rOLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES STANDARD HX64141144 QP514 .F98 1916 The problems of phys RECAP QESÜ F9B CoP. 1 Columbia Umberöttp in tfjeCttp of j&etof9orfe College of $ljpötctans ano ^»urgeonö Eef erente lUbrarp ^: Presented by DR. WILLIAM J. Gl! to enrich the library resources available to holders oi the G1ES FELLOWSHIP in Biological Chemistry Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons (for the Medical Heritage Library project) http://www.archive.org/details/problemsofphysioOOfr The price of this book is $ 6= Please state it in your review THE PROBLEMS OF PHYSIOLOGICAL AND PATHOLOGICAL CHEMISTRY OF METABOLISM FOR STUDENTS, PHYSICIANS, BIOLOGISTS AND CHEMISTS BY DR. OTTO VON FÜRTH PROFESSOR EXTRAORDINARY OF APPLIED MEDICAL CHEMISTRY IN THE UNIVERSITY OF VIENNA AUTHORIZED TRANSLATION BY ALLEN J. SMITH PROFESSOR OF PATHOLOGY AND OF COMPARATIVE PATUOLOGY IN THE UNIVERSITY OF PENNSYLVANIA PHILADELPHIA AND LONDON J. B. LIPPINCOTT COMPANY COPYRIGHT, 1916 BY J.B. LIPPINCOTT COMPANY Eleclrotyped and prir.tedby J. B. Lippincotl Company The Washington Squart Press, Philadelphia, U. S, A. AUTHOR'S PREFACE In overseeing the corrections incident to the publication of the volume herewith presented, the author has been for- tunate in having the assistance of Dr. Carl Schwarz, Docent in Physiology in the University of Vienna, and of Dr. Adolf Fuchs, Assistant in the Royal and Imperial Franz Joseph Hospital. It is a pleasure to acknowledge here to both these gentlemen the author's most cordial appreciation. Nor can the author fail to express his obligations to Mr. F. Lampe- Vischer, head of the F. C. Vogel Publishing Com- pany of Leipzig, for the readiness of cooperation which he invariably accorded, at every stage of the preparation and publication of this work. TRANSLATOR'S PREFACE The wide and cordial appreciation with which von Fürth 's ' ' Problems ' ' has been met, not alone by his students and clinicians, but by technical scientists in physiology and in pathology as well, is ample reason for its presentation in translated form. The book is based upon, and in the orig- inal text is cast in the form of twenty-five lectures addressed to students of biological chemistry, and has as its purpose the presentation of the subject of normal and pathological metabolic chemistry as a broad and connected whole. As a well-prepared and enthusiastic guide, thoroughly conversant with the topography, history, popular activities, spirit and ambitions of the land through which he is conducting a group of thoughtful travellers, seeks to point out the salient features of the landscape and the accomplishments of the people, their successes and their needs, and thus in the end leaves in the minds of the group before him a well-balanced idea of the region, so our author seeks to guide his readers through the living body, following the ingestion of the great types of food, their digestion and absorption, pointing out here and there in the unknown field of intermediate metab- olism the little which has become known, indicating their resultants, marking the points of departure of disease, pre- senting the big facts which we know in connection with the metabolic affections, and at all times suggesting the possibil- ities of further investigation and of orienting our thoughts into conformity with the general plan of nature's chemical performances. The book is thus rather a guide to thought than to the technicalities of the laboratory, and in this ap- peals alike to students, chemists, biologists and physicians. It is in no sense a compendium, nor