 12,5. 16 Deutsch. Arch. f. klin. Med., 1912, CVIII, 537. 186 Manual of Vital Function Testing Methods stationary or even fall. If, however, the cardiac muscle is insufficient, a rise in the venous pressure takes place which may even be considerable (4 to 7 cm.). Accord- ing to Schott, any reading above 3 cm. denotes cardiac insufficiency. Several methods have been devised to determine ven- ous blood pressure. Von Frey and Gaertner considered that the venous pressure can be determined by con- sidering it equal to the height above the angle of Ludwig at which the veins of the hand are seen to collapse when the arm is raised. Von Recklinghausen used an apparatus whereby the vein could be compressed by inflating a small rubber capsule provided with a glass window in the top of a rubber dam floor with an opening in its center. The dam is coated with glycerine to insure perfect apposition to the skin. It is placed over a vein on the back of the hand or wrist and the system inflated until the vein is seen to disappear, at which point the pressure is read off on a water ma- nometer. Eyster and Hooker modified the method by using an aluminum chamber with a glass top, the two ends concave to avoid pressure on the vein. The normal venous pressure obtained by this instrument at the sterno-xyphoid articulation is 5 to 10 cm. of H 2 O. In cardiac cases it may rise to 27 cm. or more. The pressure in the lip capillaries may be estimated by using the point of blanching as the criterion. The study of venous pressure is of some importance as an index of accumulation of blood in the veins and may therefore become to some extent an index of heart failure. Tests of Heart Function 187 II. APPLICATION OF CARDIAC REFLEX ESTIMATIONS IN DETERMINING HEART FUNCTION MerTden's Test The best known cardiac reflexes are those of Abrams and Livierato. Abrams' reflex consists of a diminution of the area of precordial dullness following energetic friction over the heart. Livierato's reflex consists of an increase of the area of cardiac dullness following percussion over the epigastric region. In Abrams' reflex the left ven- tricle is chiefly affected and in Livierato's reflex the right ventricle. The heart is so much less meiopragic (weakened) in proportion to its capacity to give a positive Abrams and a negative Livierato. Technique of Reflex Test. Map out the area of precordial dullness carefully by light percussion and mark with dermographic pencil. Make precordial fric- tion for one minute, using rough cloth or a rubber eraser. Follow this by rapid percussion of the pre- cordial area. After three to five minutes wait, map out the area of dullness by light percussion. If the reflex is normal, the area will be smaller than before. In using Livierato's reflex the technique is the same to determine the area of cardiac dullness. The reflex is elicited by making a series of rapid rather forceful strokes for one minute over the median line of the ab- domen. After three minutes' interim, the area of dull- ness is again made out and if the reflex is positive the right border of cardiac dullness will be found increased. The two reflexes should not be applied to the same pa- tient on the same day. 188 Manual of Vital Function Testing Methods III. ESTIMATION OF SODIUM CHLORIDE ELIMINATION AS A TEST OF CARDIAC SUFFICIENCY Vaquez-Digne Test This test is based upon the supposition that the elimination of sodium chloride is affected by the suf- ficiency or insufficiency of the heart muscle. In indi- viduals in whom the integrity of the heart muscle is normal any excess of salt ingested should be promptly eliminated from the circulation and passed out thro the kidney. In cases of cardiac insufficiency even when latent, it is contended that the salt elimination is defec- tive. In applying this test the individual is put for some days on a fixed sodium chloride ration and when an equilibrium is established the amount of salt injected is doubled and a quantitative estimation of sodium chloride in the urine made. In cases of cardiac in- sufficiency there will be defective elimination and if the diminution of function is considerable there may be oedema and signs of partial decompensation set up. The integrity of the kidney function must be previously determined. IV. MODERN CLINICAL AND INSTRUMENTAL METHODS OF INVESTIGATING CARDIOVASCULAR CONDITIONS: THEIR APPLICABILITY TO ESTIMATING CARDIAC FUNC- TION 1. Spliygmomanometer as an Index of Cardiac Func- tion. Work-Velocity Ratio The sphygmomanometer is the instrument in vogue of our day. The chief value of this instrument is to Tests of Heart Function 189 register the height of the blood pressure and since its introduction it has no doubt contributed to a clearer differentiation of states of hyper- and hypotension. Like the sphygmograph, it is teaching physicians to become more expert in the use of their sense of touch and just . as the latter instrument (sphygmograph) taught physicians to differentiate the arhythmias with- out the use of the instrument in many cases, so, too, it may come to pass that careful comparison of pal- patory pulse estimations of pressure with instrumental readings of pressure carried out day by day in cases of hyper- and hypotension will finally educate the physician to make correct deductions in many cases without the use of the instrument. There will always remain a certain proportion of cases, however, in which, owing to various physical factors, an accur- ate digital estimation of the systolic blood pressure is impossible. A distinguished clinician who has cul- tivated this perception to a remarkable degree is quoted as saying: "I can estimate the blood pressure with the fingers alone quite accurately in about eight cases out of ten, but those in which it is of real im- portance are always the other two." Modern sphygmomanometry will, however, do some- thing more than show the variations in the systolic blood pressure. Recently, since the introduction of the auscultatory or auditory method of using the sphygmomanometer, the method of Korotkof, a more accurate means of finding the exact diastolic pressure has been found than could be had by means of the older visual method with the vertical mercury or other manometers. With accurate data concerning the systolic and diastolic blood pressure, we are in a better position to interpret results in terms of cardiovascular function 190 Manual of Vital Function Testing Methods than we are by means of the systolic pressure alone. In order to fully appreciate just what may be ex- pected from such data as the above in the interpretation of cardiac function, we must bear in mind of course a few simple physiological facts. The blood pressure, that is, the systolic blood pres- sure, depends mainly upon the contractile powers of the heart muscle which enables it to pump the blood into the arteries, against the peripheral resistance caused by the friction of the blood on the vessel walls. The peripheral resistance depends upon the tonicity and physical state of the vessel walls. Under normal circumstances the elasticity of the coats of the arteries provides for a continuous instead of an intermittent flow of blood which would be the case if the arteries were rigid tubes. The systolic or maximum pressure will approximately show the actual pressure or work developed by the heart at the moment of systole. The diastolic or minimum pressure will show the degree of the peripheral re- sistance which the heart has been able to overcome and which is maintained in the peripheral circulation during the time of heart refilling. The difference be- tween the highest and the lowest pressures in the larger arteries, that is the difference between systolic and diastolic pressures, is known as the pulse pressure. The pulse pressure, therefore, is the measure of the amount of force exerted by the heart in maintaining the blood pressure over and above the arterial or peripheral re- sistance. To this extent then the pulse pressure is a measure of the pumping capacity of the heart and hence is of some importance in estimating the state of cardiac function. Gibson has called attention to the fact that there are certain normal arithmetical relations which are Tests of Heart Function 191 discoverable in a study of the three factors systolic pressure, diastolic pressure and pulse pressure. The relation of the diastolic pressure to the systolic pres- sure is normally as two is to three. The relation of the pulse pressure to the systolic pressure is as one is to three. If the systolic pressure is 150 the normal diastolic pressure will be approximately 100. With a systolic pressure of 150, the normal pulse pressure will be 50. Of course these figures represent approxi- mate and not absolute relations. They are of some service in estimating cardiac function because patho- logical relations become evident and the presence and also to a certain extent, the degree, of cardiac overload may be appreciated. The normal arteries will apparently withstand a con- tinual variation in pressure, that is a pulse pressure of 35 to 50 mm. of mercury without deterioration. Any great increase of the pulse pressure over these figures is pathological and at least indicates cardiac overload and consequently justifies suspicion that perhaps the myocardium may not long succeed in maintaining it. The heart manages to do so by undergoing hypertrophy and when this has been accomplished the organ may be regarded as at least a locus mlnoris reslstentice. A quantitative idea of the undue stress may be ob- tained by taking the difference between the normal and pathological pulse pressure. When this is multi- plied by the pulse rate and this by 60 (hour) and again by 24 (day) a concrete idea may be gained of the enormous excess of energy required to be expended by the heart in a day to overcome pathological peripheral resistance. Such a case, however, may go on and on and we have no exact method of predicting just when the break will come, with its attendant consequences of cardiac in- 192 Manual of Vital Function Testing Methods sufficiency. As it approaches, however, the diastolic pressure will fall, indicating the approaching collapse of the cardiovascular mechanism. The larger the pulse pressure in relation to the diastolic pressure, the greater the strain on the heart will be and the more imminent, therefore, is decompensation. A high systolic pressure with a relatively low diastolic pressure indi- cates, therefore, impending collapse of the heart muscle. If after treatment the diastolic pressure rises and the pulse pressure falls the indication is that recompensa- tion is taking place. Technique of Sphygmomanometry. A brief account only of the method of obtaining the systolic and dias- tolic blood pressure will be given. A more complete description is properly found in texts devoted to the subject of blood pressure. The arm band is applied to the bared arm above the elbow by placing the broad end containing the rubber bag over the region of the brachial artery. Wrap the band bandagewise around the arm and tuck in the narrow end. Connect up the indicator and pump. Apply the sphygmometroscope or ausculoscope or stethoscope over the brachial artery at the bend of the elbow. Increase the pressure until all sounds are gone, then gradually admit air. The sounds which are heard are divided into four phases. First, a loud clear-cut snapping tone. This is caused by the first and the early pulse waves that break through the constriction and its beginning repre- sents the systolic pressure. Owing to the greater sensitiveness of the ear than the fingertip it is usually heard some 5 to 10 mm. above the point where the radial pulse is first felt. In normal cases, it is said to last during the fall of about 14 mm. of pressure. Second, a murmur or succession of murmurs lasting during the fall of about 20 mm. This phase is not Tests of Heart Function 193 always present and if absent the first and third phases merge into one another. Third, a clear tone resembling the first, sometimes less well marked but often louder. This lasts during a fall of about 5 mm. Fourth, a rather sharp transition from the loud to the dull tone recently proved to be the time of diastolic pressure. As there had been considerable discussion as to whether the diastolic pressure corresponded to the be- ginning or ending of the fourth phase, Warfield under- took its investigation some years ago through a series of animal investigations and comparison of cases with accurate graphic records. These have been confirmed by others and the point now seems to be settled, that diastolic pressure coincides with the beginning of the fourth phase. Functional Tests Based on Direct Blood Pressure Determinations. I. The Cardiac Efficiency Factor of Tiegerstedt. The pulse pressure shows approximately the systolic output in energy and the velocity of the blood stream will be the product of this energy into the number of cardiac cycles (pulse beats) per minute. In other words, pulse pressure (PP) times pulse rate (PR) will equal velocity of flow. Since the interventricular pressure is almost con- stant throughout systole, it is evident that the work done by the heart is tolerably constant throughout the period. The work done by the heart in a unit of time will be the product of its maximum energy, systolic pressure (SP), multiplied by the pulse rate (PR), mul- tiplied by the duration of systole. Since the inter- ventricular pressure is constant, the factor duration of systole may be eliminated and work done equals product 194 Manual of Vital Function Testing Methods of SP X PR (systolic pressure times pulse rate). The reason why interventricular pressure is constant is because of the fact that the heart liberates all avail- able energy at each contraction. A concrete example will readily show how the velocity work ratio is ob- tained. If the SP = = 130, DP == 85, then PP == 45. If PR = 70 then PP 45 X PR 70 = 3,100 (velocity), and SP 130 X PR 70 == 9,100 (work), the ratio then becomes PP ^jjj = Blood pressure coefficient (Tiegerstedt). fe-i PP X PR _ Velocity SP X PR ~ Work This is the velocity work ratio or coefficient of heart pumping efficiency. The velocity work relation in this example is one to three and this is about the normal ratio. Expressed in percentages, it varies normally from 25 to 35 f /c . Increase in this ratio may indicate cardiac insufficiency. II. The Cardiac Strength, Cardiac Weakness Ratio of Goodman and Howell. These authors have studied the duration of the four-tone phases of auscultatory blood pressure estimation in a series of normal and pathological cases. Their test is based on the ratio of these phases to the pulse pressure and to one an- other. They set forth their views as follows : 1. "The first phase or tone phase serves principally as an index as to how far the pressure must fall before the blood current can be sustained past the obstruction in the vessel caused by the cuff at a sufficient velocity and for a sufficient duration to produce the murmur. Hence the information it affords is of negative rather than of positive value. In other words, its normal Tests of Heart Function 195 duration is of no value but an increase or decrease in length is of importance. 2. The second or the murmur phase seems to be espe- cially dependent upon cardiac effectiveness, for it is in this phase alone that the individual sounds possess a distinct element of duration and this protracted energy, for so it must be regarded, must evidently come from the heart. 3. The third phase or second tone phase depends not alone on cardiac efficiency but also on the character of the vessel wall. The more sclerotic the vessel and the greater the cardiac hypertrophy, the more favorable are the conditions for the production of a clear tone. 4. As the fourth phase or dull tone may be pro- duced by a resilient vessel, receiving a normal pulse shock, or by a rigid vessel receiving a weakened shock, its interpretation is more difficult. If our assumptions are correct it is evident that increases in the second and third phases are dependent on cardiac strength and circulatory deficiency, while the first and fourth phases suffer increase when there is cardiac weakness. Furthermore, in dealing with increases or decreases in any particular phase it is important to know at the expense of what adjacent phase this has occurred. It is apparent that an increase in the third phase for example at the expense of the second has not the same signifi- cance as an increase of this phase at the expense of the fourth. In the first instance the unit of cardiac strength which we obtain by adding the lengths of the second and third phases has not been materially changed while in the latter it has been increased. For this rea- son we recommend that the sum of the second and third phases be compared with the sum of the first and fourth phases in order to determine whether the ele- ments of force or those of weakness are predominating. 196 Manual of Vital Function Testing Methods Aside from the value of the persistence of the fourth phase in aortic insufficiency little of diagnostic value has developed in regard to the length of any individual phase. Advantage has been derived, however, from studying the changes in the sequence reading, specially in decompensating cardiac lesions as the patient im- proves or not. In these cases changes in the percent- ages of the various phases are not the only significant feature but internal peculiarities appear. Or to put it another way, sequence readings have a functional rather than an organic significance. Our results uni- formly show that with decompensation or circulatory disturbances of lesser degree, the element of heart weak- ness (the sum of the first and fourth phases) progres- sively encroaches upon that of heart strength (the sum of the second and third phases). The second phase appears to be the one which is with most difficulty sustained. The fourth phase as weakness gains the ascendency, is usually the first to lengthen the element of cardiac weakness by its encroachment on the third phase, but encroachment of the first phase on the sec- ond soon adds its share to the total." 17 The average duration in mm., the fall of and per- centages of the pulse pressure of the different phases in normal individuals are: mm. per cent. First phase 14 31.1 Second phase 20 44.4 Third phase 5 11.1 Fourth phase 6 13.3 The cardiac strength (second and third phases) : car- diac weakness (first and fourth phases) :: 55.5:44.4. "Amer. Jour, Med. Sci., 1911, CXLII, 336. Tests of Heart Function 197 Marked increase of the cardiac weakness factors indi- cates cardiac inefficiency. ///. Previously to Goodman and Howell's work many observers 18 have recognized that the duration of the second phase of the auscultatory tones indicated cardiac strength. A considerable reduction of its normal per- centage of the pulse pressure is therefore taken as a test of cardiac insufficiency. IV. The Cardiac Overload Factor of Stone. A paper on the clinical significance of high and low pulse pressure with special reference to cardiac load and overload with a report of 170 cases was presented by Stone at the meeting of the American Med. Assn. in 1912. 19 The ratio of the pulse pressure to the diastolic pres- sure representing the load of the heart has since been used as a test for cardiac efficiency. Stone says, "the pulse pressure measures the energy of the heart in systole in excess of the diastolic pressure. For clinical purposes it represents the load of the heart. The myocardiac load may therefore be expressed by the frac- pulse pressure tion . - and under normal conditions is diastolic pressure approximately 50%." Anything in excess of this is an overload. The average load in these cases was 71, an overload of 21%. Naturally in this group of cases some did well and some badly. To quote again, "judg- ing from this small series of cases it would appear that when the overload factor exceeds 50% the patient may be in danger of myocardial exhaustion at any time of slight overstrain. As a rule the greater the overload the greater the danger." That is to say, an overload 18 Forman, Ztschr. f. diet, und physik. Therap., XIII, 809; Fisher: Deutsch. med. Wchnschr., 1908, XXXIV, 1141. 19 Jour. Amer. Assn., 1913, LXI, 1256. 198 Manual of Vital Function Testing Methods of 50% or more (the pulse pressure equal to or greater than the diastolic pressure) indicates cardiac insuf- ficiency of a considerable degree with impending de- compensation. Whereas an overload of 25% would seem to indicate a mild degree of insufficiency. Swan 20 has recently published a study of the above four tests with a series of observations on 40 patho- logical cases. His conclusions are as follows : "It appears to me legitimate from the study of the cases reported to conclude that all four of these factors have some value in determinkig the efficiency of the myocar- dium. I am inclined to think' at present that the cardiac efficiency factor of Tiegerstedt and the percentage of the pulse pressure formed by the second phase are the most important. A cardiac efficiency factor of 40% or over would seem to point out distinct myocardial inefficiency. A second phase of 30% or under would seem to indicate the same condition. The CS to CW (cardiac strength to cardiac weakness) ratio is less important I think because it so often cannot be deter- mined and again because a small second phase is very frequently made up by a large third phase. On the other hand, CS : CW ratio in which the CW factor is greater than the CS factor is indicative of disturbance of the myocardium, functional if not organic. I am in- clined to think at present that the overload factor of Stone is indicative more of peripheral resistance than of myocardial weakness. A cardiac load below 50% as determined by this method giving a negative overload may have some significance, but it will require further study to determine its nature." 20 Archives Int. Med., 1915, XV, 269. Tests of Heart Function 199 #. Rontgenoscopy and Rontgenography as Indices of Cardiac Function The form, position and movements of the heart as a whole and its different chambers, also the great vessels at its base, can be very successfully examined by the X-ray. For several reasons we need only touch upon this interesting and remarkable method of cardiac ex- amination. In the first place, the application of the X-ray to the study of the heart concerns more par- ticularly the examination of the organ from a diag- nostic and anatomical point of view. From this stand- point it constitutes a valuable addition to the older methods of heart exploration. It cannot be said, how- ever, that a Rontgen ray examination sheds much light upon the question of cardiac function. Its chief use is to denote changes in the shape of the organ, hypertrophy and dilatation of its cavities, aneurysm, pericardial effusions, etc. By orthodiagraphy, the posi- tion and topography of the heart can be accurately delineated. But neither ordinary Rontgenoscopy nor orthodiagraphy of the heart shed much light upon the problem of estimating the exact efficiency of the cardiac function. 3. Sphygmocardiography and Electrocardiography; Their Relation to Cardiac Functional Capacity In many text books the phrase, functional disease of the heart, is often used synonymously for pulse irregu- larity. The phrase, cardiac function, is used in an entirely different sense here. Cardiac function, so far as the present discussion is concerned, relates to the capacity of the heart to perform its work, with the 200 Manual of Vital Function Testing Methods adequate maintenance of its reserve. An irregularity of the cardiac rhythm does not necessarily mean any serious deterioration of function. For example, an individual may have a sinus arhythmia or an occasional premature contraction and possess an absolutely normal cardiac reserve. On the other hand, the discovery of certain other types of irregular rhythm always indicates a serious disturbance of heart function. The presence of true heart block, for example, denotes a lesion of the con- ducting system and hence a deterioration of function. The same may be said of auricular fibrillation and to an even greater extent of pulsus alternans. But the detection and identification of irregularities in the cardiac rhythm is a part of the general semi- ological investigation of that organ and while of great importance to the clinician who is examining a case for heart disease, does not properly come within the scope of an investigation into the methods of estimating cardiac function. The study of cardiosphygmography and electro- cardiography has undergone a tremendous develop- ment in recent years. The names of Marey, Franck, Gaskell, Engelmann, Wenkebach, Herring, MacKenzie, Lewis, Erlanger, His and many others are prominently identified with the former and those of Waller, Ein- thoven, Kraus, Nicolai, Edclmann and others with the latter. The recognition of nearly all the varieties of arhyth- mia may be determined by the skilled clinician without the use of any technical apparatus. Unfortunately, this is not always the case and there are types of irregu- larity of the heart rhythm which can only be posi- tively recognized by the use of some form of instru- mental registration. Pulsus alternans is the best ex- Tests of Heart Function 201 ample of this fact. This variety of arhythmia cannot be recognized with certainty without pulse tracings. Its importance from a prognostic standpoint, as Lewis points out, is extremely great. This fact alone will al- ways make the polygraphic study of the pulse a matter of necessity in all cases in which there is a suspicion that pulsus alternans may be present. The ordinary methods of examining the heart, em- ployed in clinical diagnosis, are exceedingly well adapted to disclose diseases of the organ. By inspection, palpa- tion, percussion and auscultation, properly performed, not only can it be determined that disease of the heart is present, but the precise location and often the nature of the lesion can be made out. As Cabot has well expressed it, "we are very well satisfied with the ordi- nary methods of examination, when we find something such as valvular disease, obstructions, accumulations and degenerations. But in many cases in which we fear that the heart is diseased and its functional power diminished, the ordinary methods of investigation do not show anything. Even the more technical and re- fined instrumental methods are negative only too often in such cases. The heart has passed a good physical examination and yet may be insufficient. We desire to know what the heart can do, what is the condition of its reserve. The necessity of supplemental methods becomes manifest. This is the proper field for experi- mental inquiry into the heart function. It is here that the functional tests become especially useful. We give the heart some work to do and see how it reacts, how fast it tires, how slowly it recuperates. We sub- ject the patient to extra effort and note the general symptoms produced, particularly dyspnoea. We pro- ceed to a careful analysis of the history of our patient with respect to his subjective reaction to all of his 202 Manual of Vital Function Testing Methods environment. Already we are working with problems of cardiac function in a fundamental manner." General Conclusions as to Tests for Cardiac Function Hirschfelder, speaking upon the importance of func- tional tests or studies in borderland cases between func- tional sufficiency and cardiac failure, emphasizes the importance of careful observation and says : "It must be admitted that in order to be decisive, all tests have to be pushed to a point at which the appearance, sensa- tions and signs of the patient are in themselves per- fectly characteristic of cardiac insufficiency and at which, for diagnostic purposes, a little common sense observation is at least as unambiguous as observation with elaborate apparatus. This does not mean that exercise tests are unimportant. On the contrary, they are of the greatest value and no change in the patient's mode of living during convalescence or during after life should be undertaken without them. "But their importance depends more upon the care with which the physician watches the general appear- ance and condition of the patient, the rapidity with which he recovers from the exercise, his general condi- tion and whether nervousness, irritability, cough or in- somnia have set in during the 24 hours following it, than in the numerical changes which occur at the mo- ment of exercise. The symptoms to be looked for as evidence of overwork are well known. These are subtler manifestations resulting from smaller changes than may be detected by even the most refined observation by mechanical methods and which are less easily masked by ambiguities. "Moreover, it must be realized that any one form of exercise furnishes data which may depend as much Tests of Heart Function 203 upon the condition of the skeletal muscles as upon the heart. The blacksmith with a diseased heart may be able to do more work than the bookkeeper with neuras- thenia and yet under the conditions in which he lives even if not under the strength test arranged for the average man, the blacksmith's heart may be failing. In diagnosis, prognosis and therapy the testing of func- tional insufficiency is a matter of sociology as well as physiology. The important question is not what the person can do in a gymnasium, but what he can do and what he cannot do in everyday life. Each man must be fit for his own mode of life or must be made to change it. His cardiac power must be studied with reference to that mode of life rather than with refer- ence to a rigid scheme." 