is it a book of reference for details, technical or otherwise ; but is a presentation of the well-ordered thought of a master of biochemistry, the vi TRANSLATOR'S PREFACE result of well-considered culling of facts and their align- ment in probable sequence. A satisfying series of page- references serves to direct the reader to the literature in which are recorded the minutiae of details which such a work cannot reasonably be expected to embody. In the three years since the appearance of the original edition there have, it is true, appeared in current literature facts which the author would doubtless have noted had the times been more fortunate ; but the translator has felt that the book is so peculiarly one man's own that to annotate would be meddlesome and intolerable. The possible changes in statement at any rate would have been no more than minor, and the additions, mainly in connection with the sections dealing with proteins and diabetes, would surely not materially modify the general picture which the author has drawn. In the original German edition Professor von Fürth 's "Probleme der Physiologischen und Pathologischen Chemie" appeared in two separately complete volumes; a translation of the second of which, dealing with the chem- istry of normal and pathological metabolism, appears in the following pages. The first volume of the series deals with and is entitled ' ' Chemistry of Tissues ' ' ; reference to which from place to place appears in the following text. The trans- lator can only regret that the conditions of publication have permitted of the presentation of no more than a single one of these ; but it should be clearly understood that the com- pleteness of the present volume is in no sense involved by the fact that its companion volume remains untranslated. The translator has endeavored to preserve the spirit of the book, but has not attempted to maintain an absolutely literal translation. Doubtless defects of form and perhaps, unfortunately, of sense as well, may be detected in the trans- lation ; if so no one will regret them more than the translator. But these aside, he can and does frankly commend the book as an orderly and masterful delineation of the important TRANSLATOR'S PREFACE vii features in the plan of chemical function in the animal economy. To the publishers he would here express his ap- preciation of their unfailing kindness and consideration in spite of many annoying delays in the preparation of the material for publication, arising from the pressure of de- mands in other lines which at the time seemed insistent. A. J. S. CONTENTS CHAPTER PAGE I. Introduction to the Study of Metabolism. Protein Digestion in the Stomach 1 Introduction. Protein Digestion in the Stomach. "Mock Meal" and Pawlow's Ventriculus. Nervous Mechanism of Secretion. Action of Psychic Influences. Excito-secretory Stimuli. Gastric Secretin. Inhibition of Secretion. Origin of Free Hydrochloric Acid in Gastric Mucous Membrane. Physico-chemical Explana- tions. Acid-production by Marine Snails. Determination of Acidity of Gastric Juice. Binding of Hydrochloric Acid by Proteins. Extent of Protein Digestion in the Stomach. Comparative Physio- logical Considerations. Efforts to Isolate Pepsin. Methods of Pepsin Determination. Law of Pepsin Fermentation. Propepsin. Pseudopepsin. Passage of Pepsin into Intestine. Resistance of Stomach to Autodigestion. Origin of Round Ulcer of the Stomach. Chymosin. Ultramicroscopic Studies of Lab-process. Association of Bacteria in Lab-process. Plastins. Question of the Identity of Pepsin and Rennin. II. The Proteolytic Pancreatic Ferment 33 Transfer of Food from Stomach into the Intestine. Passage of Intestinal Contents into the Stomach. Pancreatic Fistulas. Secretin. Secretin and Cholin and the Vasodilatins. Entero- kinase. Activation of Trypsinogen by Calcium Salts, etc. Indi- viduality of Trypsin. Conversion of Trypsin into Zymoid. Action of Trypsin on Polypeptids. Adaptation of the Pancreatic Secretion to the Food. Quantitative Determination and Ferment Law of Trypsin. Toxicity of Parenterally Introduced Trypsin. III. Protein-Digestion in the Intestine 50 Erepsin. Extent of Protein Dissociation in the Intestine. Passage of True Proteins and High-Molecular Protein-Derivatives into the Blood. Resorption of Iodized Proteins. Objections to the Resorption of Albumoses and Aminoacids. Residual Nitrogen. Synthesis of Digestive Products. Parenteral Introduction cf Protein. Protein Synthesis from Advanced Cleavage of Protein. Abderhalden^ Experiments. Application of these Results in Sickroom Dietary. Relation of Amides in Metabolism in Vege- tarians. Protein Synthesis from Ammonium Salts. London's Studies. Summary. IV. Proteolytic and Peptolytic Tissue Ferments 75 Autolysis a Complex Process. Antiseptic Difficulties in Autolysis Experiments. Is Autolysis the Continuation of an Intravital Process? Deamidizing Tissue Ferments. Importance of Auto- lysis in Various Pathological Processes. Autolysis and the Re- gressive Changes in the Living Body. Relation to Bacterial Proc- esses. Proteolytic Ferments of Leucocytic Origin. Effect of Ex- trinsic Factors upon Autolysis. Abderhalden^ Investigations upon Proteolytic Tissue Ferments. Detection of Proteolytic Tissue Ferments by Means of Glycylt3 r rosin, Silk-peptone, and Glycyltryptophane. Optical Method. Inhibition of Proteolytic Processes by the Products of Protein Cleavage. Classification of Proteolytic Ferments. Peptolytic Power of Blood Serum. x CONTENTS V. Urea. Hippüric Acid Excretion of Aminoacids 97 Urea. Theories of the Formation of Urea in the Living Body. Uraminoacids. Mechanism of Vital Oxidation of Nitrogenous Substances. Ammonium Carbamate. Place of Urea Formation. Exclusion of the Liver. Alkalosis and Acidosis. Arginase. Post- coenal Urea Excretion. Quantitative Estimation of Urea. Hip- puric Acid. Source of Benzoic Acid. Hippuric Acid Elimination in Carnivora and in Man. Hippuric Acid Formation in Herbivora. Behavior of Benzoylated Aminoacids. Synthetic Source of Gly- cocoll from Acetic Acid and Ammonia. Glycocoll and Ornithin as Detoxifying Agents. Quantitative Estimation of Hippuric Acid. Elimination of Aminoacids. Aminoacids in Normal Urine. Elimi- nation of Aminoacids in Disease. Cystinuria and Diaminuria. Quantitative Determination of Aminoacids. VI. Creatin and Creatinin. Other Urinary Bases. Oxyproteic Acids. Urochrome 122 Creatin and Creatinin. Quantitative Estimation. Relation Between Creatin and Creatinin. Creatase and Creatinase. Endo- genous and Exogenous Distribution of Creatin-Creatinin Elimina- tion. Relation of Creatin-Creatinin Excretion to Decomposition of Tissue Protein. Muscle as Source of Creatin. Creatin Cleavage from Other Tissues. Test of Hepatic Function. Relation of Crea- tin Metabolism to Processes in the Female Sexual Organs. Pos- sible Origin of Creatin from Arginin. Other Urinary Bases. Methylguanidin, Dimethylguanidin, Vitiatin. Novain. Trime- thylamine. Arnold's Reaction. Urinary Rest-Nitrogen. Oxy- proteic Acids. Fractionation of Oxyproteic Acids. Chemical Position of Urochrome. True Urochrome and Dombrowski's Urochrome. Quantitative Estimation of Oxyproteic