21 21 Diseases of Heart and Aorta, Phila., 1913, 199. CHAPTER V THE DUCTLESS GLANDS GENERAL CONSIDERATIONS THE clinical examination of function of the endoc- rinous glands is a subject capable of great future growth. Only the first steps have so far been taken in developing this mine of hidden riches. To the physi- ologist, pathologist and clinician the subject offers a fertile and tempting field of investigation. In the following account of the functional diagnosis of the endocrinopathies but little can be given of the enormous mass of experimental material, and, as Barker 1 has said, "the greater mass of theories" pro- pounded, concerning the physiology, semiology, pathol- ogy and interrelations of the glands of internal secre- tion. These subjects with complete bibliographic and historic references can be found in the classical works of Biedl, 2 Vincent, 3 Falta 4 Levi-Rothschild, 5 Paton, 6 Sajous 7 and others. In these great works little or nothing can be found concerning the important question of functional diag- nosis. The material that has been evolved concerning Southern Med. Jour., 1914, VII, 1. 2 Internal Secretary Organs, London, 1913, Bale Danielsson (Trans, from German). 8 Ductless Glands, London, 1912, Arnold. 4 Erkrankungen der Blutdriisen Wien, 1913. 5 Endocrinologie, Paris, 1911, Dion. "Internal Secretions, Phila., 1911, Davis. 7 Regulators of Metabolism, London, 1913, Macmillan. 204 The Ductless Glands 205 the investigation of function of the ductless glands or glands of internal secretion is not only scant but scat- tered widely in the literature from whence so far as we know it has never been gathered. Not a single article devoted to the general question of the functional diag- nosis of the endocrine glands exists in the whole medical literature. In isolated instances where the subject of functional diagnosis is mentioned, upon investigation it is found that the question is treated upon an almost purely semiological basis. The semiological method of diagnosis of diseases of the ductless glands has there- fore reached a higher degree of development than the functional method, among clinicians up to the present time. Notwithstanding all this, it is admittedly true that the clinician is much in need of functional tests, to enable him to discover the various endocrinopathies in their latent stages, or, as the French say, in the forme fruste, when the symptomatic picture may be incomplete or confusing. Therefore, while at the present time it cannot be said that satisfactory chemical or biological functional tests have been discovered, capable of dis- closing with certainty the existence of latent disease of the thyroid, parathyroid, thymus, pituitary or adrenal organs, nevertheless some advance has been made in this direction and the great need for such tests, in this im- portant tho subtle field of clinical medicine, will always constitute a sufficient inspiration for further discovery. As will be developed, the principal tests which have been devised up to the present time with the object of testing endocrinous function, relate to the thyroid gland and particularly with that aspect of thyroidop- athy which is connected with an increased activity of the gland. Function testing of the adrenals has received some attention and development. The other ductless glands remain so far a terra incognita to the functional 206 Manual of Vital Function Testing Methods method. When we stop to consider how much remains to be known concerning the functions and interrelations of the glands of internal secretions, the fact will not be surprising that the subject of functional diagnosis of their diseases has not received a greater develop- ment. Regarding the ductless glands as a whole, we cannot fail to be impressed with their intimate relation to the processes of body metabolism. The organs of internal secretion or ductless glands (blutdriisen) are important regulators of metabolic processes. It is agreed that the pancreas normally inhibits carbohydrate metabolism and that on the other hand the thyroid and suprarenals normally increase carbohydrate metabolism. The thy- roid has an important effect upon proteid metabolism not shared by the other glands. Increased function of the thyroid is accompanied by increased proteid metab- olism while hypofunction of the thyroid produces a dim- inution of proteid exchanges. The parathyroids and thymus are intimately concerned with normal calcium metabolism but their exact relation to this process is unknown. The gas exchanges of the organisms are also fundamentally controlled by the ductless glands. In hypcrthyrcosis there is an increase, and in myxedema a decrease, of the oxygen absorption and C0 2 exchange. These facts have formed the basis of certain experi- mental methods of determining the functional capacity of the ductless glands and were it not for the fact that the performance of metabolic experiments is so com- plex and requires so extensive an instrumental equip- ment, the examination of these processes as aids to the functional diagnosis of the endocrinopathies would have a much wider application. The Ductless Glands 207 THE THYROID GLAND Tests of Functional Activity Without entering debatable ground, it may be con- fidently asserted that there are certain facts regarding the physiology and pathology of the thyroid gland which are universally admitted. It is clear, for in- stance, that the thyroid is of great importance in the economy of the human organism, and that certain lesions of this gland give rise to symptoms which when they are outspoken may be definitely correlated with thyroid disease. However, despite all the work, experimental and clinical, which has been done upon this gland, there is even now no absolute unanimity of opinion of the pre- cise function or functions of the thyroid. The thyroid function is probably not simple but complex. As in the case of the other ductless glands, many physicians feel convinced that there is an antitoxic function of the thyroid, which consists in collecting exogenous iodine and in some mysterious and unknown way neutralizing certain hypothetical products of intermediary metab- olism. Naturally this idea is a pure speculation and has been arrived at by indirect reasoning. The most usually accepted theory of thyroid function is, how- ever, that the thyroid manufactures an internal secre- tion, possibly an iodine proteid, which is essential to the proper growth and normal metabolism of the entire body. Baumann 8 made an epochal discovery in 1895 when he showed that the thyroid gland contains iodine. Although speculation and investigation have since been rife in respect to this discovery, its precise meaning is yet unknown. 8 Zeitsch. f. physiol. Chem., 1895, XXI, 319. 208 Manual of Vital Function Testing Methods The vast amount of work which has been done upon experimental extirpation of the thyroid can only be mentioned. If the thyroids are removed from young animals, growth is retarded. Total extirpation of the thyroids in human beings is well known to be followed by serious symptoms, the so-called cachexia strumipriva. If the individual is young there will be retarded growth, faulty ossification, mental and metabolic enfeeblement. Similar symptoms are seen in children with congenital thyroid aplasia. In human beings who are deprived of their thyroids, a phenomenon appears which is not seen in lower animals, namely myxedema. Spontaneous myxedema also occurs in the adult human being as a result of thyroid insufficiency the so-called Gull's 9 disease. Murray 10 discovered that the administration of thyroid extract will eliminate the symptoms of myxedema. If thyroid substance be fed to normal animals, symp- toms will develop which are similar to those that occur in Graves' or Bascdow's disease in the human subject and are supposed to be due to a hyperthyreosis or hyperthyroidization in other words, an increase of function of the thyroid. This brings us to the important induction that in the human subject we may have two different or rather two opposite pathological states to consider in respect to the thyroid gland and its functions, namely A. a hyperthyreosis and B. a hypothyrcosis. In the well developed state these two symptom groups constitute definite and tangible clinical syndromes. A. Hypcrf unction of the Thyroid Gland. To the syndrome of hyperthyreosis, the name of Graves' or Basedow's disease is attached. The symptoms of hyper- 9 Trans. Clin. Soc., London, 18T4, VII, 180. 10 Brit. Med. Jour., 1891, 7.96. The Ductless Glands 209 thyreosis may be placed in the following categories : 1, enlargement of the gland; 2, signs of heightened ex- citability of the vegetative or sympathetic nervous system ; 3, signs of secondary or concomitant dis- turbances in other cndocrinous glands ; 4, symptoms of profound disturbance, usually of excess metabolism ; 5, a variety of disorders of the central nervous system ; 6, a peculiar picture in the blood (leucopenia with lymphocytosis). Naturally in this review of functional diagnosis we cannot go into a detailed account of the semiological data which might be collected under each of the above headings. It is interesting to observe that the symp- toms of Graves' disease which come under the category of the vegetative nervous system involve both the auto- nomic and sympathetic portions of that system. The autonomic and sympathetic innervations are both in- volved to a certain extent in every case, but in one, the former, and in another case, the latter, system will be predominantly affected. The eye, heart, blood vessels, skin, digestive, respiratory and urogenital apparatus are all supplied with innervations of both kinds, usually reciprocally or antagonistically, and consequently in Graves' disease there are symptoms referable to disturb- ances in sympathetic or autonomic innervations in sev- eral or in all these different organs. We shall not enu- merate all the various symptoms of Graves' disease, since such an enumeration will be readily found in books or articles dealing with the semiology of this condition. Whenever a sufficient number of these symptoms can be found in a given case the diagnosis of Basedow's disease can be readily made and a quantitative idea of the se- verity of the case may be gained by the actual number or the seriousness of the symptoms. In other words, when the classical semiology of Graves' disease is pres- 10 Manual of Vital Function Testing Methods ent, naturally no functional tests will be needed. A striking phenomenon of Graves' disease is the ac- celeration of all the metabolic processes. This accelera- tion includes the total combustion in calories, the pro- tein, carbohydrate, fat and mineral metabolism. In states of hypothyroidism the opposite condition of re- tardation of metabolic processes occurs. Functional tests have therefore been proposed as a criterion of the existence of states of hyper- or hy- pothyreosis on the basis of increased or diminished oxy- gen consumption and protein, carbohydrate, fat and mineral metabolism. As simpler methods of determining the activity of these various processes are developed we may hope that this kind of investigation will gradually enter more and more into the diagnostic armamentarium of the clinician who is interested in measuring the functional integrity of the thyroid. But as was said before, the technical difficulties which have so far usually surrounded the methods of determining and measuring the various processes of metabolism have prevented their general introduction into clinical medicine. The symptoms of Graves' disease which are refer- able to concomitant or reciprocal disturbance of func- tion of the other endocrinous glands have contributed somewhat to the diagnosis of hyperthyreosis from a semiological standpoint. This circumstance we shall not attempt to develop. But from the standpoint of functional diagnosis of the thyreopathies, these disturb- ances acquire a considerable importance since they open the way, though indirectly, to the development of means for testing the functional activity of the thyroid. It is quite generally held that the thyroid and the pan- creas mutually inhibit one another's activity. The pancreas and chromaffin system (adrenals) are likewise The Ductless Glands 211 mutually inhibitory. The thyroid and the adrenals ap- pear, however, to reciprocally favor each other's activ- ity, that is, an under function of one leads to an under function of the other, while an over function of the one will lead to an over function of the other. This is the teaching of the present Vienna school of endocrinolo- gists represented particularly by Eppinger, Hess, Falta, Rudinger, and others. According to