Acids. Elimination of Oxyproteic Acids in Normal and Pathological Conditions. Other High-Molecular Residual Substances. VII. Physiology of Purin Metabolism 148 Exogenous and Endogenous Uric Acid Formation. Conversion of Adenin and Guanin into Uric Acid. Guanin Gout in the Hog. Nucleases and Deamidases. Nuclear Disintegration and Urinary Purins. Relation of Purin Metabolism to Muscular Activity. Synthetic Formation of Uric Acid in Birds and Reptiles. Synthetic Purin Formation in Mammals. Allantoin as an End-product of Mammalian Purin Metabolism. Fate of Intermediary Uric Acid in Man. Absence of Uricase in Human Tissues. Fate of Experi- mentally Introduced Purins in Human Metabolism. Decomposi- tion of Purins in the Intestine. Reconstruction of Uric Acid. Methylated Purin Derivatives. Quantitative Estimation of Uric Acid. Wiechowski's Method of Estimating Allantoin. VIII. Pathology of Purin Metabolism 170 Increase of Uric Acid in the Blood of the Gouty. Question of Increase in the Formation of Uric Acid. Question of Reduction of Uric Acid Decomposition in Gout. Curve of Uric Acid Excre- tion in the Acute Gouty Exacerbations. Delayed Nuclein Ex- change in the Gouty. Gout and Nephritis. Uric Acid Retention. Affinity of Tissues for Uric Acid. Alkali Compounds of Uric Acid. Part Taken by Changes in Alkalescence. Lactam and Lactim Forms of Urates. Nucleinic Acid Combination With Uric Acid. Complex Conditions of Solubility of Uric Acid in Relation to Uric Acid Diathesis. Localization of Uric Acid Depositions. Alkalinity of Tissues in Relation to Uric Acid Deposit. Attempts to Produce Gout Experimentally. Alcoholism. Chronic Plumb- ism. Influence of Excessive Meat Diet. Protracted Feeding of Nucleinic Acid to Dogs. Radium Therapy in Gout. Medicinal Treatment of Gout. The Diet in Gout. CONTENTS xi IX. Digestion of Carbohydrates. Blood Sugar. Diastasic Ferments 194 Carbohydrate Digestion. Ptyalin. Carbohydrate Digestion in the Stomach. Carbohydrate Digestion in the Intestine. Role of Pancreas in Production of Carbohydrate-splitting Ferments. Fate of Disacch arides. Dilution of Sugar Solution in the Intes- tine. Disappearance of Coarse Vegetable Fibres from the Diges- tive Tract. Determination of Cellulose. Cytases. Part Taken in Digestion by Enzymes Contained in the Food. Importance of Symbiotic Microorganisms. Marsh-Gas Fermentation. Food Value of Cellulose. Importance of Infusoria in Cellulose Digestion. Blood Sugar. Technic of Estimating the Sugar in the Blood. Ex- istence of Free Sugar in the Blood. Sucre Immediat and Sucre Virtuel. Does the Blood Contain Other Carbohydrates in Addi- tion to Glucose? Sugar Content in the Red Blood Cells. Sugar in the Aqueous Humor. Carbohydrate-splitting Ferments. Quantitative Determination of Diastase. Wiechowski's Tissue- Powder Method. Hepatic Diastase. Muscle Diastase. Diastases in Embryos. Origin of Diastase. Role of Pancreas in the Produc- tion of Blood Diastase. Maltases, Invertases and Glucases in the Blood-serum. Action of Diastase on Various Forms of Starch. X. Glycogen. Formation of Sugar from Protein and Fat 221 Glycogen. Quantitative Determination of Glycogen. Chemistry of Glycogen. Relation Between Consumption of Sugar by the Muscles and the Disappearance of Glycogen in the Liver. Pro- duction of Glycogen Impoverishment in the Body. Glycogen Formation in the Perfused Liver. Glycogen Formation from Glucose, Fructose, and Galactose. Behavior of Disaccharides and Polysaccharides. Other Substances of the Sugar Series. Glycogen Formation from Formaldehyde. Limit of Saturation and Utili- zation. Formation of Sugar from Protein. Carbohydrate Group in the Protein Molecule. Elimination of Sugar and Protein De- composition. Respiration Experiments. New Formation of Car- bohydrate in Glycogen-Free Tissues. Formation of Sugar from Various Proteins. Origin of Sugar from Animoacids. Formation of Sugar from Fat. Sugar Formation from Glycerine. Seegen's Experiments. Respiratory Quotient in Diabetes. Sugar-Nitrogen Quotient. Fat Impoverishment in Phloridzin Animals. Influence of the Introduction of Higher Fatty Acids upon Sugar Elimina- tion, XI. Pancreatic Diabetes. Human Diabetes 247 Pancreatic Diabetes. Discovery of Pancreatic Diabetes. Inter- rupted Extirpation of the Pancreas. Function of Islands of Lan- gerhans, Duodenal Diabetes. Does the Internal Secretion of the Pancreas Pass with the Lymph through the Thoracic Duct into the Blood? Blood Transfusion and Parabiosis. Glycogen Forma- tion, Diastasic Power and Formation of Sugar from Carbohydrate- free Material in the Liver in Pancreatic Diabetes. Glycolysis. Oxidation Processes in the Diabetic Economy. Metabolism in Pancreatic Diabetes. Partial Extirpation of Pancreas. Antipan- creatin Serum. Isolation of the " Pancreatic Hormone." Human Diabetes. Degeneration of the Pancreas in Human Diabetes. Glycogen Content of the Liver. Hyperglycemia. Methylene- Blue Reaction of the Blood. Urinary Dextrine. Protein Destruc- tion. Diabetic Lipsemia. Respiration Experiments in Diabetes. Substitutes for Bread for Diabetics. Oat Treatments. Influence of Mineral Waters. Medicinal Treatment of Diabetes. XII. Phloridzin Diabetes. L^evulosuria. Lactosuria. Pento- suria. Experimental Glycosurias of Different Kinds. . . . 275 Phloridzin Diabetes. Lack of Hyperglycaemia. Role of the Kidney. Formation of Sugar in the Kidney. Fate of Phloridzi a xii CONTENTS in the Body. Laevulosuria. Detection of Laevulose. Trans- formation of Glucose into Laevulose. True Laevulosuria. Ali- mentary Laevulosuria. Lactosuria. Lactosuria of Puerperal Women. Lactosuria in Infants. Alimentary Galactosuria in Disturbances of Hepatic Functions. Pentosuria. X-xylose. Alimentary Pentosuria. Pentosuria in Diabetes. Chronic Pen- tosuria. Detection of Pentoses. Cammidge Reaction. Experi- mental Glycosurias of Various Kinds. Renal Glycosurias. Sugar Puncture. Toxic Glycosurias. Salt Glycosuria. Gly- cosuria from Chill. XIII. Relation of Glands with Internal Secretion to Carbohy- drate Metabolism. Glycuronic Acid 300 Relations of the Adrenals to Carbohydrate Metabolism. Nature of Adrenal Diabetes. Dependence of Adrenin Glycosuria upon the Renal Function. Hypothesis of the Regulating Influence of Adrenin upon Normal Carbohydrate Metabolism. Does the Sugar Puncture Act through the Adrenals to Cause Glyco- suria? Failure of Effect of Glycosuric Puncture After Exclusion of the Adrenals. Chromium Affinity of the Adrenals. Question of Increase of Adrenin after Sugar Puncture. Do the Adrenals Have a Regulating Influence upon the Normal Carbohydrate Metabolism? Relations of the Thyroid Gland to Carbohydrate Metabolism. Removal of the Thyroid and of the Parathyroids. Hyperthyroidism. Interaction of the Internal Secretory Glands. Relation of Hypophysis to Carbohydrate Metabolism. Gly- curonic Acid. Constitution. Conjugation Conditions. Origin of Glycuronic Acid from Oxidation of Sugar. Oxaluria. Con- version of Glycuronic Acid into X-xylose. Occurrence of Gly- cyronic Acid in the Body. Importance of Glycuronic Acid in Diagnosis of Intestinal Disturbances and Diseases of the Liver. Detection and Estimation of Glycuronic Acid. XIV. Sugar Destruction in the Economy 327 