the teaching of this school, a hyperthyroid function will be accom- panied by an insufficiency of pancreatic function (inter- nal secretion) and by an increased activity of the chromaffin or adrenal system. Hypothyroidism, on the contrary, will be followed by over function of the inter- nal secretion of the pancreas and diminution of adrenal functional activity. On the basis of these hypotheses, certain tests have been devised to disclose a hyperfunction of the thyroid gland. One of these, the so-called adrenalin-mydriasis test of Loewi, is used to disclose on the one hand an insufficiency of the internal secretion of the pancreas and on the other a hyperthyreosis. So far as its ap- plication to the investigation of pancreatic insufficiency is concerned, the test has already been described (v. s.) With reference to the second application, namely that of disclosing a hyperthyreosis, details will be later given. Tests founded upon the existence of a glyco- suria, either spontaneous or following the injection of adrenalin, will be considered under the heading Adrenal Glands. The diagnosis of hyperthyroidism, or Graves' disease, is easy in typical cases. The enlarged thyroid, the tachycardia, the disturbances of the sympathetic nervous system, the tremors, the mental state, the ac- celerated metabolism and the blood findings make a definite and indubitable diagnostic picture. Manual of Vital Ftvnction Testing Methods There are, however, many cases which elude the clini- cian because they are atypical. To these latent or atypical cases the French have given the expressive title of formes frustes. Barker 1X has warned us very properly that any one of eight different symptoms should make the clinician suspicious of Graves' disease. The symptoms are: (1) persistent tachycardia (pulse above 85); (2) rapid emaciation without apparent cause; (3) excessive sweating; (4) persistent watery diarrhoea; (5) neurasthenic and psychasthenic states; (6) outspoken lymphocytosis ; (7) fine tremors of the fingers; (8) one or more of the usual ocular symptoms of the disease, namely protrusio bulbi or positive Dal- rymple, 12 von Graefe, 13 Moebius', 14 Stellwag, 15 Jelli- nek 16 and Rosenbach 17 signs. Suppose, however, one of these suspicious signs be present. How shall it be determined whether or not there is actually present a condition of hyperthyroid- ism? It is here that a satisfactory functional test would be invaluable. Frederich Miiller first called attention to its necessity under such circumstances. Several functional tests are at present available for this pur- pose. Unfortunately, they have not been found entirely adequate. Nevertheless, they are of a sufficient degree of assistance to warrant their retention by the clinician, especially as they form an important nucleus upon which future investigators may build new theories and points of departure for renewed attempts at explora- tion. Some of them have not been sufficiently developed as yet to allow a final opinion to be formed. The tests of hypcrfunctional activity of the thyroid gland are as follows : "Widened eye slit; "lagging upper lid; "insufficient converg- enee; "infrequent incomplete winking; w pigmentcd eyelids; 17 tremor of closed lids. The Ductless Glands 213 1. Hypophysis Test of Claude, Baudouin, and Porak. 2. The Adrenalin Mydriasis Test of Loewi. 3. Induction of Experimental or Artificial Hyperthy- roidism as a Functional Test. 4. The Aceto-Nitril Test of Reid Hunt. 5. Metabolic Studies in the Functional Diagnosis of Hyperthyroidism. 6. The Complement Fixation Reaction as Functional Test of Hyperthyroidism. 7. The Specific Ferment Reaction of Abderhalden, as Functional Test of Hyperthyroidism. 1. THE HYPOPHYSIS-EXTRACT TEST FOR HYPERTHYREO- SIS. CLAUDE, BAUDOUIN, PORAK TEST 18 Recently Claude, Baudouin and Porak have published some interesting researches upon the use of extract of the posterior lobe of the hypophysis, in disclosing the presence of latent hyperthyroidism. In their experiments they made use of a hypophyseal extract of posterior lobe of such strength that 1 c.c. of the substance to be injected was equivalent to 1/2 of a posterior lobe of a beef's hypophysis. This they say corresponds to .05 of hypophysis powder. This extract was obtained by the action of alcohol at 70 upon the hypophysis powder, dried and freed from fat. The alcohol is evaporated and the residue re- dissolved in normal salt solution. They also employed occasionally a watery extract, obtaining results with the latter which were practically similar to those ob- tained from the former. They found that subcutaneous injections of both watery and alcoholic extracts of hypophysis produced a marked reaction, pallor, glycosuria and diarrhoea, the 18 Bull, et Mem. Soc. Med. d. Hop. de Par., 1914, XXX, No. 22, 1904. 214 Manual of Vital Function Testvng Methods greater effects being produced by the watery solution and accompanied by greater pain. The alcoholic ex- tract was found to be less painful and the pain less lasting. When a quantity of alcoholic extract corresponding to one whole lobe (beef) was injected, the authors noted complex effects, such as an action upon smooth muscle fiber, a cardiovascular effect and an action on nutrition. The diuretic effect was doubtful. The action on nutrition was characterized by gly- cosuria, the cardiovascular effect consisted of accelera- tion of the heart. In these complex effects of the hypophysis there appears to be an excitation of the sympathetic system shown by the cutaneous vasocon- striction and the glycosuria. The accelerator fibers of the heart being likewise of sympathetic origin, one would naturally expect to find acceleration of the beat. One would also expect that with concomitant constriction of the peripheral vessels that the blood pressure would rise. This phenomenon did not appear. The blood pressure remained the same or was lowered. The au- thors attribute this to a depressing effect of hypophysis extract on the myocardium. They noted occasionally a galop rhythm following the injections. With normal individuals the authors noted acceleration of the pulse. They then proceeded to experiment upon cases of Graves' disease. The patients were kept under observa- tion free from emotional excitement until the normal pulse rate was accurately determined. Injections of plain salt solution were tried as controls. Thirteen typical Basedowians were used in their ex- periments. With the exception of the cardiovascular effects the results of injections did not differ from those of normal cases. The symptoms, pallor, contraction of smooth muscle fiber of intestine The Ductless Glands 215 and uterus, and glycosuria, the latter fairly well marked, were noted in the case of Graves' disease. Alimentary glycosuria was more readily provoked after the injections than before, showing a diminution of the already lowered carbohydrate tolerance. The cardio- vascular effects of the injections were highly significant. In normal subjects the pulse becomes accelerated. The acceleration commences two or three minutes after the injection. It reaches a maximum in 10 or 15 minutes. Then the frequency rapidly diminishes and in about 20 minutes the pulse is normal. In the cases of Graves' disease, however, the results were found to be diametrically opposite. The pulse which is accelerated before the injection of hypophysis extract, becomes quickly slowed. In one of these cases the pulse dropped 42 beats. Usually the diminution in number of beats is much less, averaging 8 or 10. The maximum lowering is reached in about 2 minutes, some- times 4 or 6 and rarely even 10. The bradycardia is usually ephemeral. Usually in 7 or 8 minutes the pulse becomes fast again. Sometimes it was found to return to the original number previous to the injection. In the majority of instances, however, it remains not- ably beneath this point. It would appear as a result of these interesting ex- periments that extracts of the hypophysis, which nor- mally produce tachycardia, bring about an opposite effect, namely, bradycardia in case of Graves' disease. The authors believe that the extract of hypophysis contains principles which simultaneously excite the terminations of both sympathetic and vagus fibers. This is the only explanation of the complex effects of the substance, differing from those of adrenalin, which is a pure sympathetic stimulant. Adrenalin, when in- jected, causes both glycosuria and tachycardia, but it 216 Manual of Vital Function Testing Methods does not produce, ordinarily, pallor of the skin, nor contraction of intestine and uterus, as does hypophysis. The pneumogastric is generally considered the nerve which produces intestinal peristalsis. Since hypophysis produces peristalsis, it must stimulate the 10th pair. If now we consider the effects of hypophysis in Graves' disease, we may begin by admitting that in this condition there is a general state of erethism or hyper- excitation of the entire sympathetic and parasympa- thetic (vagus-autonomic) systems. That the sympa- thetic is excited is proved by the exophthalmus, glyco- suria and tachycardia. On the contrary, the symptom diarrhoea which is so constant and characteristic a symptom in Basedow's disease is explained by Ep- pinger and Hess and others on the theory of a hyper- vagotonia. When one injects into a Basedowian an extract of hypophysis, with the exception of the pulse rate, the effects are generally similar to those obtained upon the normal subject. The heart slowing phenomenon, however, found by Claude, Baudouin and Porak, in Basedowians following injections of hypophysis extract remains to be ac- counted for. The authors believe that the slow- ing is due simply to stimulation of the vagus nerve or 10th pair. They think that hypophysis extract acts on the cardiac rhythm of the non- Basedowian by stimulating the accelerator sym- pathetic. In the Basedowian, however, there is al- ready a tachycardia which is due to the continual hyper- excitation of the sympathetics. These nerves being already in a state of hyperexcitation do not react to hypophysis extract. The terminations of the vagus which are not excited, therefore, feel the full effect of the hypophysis stimulation and the heart is temporarily The Ductless Glands 217 slowed while the effect lasts. Naturally, the authors of the "hypophysis test" mention the possibility of its use in the diagnosis of latent forms of Graves' disease. If future use of this test should corroborate the early work of Claude, Bau- douin and Porak in this regard the "hypophysis test" will become an important adjunct to the functional diagnosis of the hyperthyreopathies. With respect to its applicability to the diagnosis of the forme fruste or latent form of Graves' disease, the authors report two very instructive cases. In one a woman 27 years of age, of neuropathic taint, suffering from tachycardia, dysmenorrhoea and slight hand tremor without thyroid enlargement, the injection of 1 c.c. of hypophysis extract produced a slight increase in the rate from 120 to 126 beats. The test was there- fore negative. In the second case, that of a nervous man 47 years old, with a tachycardia (100 to 105 pulsations) with slight exophthalmia and slight hand tremors, with no apparent thyroid enlargement, the in- jection of hypophysis extract slowed the pulse from 100 to 84 in four minutes. The test was, therefore, positive. The case was a true latent form of Graves' disease, that is, the syndrome of hyperexcitation of the sympathetic nervous system presented by the patient was truly connected with and due to a hyperfunctiona- tion of the thyroid gland. The authors likewise found, which may be mentioned for its scientific interest only, that in cases of paroxys- mal tachycardia the test is negative, as would on a priori grounds be expected. In this condition the pathogenesis resides not in the sympathetic nervous sys- tem as a whole nor in any dysfunction of the thyroid nor any other endocrinopathy but in changes that have taken place in the cardiac musculature. 218 Manual of Vital Function Testing Methods 2. THE ADRENALIN MYDRIASIS TEST OF LOEWI 19 In 1907 Loewi found that in pancreatcctomized ani- mals the instillation of 1-1000 solution of adrenalin pro- duced marked dilation of the pupil. In human beings with diabetes Loewi found the same effects. In 30-60 minutes a marked dilation occurred in diabetic cases. The application of this phenomenon to the detection of pancreatic insufficiency has already been mentioned. Loewi also made a simultaneous observation that the instillation of 1-1000 solution of adrenalin into the con- junctional sac in cases of Basedow's disease, likewise resulted in dilation and proposed the method as a test for hyperfunction of the thyroid gland on the ground that the internal secretion of the thyroid and suprare- nal are synergistic, both acting by stimulating the sym- pathetic nervous system. In cases of hyperthyroidism, the sympathetic nervous system is in a state of increased irritability, therefore the dilator fibers of the iris which are governed by sympathetic nerves respond with ab- normal alacrity to the instillation of adrenalin. Loewi's findings were corroborated by Falta 20 and Zak. 21 Eppinger, Falta and Rudinger, 22 found an increased adrenalin mydriasis in dogs which had been fed with thyroid extract. In depancreatized and thyroidectom- ized animals the reaction was absent. Eppinger and Hess 23 also reported the test positive in Basedow's disease. The interesting and extremely simple test of Loewi 19 Wien. klin. Wchnschr., 20, 1907, 782; Archiv f. exper. Path, und Pharm., 59, 1908, 83. ^Wien. klin. Wchnschr., 20, 1907, 1559. "Verhandl. d. 25 Kong. f. inner. Med., 1908, 392. 22 Wien. klin. Wchnschr., 21, 1908, 241. 23 Verhandl. des 26 Kong. f. inner. Med., 1909, 385. The Ductless Glands 219 has not been much discussed in the literature in recent years. It would be interesting to determine whether an increased susceptibility of the iris sympathetic as shown by mydriasis exists in cases of latent Graves' disease. 3. TEST OF EXPERIMENTAL HYPERTHYROIDISM. ADMIN- ISTRATION OF THYROID EXTRACT, IODINE AND IODIDE OF POTASSIUM AS A MEANS OF DISCLOSING FUNCTIONAL HYPERACTIVITY OF THE THYROID Fr. v. Mueller, who criticized the metabolism tests for hyperthyroidism as being too complex, suggested the administration of iodine as a means of disclosing hyperthyreosis. But apparently Mueller only made the general suggestion and did not elaborate any speci- fied technique. Since no one else has done so, it can- not be said that an "iodine test" exists for determining the presence of a latent hyperthyroidism. Patients with hyperthyreosis often show intolerance to iodine by developing emaciation and tachycardia, after its admin- istration. Many attempts to produce experimental thyroidism in animals by feeding thyroid substance have been made, and there seems to be great variation in the resistance of different genera to thyroid ingestion. When symp- toms appear in healthy animals the most constant signs seem to be emaciation and diarrhoea (Carlson, 24 Bal- let 25 ). Kraus and Friedenthal 2G found that the intravenous injection of thyroid juice in rabbits also produces en- largement of the palpebral fissure, projection of eye- M Prac. Am. Physiol. Soc., 1910-11, XXVII, p. XIII. 25 Limousin Med., 1896, XX, 69. * Berl. klin. Wchnschr., 1908, 1709. Manual of Vital Function Testing Methods balls and enlargement of the pupil. Other authors have succeeded in obtaining similar results. Since the introduction of thyroid preparations into clinical medicine, artificially produced hyperthyroidism has been observed following their indiscriminate admin- istration. The continued injection of thyroid extract is frequently followed by symptoms of intolerance such as subjective sensations of heat, perspiration, palpita- tion or tachycardia and occasionally glvcosuria, all of which denote hyperthyroidism. A few cases have been reported in which a typical Graves disease syndrome has been produced by the administration of thyroid extract. The symptoms dis- appeared after a suspension of the treatment. It is a well known fact that the administration of iodides over long periods to cases of goitre may pro- duce symptoms of hyperthyroidism (Kocher). 27 To these cases the name iodine-Basedow has been given. The actual administration of thyroid extract, iodine, and iodide of potassium to disclose a latent hyperthy- roidism or Graves' disease is not to be recommended as a routine procedure. Most writers advise against the use of iodine, iodides or thyroid extract in any case where there are signs of emaciation (Krecke 2S ). Taking all the above facts into consideration, it will no doubt be admitted that there is little, if any, justi- fication for the administration of either iodides, iodine or thyroid extract in cases of suspected Graves' syn- drome with a view of thereby developing indubitable signs of the disease. It cannot be said to be justifiable under any circumstances to attempt to convert a latent or doubtful into an outspoken case of Graves' disease for purposes of diagnosis. 27 Verhandl. d. Dcutsch. Ges. f. Chir., Berl., 1910, 396. 28 Munch, mod. Wchnschr., 1911, LVIII, 1601 and 1676. The Ductless Glands There appears to be among many medical men a lack of appreciation of the dangers which are attached to the indiscriminate use of thyroid extract and some surgeons have stated that many cases of Graves' dis- ease coming under their observation for operation give a history of previous ingestion of thyroid extract. It would certainly seem rational to assume that noth- ing but harm can come from such a practice. These facts are, of course, well known and appreci- ated by a very large majority of medical men, and be- cause of this knowledge no systematic attempt has ever been made to develop a test of experimental thy- roidism. The use of thyroid extract, iodides, or iodine for such a purpose can only be mentioned to be con- demned. 4. THE ACETO-NITRIL, TEST OF REID HUNT It was in the effort to develop a quick and satisfac- tory method for comparing the physiological activity of different thyroid preparations that Hunt 29 dis- covered the remarkable fact that mice when fed upon thyroids develop an increased resistance to aceto-nitril or methyl cyanide, CH.CN. This substance produces toxic effects chiefly through the slow liberation of hydrocyanic acid in the body. Since thyroid feeding does not alter the resistance of mice to hydrocyanic acid, it is probable that its action so far as aceto-nitril is concerned, is exerted upon the processes by which the substance is decomposed in the organism. Hunt found that when small amounts of thyroid are fed to mice for a few days these animals acquire ^Amer. Jour, of Physiol., 1899, III; Proc. Soc. Exp. Biol., N. Y., 1905, Oct. 18; Jour. Biol. Chem., I, 33, Oct., 1905; Jour. Amer. Med. Assn., 1906, XLVII, 790; Hygien. Lab. Bull., No. 47, 1907. 222 Manual of Vital Function Testing Methods a markedly increased resistance to aceto-nitril. This is true for both white and gray mice, although most of his experiments were performed upon the former va- riety. A mouse which had received thyroid in the form of cakes, recovered from 17 times the relative amount of aceto-nitril fatal to a control. Another mouse recovered from 16 times the relative dose fatal to con- trols. A third mouse recovered from 11 times, a fourth from 6 times and a fifth from 2V2 times the fatal dose to controls. Hunt suggested this reaction as a delicate test for thyroid substance. 30 He found no other substance with an effect upon the resistance of mice to aceto-nitril at all comparable to thyroid. The test is more delicate than any chemical test. Hunt suggested in 1907 that this method is adapted to throw light on the question as to whether there is an excessive amount of thyroid secretion in the blood in cases of Graves' disease. He applied the test in three cases. In one of these the blood of the patient had a marked effect in increasing the resistance of mice to aceto-nitril, indicating thereby an excess of thyroid secretion. In a second case the results were doubtful and in a third case, negative. Hunt suggested that the test might have some diag- nostic value though he points out that it is not neces- sary to assume that in Graves' disease there is always an excess of thyroid secretion present in the blood at all times. To carry out the method, he suggested that the best results might be obtained by administering to mice 1 or 2 c.c. of blood made up with meal in the form of cakes, for 9 or 10 days before testing with 30 Jour. Amer. Mecl. Assn., 1907, XLIX, 240. The Ductless Glands nitril. Controls are indispensable. One-fourth of a milligram of aceto-nitril per gram of body weight of mouse may be fatal to a normal animal in a few hours. Hunt used doses of a fraction of a milligram, one- fourth, to several milligrams, 1, 2, 4, in his experi- ments. Hunt believed as a result of his researches that the activity of a given thyroid preparation or substance is parallel with its iodine content. The test suggested by Reid Hunt has not been extensively developed. His findings were, however, sub- stantially corroborated by several authors, among whom may be mentioned Trendelcnburg in 1910, 31 Ghedeni in 1911. 32 Before a final judgment as to the value of this pro- cedure can be formed it will be necessary to determine the resistance of mice to aceto-nitril after feeding with the blood of patients with Graves' disease in a large series of cases. Also experiments should be done to determine the resistance of mice to aceto-nitril after feeding with normal blood, for perhaps the test is so delicate that even the amounts of thyroid secretions present under normal circumstances may be sufficient to increase the animal's resistance. Of course the vari- ations in natural resistance of the animals both as regards species and seasons which have already been demonstrated as well as the possible variations in the blood content of thyroid substance even under normal circumstances may so complicate and obscure results that the test may be found impracticable. There is, however, something extremely suggestive about this type of biological experimentation which makes it seem probable that some such test will be 31 Biochemische Zeitschr., 1910. S2 Wien. klin. Wchnschr., 1911, XXIV, 736. Manual of Vital Function Testing Methods discovered in the future of real value in the diagnosis of hyperthyreosis. 5. METABOLIC STUDIES AS CRITERIA OF HYPERTHYROIDISM In Graves' disease, as was above mentioned, the metabolic changes are increased. Biedl says they are so characteristic of the condition as to constitute an important diagnostic criterion. The metabolism of Graves' disease is accompanied by a pretty constant increase in the expenditure of energy. The respiratory gas interchange shows an increase of 50%, sometimes 70% to 80% in the amount of oxy- gen consumed, according to Magnus-Levy, 33 Salomon, 34 and others. The increased production of heat is usually accom- panied by an augmented metabolism of albumen and fats. The assimilation of carbohydrates is diminished in Graves' disease and it is for this reason that ali- mentary glycosuria is readily produced. Kraus was the first to suggest that determinations of the respiratory metabolism (increase of CO 2 and N), by the use of the Zuntz-Geppert apparatus may be use- ful in the functional diagnosis of latent or outspoken hyperthyroidism. Fr. v. Mueller, however, observed that the method is too complicated for practical work. Studies of basal metabolism can be calculated by indirect calorimetry from the oxygen absorption and the respiratory quotient, using a Benedict unit appar- atus (mouthpiece and spiromcter). At least three ten-minute periods are run and the average taken for 33 Bcrl. klin. \Vclmsrhr., 1S95; /cit. f. klin. Mod., 33, 1897; Noor- di-m's Handbook d. Path. Stoffwi-chs., II, 3.52, 1907. 31 Bcrl. klin. Wdmschr., 1901. The Ductless Glands 225 that day's basal metabolism. Means 35 has recently reported some studies of basal metabolism and its relation to body surface in obesity, myxedema and pituitary disease. He found a diminu- tion of 27% below normal in myxedema. The emaciation which occurs in many cases of Graves' disease naturally points to an increased katabolism. Kocher found reduction in weight in 88% of his cases. As much as 15 to 20 kgs. may be lost in a few months. The loss of weight is an early symptom. The basic cause for this loss in weight is the remarkable increase in the caloric production which occurs in Graves' disease. By using the Zuntz-Geppert appa- ratus many authors have made the demonstration of increased oxygen consumption and increased CO 2 elim- ination. Magnus-Levy and Salomon have already been mentioned. Experiments have also been made in the Voit-Petten- kofer apparatus which give like results. The increase of caloric production as was before stated may reach as high as 70% or more above normal. In some cases (Magnus-Levy) the oxygen consumption has been found from over 5 to nearly 7 ccm. per kilogram of body weight. Salomon has shown that these metabolic dis- turbances occur and may be demonstrated in the latent cases. It is to be hoped that the gradual simplification of methods for studying metabolism will lead to practical clinical results which will undoubtedly find a rich field of application in the functional diagnosis of thyroid diseases. ^Proc. Soc. fr. Exp. Biol. and Med., 1914, XII, 1913. 226 Manual of Vital Function Testing Methods 6. APPLICATION OF THE PRINCIPLE OF COMPLEMENT DEVIATION TO FUNCTIONAL DIAGNOSIS OF HYPER- THYROIDISM. MARINESCO-ROSEO TEST Marinesco 36 in 1911 and Roseo 3T in 1912 appear to have been the first to suggest that in Graves' disease there is thrown out into the blood serum sufficient thy- roid substance (antigen) to give rise to the formation of antibodies (amboceptors) in the patient's blood. They therefore proposed to test for the presence of these antibodies in the blood serum of suspected cases of Graves' disease by means of an antigen prepared from thyroid tissue removed at operation from an outspoken case of Graves' disease. Both Marinesco and Roseo have studied the reaction of fixation of alexine (complement) in cases of Base- dow disease and they believe that the positive results obtained proved the existence of true specific antibodies in the blood in this condition. This test may in future be found useful from the standpoint of functional diag- nosis. The principle of the complement fixation or deviation test depends, as is well known, upon the observation that the injection of the living organism with bodies of a proteid nature, cells, bacteria, organ extracts, etc., results in the formation by the organism of certain an- tagonistic bodies called antibodies. Perhaps these anti- bodies are ferments. The bodies that are inoculated in order to produce antibodies are called antigens. Some antibodies such as the agglutinins and precipitins act directly on the specific agent or antigen which produces them. Other antibodies, such as cytolysins and hcmolysins, act only in the presence of a third ^Deutsch. Zeits. f. Nervenheilk., 1911, XLI, 268. "Biochem e terap. sper., Milan, 1912-13, IV, 1. The Ductless Glands 227 body, which is always present in blood serum or tissue juices to which the name complement has been given. It is upon this state of facts that the now famous Was- sermann reaction is founded. The performance of the complement fixation test particularly in the diagnosis of syphilis has become a part of the routine work of almost every well equipped pathological clinical laboratory. Consequently there will be no difficulty in a well equipped hospital for the clinician to have complement fixation tests performed. I have been unable to obtain the exact results of Roseo's work. Marinesco's 38 observations upon the reaction of fixation of complement included two series of experiments. In the first he used an aqueous extract of goitre from a classical case of Graves' disease as antigen and the serum of the same patient for antibodies and that of four other patients with the same disease. For controls he used the serum of normal persons. The fixation was complete in the first case whose goitre furnished the