Glycolysis. Distribution of Zymases in the Vegetable Kingdom. Supposed Occurrence of Zymases in Animal Tissues. Cohn- heim's Pancreas-Muscle Experiments. Objections of Claus and Embden. Possible Existence of Glycolytic Tissue Ferments. Experiments upon Surviving Tissues. Glycolysis in Blood. Importance of Leucocytes in Blood-Glycolysis. Sugar Catabol- ism from the Influence of Alkali. Importance of Catalyzers in the Catalysis of Sugar. Electrolytic Cleavage of Sugar. Mech- anism of Alcoholic Fermentation of Sugar. Butyric Acid Fer- mentation. Citric Acid Fermentation. XV. Digestion and Resorption of Fats 350 The Lipase of the Stomach. Problems of Fat Resorption. Fat Cleavage and Solution of Cleavage Products. Fat Synthesis in the Intestinal Wall. Behavior of Non-Saponifiable Emul- sions. Histological Observations Bearing upon Fat Resorption. Resorption of Soaps. Method of Study with Isolated Intestinal Loops. Influence of Pancreatic Juice and Bile upon Fat Diges- tion. Influence of Extirpation of Pancreas upon Fat Resorption. Activation of Pancreatic Steapsin by Salts of the Biliary Acids. Question of Complex Nature of Pancreatic Steapsin. Synthetic Production of Fat by Reverse Ferment Action of Lipase. Fat Cleavage in the Intestine in the Absence of Pancreatic Secretion. Solvent Power of the Bile. Lipaemia. Resorption Path of the Fat. Haemoconiosis. Masking of the Fat in the Blood. Rela- tion of the Masking of Fat to Fatty Degeneration. Pathologi- cal Lipsemias. Lipaemia from Mobilization of Fat Deposits. Diabetic Lipaemia. Lipaemia from Narcosis. Passage of the Fat out of the Blood Stream. Fetal Lipaemia. Diet Lipaemia. Pavy's Theory. CONTENTS xiii XVI. Fat Metabolism. Obesity 378 Dependence of Protein Destruction upon the Fat Supply. Im- portance of Lipoids in Nutrition. Parenteral Fat Absorption. Fat Storage in the Body. Deposit of Foreign Fat. Transfor- mation of Fat in the Body. Depot Fat and Cell Fat. Oxidative Function of the Liver in Catabolism of Higher Fatty Acids. Formation of Fat from Sugar. Transformation of Fat into Carbohydrate in the Vegetable Economy. Characteristics of Fat which is Formed de novo from Carbohydrates. Place of Origin of Fat from Carbohydrates. Chemistry of Fat Formation from Sugar. Experiments upon the Disintegration of Fat by Plants. Catabolism of Fatty Acids in the Animal Body. Nature of Obesity. Corpulence and Over-feeding. Antifat Treatments. Fattening. XVII. Fat-Splitting Tissue Ferments. Fat Formation From Pro- tein. Fatty Infiltration and Fatty Degeneration. Origin of Milk-Fat ............. 401 Fat-Splitting Tissue Ferments. Fat Cleavage in the Blood. Lipase of the leucocytes. Cleavage of Esters in the Tissues. Fat-Splitting Tissue Ferments. Formation of Fat from Protein. Fatty Degeneration and Fatty Infiltration. Fat Phanerosis in Autolysis. Nature of Fat Phanerosis. Formation of Higher Fatty Acids by Microorganisms. Hoffmann's Experiment with Fly-Maggots. Adipocere. Formation of Fat in Ripening of Cheese. Accumulation ot Fat in the Liver in Phosphorus Poisoning. Fatty Degeneration of the Liver. Fatty Infiltration in Other Pathological Conditions. Rosenfeld's Theory. Asso- ciation of Fat Phanerosis in the Phenomena of Fatty Degenera- tion. Fatty Change of the Kidney. Cholesterol-Ester Steatosis. Origin of Milk Fat. Passage of the Fat of the Food into the Milk. Origin of Milk Fat from the Carbohydrates of Food. Lower Fatty Acids in Milk. Sebaceous Glands and Coccygeal Gland. Haptogenic Membranes. XVIII. Acetone Bodies • • 431 Acetone Bodies. Relation of Acetone Body Formation to the Corporeal Fat and to that of