antigen, while in two other Basedowians there was incomplete hemolysis, and in a fourth the hemolysis was complete as in a normal control. An objection which may be urged against this first series as pointed out by Marinesco is that he did not have at his disposition an extract of normal thyroid. Later, with the assistance of Madame Papazol, he repeated his experiments, this time making an examination of 23 sera, and using different extracts from the thyroid gland of cases of Graves' disease, also from one goitrous thyroid and one normal gland. The extracts were prepared in the usual way for ether extracts. Eight grams of Basedow goitre were triturated in a mortar and 100 grams of ether added, drop by drop. The mixture was put in a sterilized 38 Loco citato. Manual of Vital Function Testing Methods glass and placed in a shaker. After shaking and filtra- tion, the mixture was kept for 48 hours in the thermo- stat. After the evaporation of the ether a little car- bolated water was added, 40 c.c. for 8 grams of sub- stance. The extract was then again shaken and filtered through cloth. The prepared extract was kept on ice in a dark bottle. The alcoholic extract was similarly prepared. In the two cases in which Marinesco made use of an autoextract of the thyroid from cases of Graves' dis- ease, the prevention of hemolysis was complete. He found the same absolute prevention of hemolysis in six other cases of Graves' disease when he employed thyroid tissues obtained from other Basedowians. Aqueous, al- coholic and ethereal extracts appeared to act about the same, but sometimes the ether extract seemed more active. In most cases of Graves' disease Marinesco obtained either a total absence of hemolysis or an incomplete or partial one. On the contrary, the serum of Graves' disease cases never fixed complement in the presence of normal thyroid body or extract of ordinary goitre. He got the same results when he used serum from normal persons and Basedowian antigen. In one case, however, Marinesco and Madame Papazol found that the serum of a syphilitic patient gave a partial hemolysis with ether and alcoholic extract of normal thyroid. Other authors have likewise found a more or less complete fixation with syphilitic serum in the presence of normal thyroid extract. Mueller, of Vienna, in a personal com- munication to Marinesco stated that in a case of Graves' disease he had noted a fixation of complement in the presence of alcoholic extract of heart. Marinesco believes that his experiments tend to show the presence of an antigen in the thyroid gland of Base- The Ductless Glands 229 dowians and that the reactions of fixation which he obtained are not simply due to an increase in the active substance (internal secretion) of the thyroid gland but to a change in the colloidal state of this substance, due to the harmful effects of a pathogenic agent. Marines- co calls attention to the difficulty in penetrating more deeply into the mechanism of his fixation reaction since authors in general are by no means in accord in explain- ing the mechanism of the fixation reaction discovered by Wassermann in the serum of syphilis. Some authors like Wassermann himself, seeing in the reaction the existence of true specific syphilitic antibodies, others on the contrary considering it as a non-specific physical chemical phenomenon. The practical possibilities for functional diagnosis, of his findings are believed by Marinesco to be worthy of mention. For the Marinesco-Roseo test it will of course be necessary to secure thyroid gland tissue at the time of operation in a case of outspoken Graves' disease. As to the clinical value of the test so little work has been done by investigators subsequent to the reports of Marinesco and Roseo that no definite opinion can be formed as to its value. This will become a matter for future investigation. It is to be hoped that the test will be carried out in a sufficient number of outspoken cases of Basedow's disease to determine in just what percentage it will be positive. If the findings are cor- roborated, the test should be applied to a series of cases of suspected latent hyperthyroidism. At the present time the final decision as to its value remains sub judice. 230 Manual of Vital Function Testing Methods 7. SPECIFIC FERMENT TEST OF ABDERHALDEN APPLIED TO FUNCTIONAL DIAGNOSIS OF THE THYROID Lampe 39 has attempted to apply the Abderhalden dialysis method to the study of the blood serum of patients with Graves' disease. He believes that in the blood serum of these patients ferments exist which are specific for thyroid tissue. This method has never been applied extensively to the functional diagnosis of incipient Graves' disease since its introduction by Lampe. The technical diffi- culties attached to the carrying out of Abderhalden's method and the many conflicting results obtained in its general use have prevented it from becoming popu- lar in clinical practice. Perhaps in the future when the method is simplified and its precise limitations defined, some practical results may be hoped for. Lampe first demonstrated in 1913 that normal blood serum obtained from healthy individuals does not con- tain any ferments capable of splitting the tissues of any of the organs. In the same year Lampe and Papazolu 40 examined the effects of the serum from cases of Graves' disease to determine the presence or absence of proteolytic fer- ments specific for thyroid tissue. Lampe thought that by the results of the dialysis method he might be enabled to throw some light upon the question as to whether in Basedow's disease there is a hyperthyroidism or a dys- thyroidism. He does not mention the possibility of employing the test as an aid towards the functional diagnosis of the disease. Lampe argued that if in Graves' disease there is an over-production of the normal thyroid secretion, there- ""Miinch. med. Wchnschr., 1913, 26. 40 Munch, med. Wchnschr., 1913, 28. The Ductless Glands 231 fore a negative result of the Abderhalden reaction (serum -}- thyroid gland) would be expected because in this case there would simply be the introduction into the blood of a purely native protein only in increased amounts and consequently no development of ferments. If on the contrary, Graves' disease is a true dysthyroid- ism, if, in other words, the thyroid gland in Graves' disease pours into the blood a qualitatively altered pro- teid secretion, produced by the pathological changes in the gland, then this secretion acting as a foreign proteid would be expected to stimulate the produc- tion of protective ferments, and the Abderhalden reac- tion would be positive. Lampe hoped also to be able to throw some light by his method upon the role which the thymus plays in Graves' disease. Lampe and Papazolu experimented with the serum from Basedow cases upon normal thyroid gland, ex- ophthalmic goitre gland, cystic and parenchymatous goitre, normal thymus, Basedow thymus and several other organs and tissues as ovary, testicle, kidney, su- prarenal, pancreas, etc. In their article they give the protocols of experiments upon the serum of twenty-five cases of exophthalmic goitre. In all cases in which the serum from the Graves' disease cases was allowed to act upon exophthalmic goi- tre tissue, the tissue was digested. In very few cases only was the reaction positive when normal thyroid tissue was used. In four out of five of the cystic goitre products, in almost all thymus and ovarian tissues they found the reaction positive. With all other substrate kidney, liver, pancreas, etc., the reaction was negative. Lampe believes that his researches demonstrate that in Graves' disease there is a true dysthyroidism and not a simple hyperthyroidism. Principle of the Abderhalden Method. The basic 232 Manual of Vital Function Testing Methods principle underlying the now much discussed method of Abderhalden 41 is the fact that albumen, being a colloid does not diffuse through animal membranes, while, on the other hand, the peptones, which are the 'first products of its decomposition, are diffusible. If albumen is put in a dialysing tube and the latter placed in water no albumen appears in the surrounding fluid, even after a considerable lapse of time. If pepsin and HC1 are added to the albumen solution peptones are formed and will appear in the dialysate. If it is desired to determine whether a liquid contains any proteolytic substance or ferments, the solution may be placed in a dialysing tube and peptone will appear in the surround- ing media. In this way blood scrum, cerebrospinal fluid, lymph, extracts of organs, etc., may be tested. The actual carrying out of the Abderhalden method is extremely difficult, so much so that the method can- not be used in the ordinary routine of clinical work. If the method is to be tried the individual who proposes to do so will find it advantageous to consult the little work of Abderhalden himself, which has recently been translated and to which reference has been given. B. Hypof unction of the Thyroid Gland (Myx- edematous States). The best concrete example of the loss of function of the thyroid in human beings is met with in those cases in which the whole gland has been removed by operation for goitre. Reverdin 42 in 1882 was the first to describe the results of goitre extirpation. In 1882 Kocher 43 pub- lished his classical report on the same condition. The 41 Defensive Ferments of Animal Organism, Abderhalden; tr. by Gavronsky and Lanchester, Lond. Bale. Co., 1914. 12 Rev. Med. de le Suisse Rom., 1882, 539. "Archiv f. klin. Chir., 1883, XXIX. The Ductless Glands 233 names operative myxedema or cachexia strumipriva were given to this condition. Inasmuch as the thyroid gland is never completely excised at the present day the subject has become of historical interest only. What is of greater practical moment is the fact that symptoms somewhat similar to the cachexia strumipriva may spontaneously arise in adult human beings and give rise to the now well known but only too often overlooked syndrome of spontaneous or idiopathic myxedema of adults, Gull's disease. The symptoms are produced by retrogressive changes in the thyroid gland. Similarly there may be congenital states of hypothyroidism and infantile types, develop- ing after birth, the so-called sporadic cretinism. Fi- nally in some countries a condition known as endemic cretinism exists. All the above types of disease are associated with a diminution of function of the thyroid gland. In all states of hypothyroidism the gland itself under- goes retrogressive changes. There are symptoms refer- able to the skin and subcutaneous tissues, the nervous system, metabolism, the bones, blood, etc. We shall not attempt to go into details in regard to the semiology of these interesting conditions. The diagnosis of myxedematous states is easy in typical cases but even here many cases are overlooked by the practitioner. In latent cases, however, the formes frustes, the diagnosis may not be easy. Some- times the edema is taken to mean Bright's disease. I have seen the nervous symptoms, speech difficulties and gait disturbance ascribed to chronic alcoholism. 44 Kocher calls the latent form of myxedema, thyropenia. All authors call attention to the great frequency with which it is overlooked. "Jour. Am. Med. Assn., 1915, LX1V, 986. 234 Manual of Vital Function Testing Methods The functional diagnosis of thyropenia is intimately bound up with the treatment for there is but one diffi- culty and that is to suspect the disease. Once sus- pected there is one infallible test, the therapeutic test. Therapeutic Test for Lowered Functional Activity of the Thyroid Gland This consists in commencing the administration of thyroid extract. If the case is one of thyroid insuffi- ciency the symptoms will magically disappear. If they are not entirely gone or improved in two weeks, the test is negative. The condition is not one of hypothyroid- ism. Thyroid gland is best given in the form of tablets of the dried gland. The tablets contain 1% to 5 grains (.1 .3 gm.) of desiccated thyroid gland. Begin by administering a small dose after each meal or less often. The patient should lie down for twenty minutes after swallowing the tablet. The dose is gradually increased until 6-10 tablets per day are given, care being taken not to produce rapid heart action, sweating, diarrhoea or nervousness, which are symptoms of intolerance. From a therapeutic standpoint, which point we can- not discuss in this place, it is well known that the ad- ministration of thyroid gland in states of hypothyroid- ism must be kept up indefinitely, for so soon as the treatment is stopped, the symptoms will invariably recur. THE PARATHYROID GLANDS The first person to specifically describe the para- thyroids was the Swedish anatomist, Sandstrom, in The Ductless Glands 235 1880. Gley practically rediscovered them in 1891. Since the latter date a very considerable literature has risen upon these interesting structures. Following the removal of two or more of the four parathyroid glands in the human subject, tetany comes on in from 2 to 5 days. All the special symptoms of tetany are present. Trousseau's, Erb's, Chvostek's and Hoffmann's signs, with irritability of the nerves of spe- cial sense and the sympathetic, irregular pulse, arterial spasm, angioneurotic oedema, spasm of gastrointestinal tract, leucocytosis and disturbance of heat regulation may occur. After incomplete extirpation or temporary injury of the glands, milder symptoms occur called tetanoid or subtetanic hypoparathyreosis (Halsted). Sometimes the sj'mptoms are latent and come on sometime after injury or as a result of pregnancy, trauma or infection. In the well-known infantile tetany lesions of the para- thyroids have been found. Other convulsive diseases such as epilepsy, paralysis agitans, myoclonus, myotonia and myasthenia have been supposed to be due to disease of the parathyroids, but the exact facts in this direction are as yet unknown. The exact relation between the thyroids and para- thyroids is not known, some thinking that an an- tagonism, others that a synergism, exists between the two. There is no method at present known of experimen- tally determining the functional activity of the para- thyroid glands. THE THYMUS GLAND The function of the thymus is as yet not definitely known. It is assumed that inasmuch as the basic 236 Manual of Vital Function Testing Methods structure of the thymus is that of lymphoid tissue in general, that there is a related function between the two, in other words, that lymphocytes and eosinophiles are formed in the gland. There are, however, present in the thymus structure some epithelial elments, the so-called corpuscles of Hassal. What their function may be is quite unknown. Many believe that in the epithelial cells an internal se- cretion is elaborated which has to do with the develop- ment of the skeleton, nervous system, sexual apparatus and general metabolism. There is a general belief that a reciprocal action exists between the thymus and testes, since castration delays the involution of the thymus while removal of the thymus causes rapid development of the testes. The fullest development of the thymus is reached at the end of the second year of life. From this time on to puberty it gradually atrophies and in adults is represented only by a small mass of fibrous tissue and fat. Occasionally, however, the thymus gland persists or undergoes hypertrophy, producing symptoms of tracheal stenosis with attacks of laryngeal stridor or asthma, and sometimes there is sudden death, the so- called mors ihymica. A condition known as the status lymphaticus may gradually develop in which there is more or less anemia, with lymphocytosis, together with rachitic and gastrointestinal symptoms. No tests have so far been devised for determining the functional activity of the thymus. The diagnosis of its diseases is strictly semiological and in the diagnosis, radiography has recently been of considerable assist- ance. The Ductless Glands 237 THE SUPRARENAL GLANDS Our more intimate knowledge of the suprarenal glands appears to date from the year 1855, when Addi- son published his famous work upon the disease which bears his name, though they have been known since 1564, the date of their discovery by Eustachius. A tremendous amount of work has been done upon the adrenals by investigators in the past half century. Much remains to be learned but certain facts appear to have been gained. These may be briefly stated as fol- lows : disease of the glands resulting in their gradual atrophy or destruction gives rise to a train of symp- toms characterized chiefly by pigmentation of the skin and extreme weakness, i. e., Addison's disease. Ex- tirpation in animals of both adrenals is an extremely dangerous operation and according to most authori- ties leads infallibly to death. Extracts obtained from the medullary or central part of the organ are toxic when administered to animals, among the symptoms being glycosuria and arterial degenerations. The same extracts when injected intravenously produce a powerful constriction of the blood vessels with rise of the blood pressure due to stimulation of the sympa- thetic nervous system. It is generally accepted by clinicians and patholo- gists that the adrenal medulla elaborates an internal secretion and that adrenin is the product of this se- cretion. The exact function of the adrenal cortex is still un- known. The cortex contains a considerable quantity of lipoid and cholinogen substances, the presence of which has given rise to the hypothesis that neutraliza- tion of toxic substances is effected here. In 1894) Oliver and Schafer noted that extracts of 238 Manual of Vital Function Testing Methods adrenal medulla produce a rise of blood pressure. In 1897 and 1898 Furth and Abel and in 1901 Takamine and Aldrich succeeded in gradually separating and finally isolating in pure form the active substance of adrenal medulla adrenin. From a clinical standpoint the functional activity of the suprarenal glands may be considered from two points of view. From the first the functional activity of the glands may be considered to be lowered and from the second it may be regarded as raised. To the first condition, the name hypoepinephria or hypo- adrcnalism has been given, and to the second hyper- epincphria or hyperadrenalism. In our brief discussion of the functional examination of the suprarenal glands this classification will be found most practical. We may properly allude here again to the antago- nism which is generally considered to exist between the function of the adrenals (chromaffm system) and the pancreas. It has already been stated that the adrenals and thyroid are functional synergists. The adrenal secretion inhibits carbohydrate catabolism and raises blood sugar, while the pancreas hormone facilitates carbohydrate catabolism arid lowers blood sugar. The effect then of lowering the adrenal function is to raise that of the pancreas, namely, to facilitate carbohydrate catabolism and lower blood sugar to lead to hypogly- cemia, oliguria and hence absence of glycosuria. The effect of raising the adrenal function will be to inhibit carbohydrate catabolism and to raise blood sugar, hence to lead to hyperglycemia diuresis and glycosuria. A. Hypofunction of the Suprarenal Glands. Sev- eral different clinical forms of hypoepinephria or low- ered adrenal function have been described, but few of The Ductless Glands 239 them have been generally recognized by clinicians as separate morbid entities. The essential features of lowered adrenal function appear to be myasthenia and hypotension. The systolic blood pressure is usually below 100 mm. Other features justifying a suspicion of hypoepinephria are hyperesthesias, lumbar pains, headache, delirium, coma, digestive disturbances and sudden death without previous symptoms. The chief clinical entity which is recognized as be- ing accompanied by a true persistent hypoadrenalism is Addison's disease. Addison's disease, first described by Thomas Addison in 1855, is a chronic condition usually appearing in the third or fourth decade of life. It is characterized clinically by pigmentation of the skin and mucous membranes, by muscular and vascular weakness, dis- turbances of the gastrointestinal tract and nervous system, and final cachexia and death. Anatomically it is accompanied by disease of both adrenals, usually a caseous tuberculosis. We shall not attempt a de- scription of the symptomatology or pathology of the disease. The clinical diagnosis of outspoken cases of Addi- son's disease is sometimes easy. If there is a definite history of weakness, vomiting, constipation and diarrhoea, abdominal and lumbar pains and there is present a pigmentation of the skin and mucous mem- branes, and when pernicious anaemia and a few other conditions which might be confused with it can be ex- cluded, the diagnosis is reasonably certain. In Addi- son's disease there is a mononucleosis and a hyper- eosinophilia, in the blood. Before the pigmentation occurs, however, the diag- nosis is extremely difficult or impossible. Latent Ad- dison's disease and other conditions of hypoepinephria 240 Manual of Vital Function Testing Methods can only be disclosed by the application of principles of functional diagnostic methods. Unfortunately, the principles upon which a functional investigation might be applied, toward the elucidation of adrenal dis- turbances, have not been as yet developed to an extent where they can be of great practical assistance to the clinician. One very evident possibility, however, suggests it- self. If states of hypoepinephria are accompanied by diminished function of the adrenals, there must be a lessened amount of the substances in the blood which represent the gland's activity. There is no practical way at present to make use of such an hypothesis. Tests for increased amounts of adrenalin in the blood have been used to discover the opposite state of hyper- adrenalism and these tests will be described below. Eppinger, Falta and Rudinger 45 showed that in cases of Addison's disease (hypoadrenalism) the sugar tolerance is remarkably high. Polak 46 was un- able to produce a glycosuria with 2 mg. doses of adrenalin in a case of Addison's disease. Similar doses in normal persons invariably produce glycosuria. Meyer and Kahn corroborated these findings. These facts form the basis for the application of various kinds of glycosuria tests to the functional diagnosis of hypoadrenalism. Tests of Increased Sugar Tolerance as Evidence of Hypoadrenal Function The so-called sugar tests have been described at length in the chapter on liver function testing. (See 45 See Erkrankungen der Blutdriisen. Falta. Wien, 1913. "Wien. klin. Wchnschr., 1909. The Ductless Glands 241 page 14.) In every case of suspected adrenal disease the sugar tolerance should be investigated. B. Hyperfunction of the Suprarenal Glands. Just what morbid conditions are associated with or produced by hyperfunction of the adrenals is by no means clearly understood. Certain tumors of the chromaffin tissues have been held to produce symptoms connected in some way with hyperadrenalism. The question as to whether other conditions besides tumors of the adrenals can give rise to states of hyperfunction is not de- cided. A number of pathological states of the organism have at one time or another been claimed to owe their origin to hyperfunction of the suprarenal glands or chromaffin tissues generally. A school of pathologists in France has for a long time endeavored to explain the heightened blood pressure of nephritis on the ground of an increased function of the suprarenal glands. Further than this it has been held that the arterio- sclerosis which accompanies the circulatory hypertonia is the result of hyperadrenal function. Finally accord- ing to some pathologists the whole process, of which circulatory hypertonia and nephritis form important parts, is to be ascribed to a primary hyperplasia of the chromaffin tissues. Certain authors have contended that they were able in such conditions to demonstrate by means of the Ehrmann-Meltzer reaction (v.i.) the presence of ex- cessive amounts of adrenalin in the blood. The question as to just what pathological processes and clinical syndromes are to be held related to hyper- adrenalism as effect to cause, is a question almost entirely open and undecided at the present time. Fur- ther than this the subject of functional diagnosis of 24$ Manual of Vital Function Testing Methods hyperactive states of the adrenal glands has only begun to be developed. The principal methods suggested are two in number. The first depends upon the generally accepted influence of hyperadrenalism upon the carbohydrate metabolism. There is said to be always a hyperglycemia. Hence the demonstration of an excess of sugar in the blood is one method of diagnosing a hyperepinephria, pro- vided, of course, that other causes of hyperglycemia can be excluded. There is unfortunately no simple method of making the test. The second class of functional tests for hyperad- renalism depends upon the demonstration of excess of adrenalin in the blood and the production of gly- cosuria following the injection of adrenalin. 1. Tests for Adrenalin in the Blood as an Evidence of Hyperadrenalism. The Ehrmann-Melt zer Reaction It has long been known that intravenous injection of adrenalin produces dilatation of the pupil. This fact has been utilized as a test for adrenalin in various fluids, as blood serum, urine, etc. Meltzer and Auer, 47 Wessely 48 and others have found that when adrenalin is applied to the frog's eye mydriasis is produced. Ehrmann 49 studied this phenomenon and suggested it as a delicate test for adrenalin. He found that adrenalin acts upon the dilator (sympathetic) fibers of the iris in a strength of 1 to 20,000,000. Dilatation of the pupil of the frog's bulbus oculi immersed in salt solution occurs when excessively minute quantities of "Centralbl. f. Physiol., 1904, XVIII, 316. 48 Zeitsch. f. Augenh., Aug. 13, 1905. 49 Archiv f. exper. Path. u. Pharra., 1905, LIII, 96. The Ductless Glands adrenalin are present, quantities as small as .000025 mg. Later investigations have shown, however, that other substances in blood serum will produce the same reaction and therefore the practical availability of the reaction as a test for adrenalin in the serum is vitiated. 2. Adrenalin Glycosuria as a Test of Hyper function of the Chromaffin System We owe to Blum 50 the discovery that the hypodermic injection of adrenalin will occasionally produce glycosuria. The reducing substance found in the urine has been proved to be glucose and there is always a hyperglycemia (Metzger 51 ). The action of adrenalin in thus producing a hyperglycemia is due to its well known stimulating effect upon the sympathetic nervous system ( Underbill 52 ) acting upon sugar storing organs and causing them to relinquish their supply of dextrose producing substances as glycogen. Soon after Blum made this discovery the interesting fact was tested and confirmed in many directions. It was discovered that the glycosuria appears after the exhibition of extract of adrenal substance as well as after that of its active principle, adrenalin. Adrenalin glycosuria appears after comparatively small doses (.Ol-.l mg. ) and is readily provoked by a subcutaneous injection. The injection of one or two milligrams of adrenalin is followed in half an hour to two hours by a glycosuria lasting three hours. The glycosuria is always accompanied lay a hyperglycemia. 50 Deutsch. Arch, f . klin. Med., 1901, LXXI, 146. 01 Munch, mecl. Wchnschr., 1902, 478. B2 Amer. Jour. Physiol., 1906-07, XVII, 42. 244 Manual of Vital Function Testing Methods 3. Deviation of Complement in Functional Diagnosis of Suprarenal Disease Polito and Corelli 53 have attempted to apply the complement fixation test to the diagnosis of suprarenal gland disease (hyperf unction), using an alcoholic ex- tract of suprarenal gland as antigen. Their results were indeterminate. THE HYPOPHYSIS The pituitary gland or hypophysis, as is well known, is composed of two portions, a larger anterior epithelial, follicular, glandular portion and a posterior lobe con- sisting of connective and vascular structures. Between the two is a partly glandular, partly vascular por- tion, the pars intermedia. The whole organ is con- tained in a bony inclosure, the sella turcica, or pituitary fossa of the sphenoid bone. Since Marie first described the disease, acromegaly, and Rogowitch noted hypertrophy of the pituitary after thyroidectomy, both of which took place in 1886, a very large literature has sprung into existence con- cerning the physiology and pathology of the hypoph- ysis. The deep situation of this interesting organ at the base of the brain makes experimental investigation very difficult. The embryological and histological differ- ences between the anterior and posterior lobe of the hypophysis, made it extremely probable early in the history of these investigations that a different func- tional activity must be attributed to the two portions. 68 La Nouva Riv. Clin. Terap., 1911, XIV, 482. The Ductless Glands 245 In this respect there is an analogy with some of the other ductless glands. The differentiation of the two systems in the hypoph- ysis, from the standpoint of pathology, is especially difficult because of the confined space in which the organ is lodged, making it almost inevitable that disease of one portion will affect the other. The name of Gushing in our country is intimately associated with our knowledge of the hypophysis on account of his extensive experimental and clinical in- vestigations. 54 There is still much to be learned concerning the physiology and pathology of the hypophysis. Certain facts, are, however, pretty well agreed upon. The pituitary is probably essential to life, i. e., it is a vital organ. After its removal, animals soon die with severe cachexia. The secretion of the posterior lobe is sup- posed to gain access to the cerebrospinal fluid and the general circulation. It is concerned in regulating metabolism, particularly that of carbohydrates. It affects also the growth of fat. The internal secretion of the anterior lobe affects the processes of general metabolism and especially growth. Oliver and Shafer in 1895 55 discovered that ex- tracts of the pituitary produce when injected into blood vessels, a rise of blood pressure, like that of the ad- renals. Three years later Howell 56 discovered that only extracts of the posterior lobe have this effect. As with the thyroid and adrenals there is the same tendency among clinicians to regard the pathology of the hypophysis as being manifested by states of hyper- and hypo-function. Acromegaly or Marie's 54 The Pituitary Body and its Disorders, Phila., 1912. 05 Jour, of Physiol., 1895, 18. 56 Jour. Exper. Med., 1898, 3. 246 Manual of Vital Function Testing Methods disease is regarded as a typical example of the former, while adiposo-genital dystrophy or Frohlich's disease is looked upon as an equally typical example of the latter. In our very brief account of the functional diag- nosis of the pituitaropathies, brief because so little of importance has been accumulated in medical literature, we shall consider the two states, opposite and distinct, producing diametrically opposite effects upon the or- ganism: hyperpituitarism and hypopituitarism. A. States of Hyperpituitarism. The most typical example is acromegaly or Marie's disease, first described by him in 1886. 57 Acromegaly is a chronic disorder characterized by an abnormal increase in the size of the nose, lips, tongue, lower jaw, hands and feet, by hyperplastic changes in the bones and soft parts, usually accompanied by considerable enlargement of the hypophysis and widen- ing of the sella turcica. Symptoms of increased intra- cranial pressure often occur. The vegetative nervous system is in a state of hyperirritability. The most common pathological finding is adenoma of the anterior part of the hypophysis. There is increase of function of the glandular hypophysis, a true hyperpituitarism. We shall not enter into the etiology and pathology or into the detailed account of the general symp- tomatology of acromegaly. As to the general diag- nosis of the disease it may be said to be quite easy in outspoken and typical cases. It is, however, difficult in the early stages, the so-called latent period of the disease. Acromegaly must be differentiated from cer- tain diseases which may partially resemble it. Brain tumor, arthritis deformans, Graves' disease, diabetes, " Revue de Med., 1886, p. 298. The Ductless Glands 247 progressive muscular atrophy, have all been diagnosed as present when acromegaly really existed. 58 Combinations of acromegaly with Basedowian or myxedematous symptoms sometimes occur in the earlier stages. X-ray examination of the sella turcica (Oppen- heim) is, as is now well known, of extreme diagnostic value. Two methods of functionally determining the exist- ence of hyperpituitarism have been suggested. They are: 1. Demonstration of Disturbed Metabolism, as shown by increase of gas exchange. 2. Demonstration of Alimentary or Spontaneous Glycosuria. 1. Demonstration of Metabolic Disturbance as Shown by Increase of Gas Exchange as an Aid to the Func- tional Diagnosis of Hyperpituitarism Very little work has been done upon the study of the gas exchanges in acromegaly. That which has been done has been carried out with the Zuntz-Geppert apparatus. Cases have been examined by Magnus- Levy, Salomon, Bernstein and Falta. In all, there are seven cases reported, in which de- tails are given as to the clinical facts and the amount of oxygen consumption and carbon dioxide production in ccm. per kilogram per minute. The figures vary for oxygen from 5.19 ccm. down to 3.55 and for C0 2 from 4.33 down to 2.73. Falta states that the results of the cases so far investigated do not demonstrate an in- evitable increase in gas exchanges in acromegaly, as is the case in hyperthyroidism. 58 Modern Med., Osler-McCrae, 1915, IV, 813. (Dock.) 248 Manual of Vital Function Testing Methods It appears to be the opinion of Magnus-Levy and Salomon that if hyperpituitarism is uncomplicated by disorder of other glands of internal secretion (as thy- roid) there is no increase in the gas exchanges. In this opinion Falta 59 concurred. 2. Demonstration of Spontaneous and Provocative Glycosuria as a Functional Test for Hyperpitui- tarism It has been known for some time that acromegaly is often accompanied by a temporary or permanent glyco- suria. Marie, who first described the disease, called at- tention to this fact. Borchard, from a study of 176 cases, from the literature found spontaneous glycosuria reported in 63, and alimentary glycosuria in 8. In 8 cases studied by Falta there was spontaneous or ali- mentary glycosuria in 5. Glycosuria appears only in the early stages of the disease, disappearing towards the end with the beginning of cachexia. The test for provocative alimentary glycosuria should be made in every case of suspected hyper- pituitarism. It will be unnecessary here to repeat the details of technique of the various tests for provocative gly- cosuria, since they have been fully dealt with under a previous chapter. The four tests there described are (1) the Cane Sugar Test, (2) the Glucose Test, (3) the Levulose Test, (4) the Galactose Test. The Glucose test has been more frequently used than the others in testing the carbohydrate powers in cases of suspected hyperpituitarism. 59 Erkrankungen der BlutdrUsen, Berl., 1913, p. 213. The Ductless Glands 249 B. States of Hypopituitarism. This state is typ- ically represented in the so-called hypophyseal dys- trophy of Frohlich, or dystrophia adiposo-genitalis. Frohlich, 60 in 1901, first emphasized the connection between destructive tumors of the hypophysis and the occurrence of the syndrome which bears his name and whose chief characteristics are a rapidly developing adiposity, infantilism of the genitalia and myxedem- atous degeneration of the subcutaneous tissues. Many authors have since reported cases. The opposite conditions with respect to gas ex- changes and glycosuria obtain in hypopituitarism as compared with hyperpituitarism. In hypopituitarism, therefore, the same functional tests are applied as were discussed above under acromegaly. The results, how- ever, will be the opposite. The gas exchanges will be diminished and glycosuria, both spontaneous and pro- vocative, will be negative. ""Wien. klin. Rundschau, 1901, XLVII, 48. < INDEX PAGE Abderhalden's test in hyperthyroidism 230 Abderhalden's method and liver function 46 Aceto-nitril test of hyperthyroidism 221 Adrinalinemia and hyperadrenalism 242 Adrenalin glycosuria and hyperadrenalism 243 Adrenalin-mydriasis test of hyperthyroidism 218 Albarran's method of testing kidney function 72 Aminoaciduria, experimental provocative 33 Aminoaciduria and liver function 33 Ammonia nitrogen, estimation in urine (formalin method) 31 Ammoniuria experimental provocative 32 Antitoxic liver function 36 Biliary liver function 47 Bilirubinuria and liver function 51 Blood coagulation and liver function 41 Blood studies and renal function 90 Cammidge reaction 162 Cane sugar test of liver function 15 Cardiac efficiency factor of Tigerstedt 193 Cardiac overload factor of Stone 197 Cardiac reflex and heart function 187 Cardiac strength, cardiac weakness ratio 194 Cell nuclei test of pancreatic function 148 Claude Baudouin, Porak test of hyperthyroidism. . . 213 Coagulation time and renal function 104 Coagulation time, test for 41 Complement-fixation in adrenal disease 244 Complement- fixation test of hyperthyroidism 226 251 252 Index PAGE Cryoscopy of blood and renal function 104 Cryoscopy of urine and renal function 86 Diastase in feces and pancreatic function 158 Diastase in urine and kidney function 85 Diuretic drug tests of kidney function 75 Ehrlich's urobilinogen test 51 Electric conductivity of urine and kidney function. . 89 i Fat digestion and pancreatic function 150 Ferment identification and pancreatic function 153 Fibrinogen test of liver function 42 Fibrinolysis test of liver function 43 Folin-Denis method of estimating incoagulable nitro- gen in blood 99 Galactose test of liver function 18 Gas exchange and hyperpituitarism 247 Ghedini's test of liver function 46 Glucose test of liver function 16 Glutoid capsule test of pancreatic function 148 Glycosuria and hyperpituitarism 248 Glycosuria and pancreatic function 166 Goodpasture's test of liver function 43 Graupner's test of heart function 175 Gymnastic test of heart function 184 Heart function tests 1 68 Herz' test of heart function 181 Hippuric acid test of renal function 108 Hohlveg-Meyer method of estimating incoagulable nitrogen in blood 99 Hunt's test of hyperthyroidism 221 Hyperadrenalism 241 Hyperpituitarism 246 Hyperthyroidism^ experimental 219 Hyperthyroidism tests for 208 Index 253 PAGE Hypoadrenalism 238 Hypopituitarism 249 Hypophysis cerebri 244 Hypophysis test of hyperthyroidism 213 Hypothyroidism 232 Incoagulable blood nitrogen and renal function 90 Incoagulable nitrogen in blood, estimation of 99 Indicanuria and liver function 38 Indigo carmine test of renal function 114 Katzenstein's test of heart function 181 Kidney function tests 64 Kjeldahl's nitrogen method 28 v. Koranyi's test of renal function 86 Lactose test of renal function 108 Levulose test of liver function 16 Lipase estimation in blood 45 Lipase in blood and liver function 44 Lipase in feces 160 Liver function tests 13 Loewi's test of hyperthyroidism 218 Loewi's pupillary test 165 Lowenhart's lipase estimation method 45 Marshall's method of urea estimation in blood 94 Marshall's method of urea estimation in urine 24 Mendelsohn's test of heart function 179 Metabolism test of hyperthyroidism 224 Methylene blue test of liver function 36 Methylene blue test of renal function Ill Morris' method of estimating incoagulable nitrogen in blood 99 Nitrogen coefficient and liver function 22 Nitrogen estimation in urine (Kjeldahl's method) . . 28 Nitrogen in urine and kidney function 80 254 Index PAGE Pancreatic function tests 142 Parathyroid glands 234 Phenolsulphonephthalein test of renal function 115 Phenoltetrachlorphthalein test of liver function.... 55 Phloridzin test of renal function 107 Polyuria experimental, and kidney function 72 Potassium iodide test of renal function 105 Protein digestion tests of pancreatic function 146 Residual nitrogen and liver function 34 Rest nitrogen in blood and renal function 90 Roche's test of liver function 37 Rontgenoscopy and cardiac function 199 Rowntree, Geraghty test of renal function 115 Rowntree, Horwitz, Bloomfield test of liver function 55 Russian test of heart function 185 Sahli's test of pancreatic function 148 Sanguinopoietic liver function 40 Schmidt's test of pancreatic function 148 Schott's test of heart function 185 Selig's test of heart function 173 Sodium chloride elimination and cardiac function. . . 188 Sodium chloride elimination and renal function 76 Sodium chloride estimation 79 Sphygmography and cardiac function 199 Sphygmomanometry and cardiac function 188 Staircase test of heart function 173 Starch digestion and pancreatic function 153 Stone's cardiac overload factor 1 97 Straus-Griinwald test of kidney function 75 Sugar tests and adrenal function 240 Sugar tests and liver function 14 Suprarenal glands 237 Thymus gland 237 Thyroid gland 207 Tigerstedt's cardiac efficiency factor 193 Index 255 PAGE Trypsin estimate in stomach contents 156 Trypsin estimate in stools 154 Urea in blood and renal function 90 Urea elimination and kidney function 82 Urea elimination and liver function 22 Urea estimation in urine (Marshall's method) 24 Urea provocative test of McCaskey 83 Ureagenetic function tests of liver 20 Urinalysis as criterion of kidney function 70 Urinary toxicity and renal function 89 Urobilinogen test of liver function 51 Urobilinuria and liver function. . . 51 Urobilinuria, tests for 54 Urochrome and kidney function 84 Venous pressure test of heart function 185 Water tests of kidney function 75 Whipple, Horwitz test of liver function 42 Whipple's lipase test of liver function 44 Work-velocity ratio and cardiac function 188 Wright's coagulation-time method 41 UNIVERSITY OF CALIFORNIA, LOS ANGELES THE UNIVERSITY LIBRARY This book is DUE on the last date stamped below 2 8 (L UC SOUTHERN REGIONAL LIBRARY FACILITY A 001 359126 8 RB37 B28m