the Food. Origin of Acetone Bodies from the Lower Fatty Acids with Even Carbon Atom Chain. Possibility of Disintegration of the Longer Fatty Acid Chains into Short Parts. Is /3-oxybutyric Acid a Product of Normal Metabolism? Derivation of Acetone Bodies from Com- pounds with Branched and Cyclical Chains. Carbohydrate Deficiency and Acidosis. Diabetic Coma. Alkaline Treatment of Coma. Antiketogenic Substances. Ammonia Elimination and Acidosis. Interrelations of Acetone Bodies. Determina- tion of Acetone and Diacetic Acid. Quantitative Determina- tion of Oxybutyric Acid. XIX. Lactic Acid. Fate of Body-Foreign Substances in the Economy 452 Lactic Acid. Quantitative Estimation of Lactic Acid by the Method of Fürth and Charnass. Determination of Lactic Acid and /3-oxybutyric Acids Together. Ryffel's Method of Lactic Acid Determination. Postmortem Formation of Lactic Acid. Origin of Lactic Acid from Sugar. Embden's Schema of Sugar Catabolism in the Living Body. Glycerol-Aldehyde as an Inter- mediate Product between Sugar and Lactic Acid. Racemic Acid as an Intermediate Product. Appearance of Lactic Acid in the Urine. Fate of Body-Foreign Materials in the Economy. Decomposition of Fatty Acids and Aliphatic Sidechains. Ca- tabolism of a-aminoacids. Oxidation of Cyclic Nuclei. Reduc- tion Processes in the Economy. Deaminization. Synthetic xiv CONTENTS Formation of Aminoacids in the Animal Body. Acetylizing Processes in the Animal Body. Alkylation. Detoxification by Sulphuric Acid and Sulphur-containing Rests. Conjugation with Glycocoll and Ornithin. Uraminoacids. Behavior of Stereoisomeric Substances in the Body. XX. Nutritional Requirements. Fasting. Parenteral Nutrition 482 Nutritional Requirements. Amount of Food. Metabolic Mini- mum. Protein Minimum. Relation Between Protein Require- ment and Total Energy Requirement. Vegetarianism. Mechan- ical Preparation of Vegetable P'ood. Nitrogen Balance. Tissue Protein and Circulating Protein. Specific-Dynamic Action of Protein. Physiological Value of Various Proteins. Heterospe- cific and Homospecific Proteins. Food Composed of Simple Sub- stances. Velocity of Protein Catabolism. Metabolism in Fast- ing. How Long May Hunger and Thirst be Endured? Loss of Weight of the Organs. Total Metabolism in Inanition. Protein Economics in Inanition. Carbohydrate Metabolism. Urine in Inanition. Respiratory Quotient. Hibernation. Rhine Sal- mon; Batrachian Larva?. Sensation of Hunger. Parenteral Introduction of Protein. Parenteral Feeding with Sugar. Parenteral Feeding with Fat. XXI. Technic of Study of Gas Exchange. Maintenance Metabol- ism and Growth. Energy Exchange from Ingestion of Food 514 Methods of Gas Exchange Studies. Pettenkofer Type of Res- piration Apparatus. Regnault and Reiset Type of Respiration Apparatus. Respiration Calorimeter of Atwater and Benedict. Method of Zuntz and Geppert. Other Methods. Question of Part Taken by Elemental Nitrogen and Hydrogen in Metabol- ism. Maintenance Exchange and Growth. Significance of Sur- face Development and Volume of Body Protein. Energy Cal- culation in Infancy. Laws of Growth. Energy Exchange after Ingestion of Food. Physiological Utilization Value. Range of Metabolic Increase. Work of Digestion. Specific-Dynamic Effect of Proteins. XXII. Oxidation Ferments 534 Theory of Action of Oxidases. Peroxidases and Oxygenases. Sources of Error in Study of Peroxidases. Iodine Reaction. Oxi- dation of Formic Acid. Purpurogallin Method. Phenolphtha- lin Method. Leucomalachite-green Methods. Measurement of Oxygen Consumption. Limit of Availability of Above Meth- ods. Peroxidase-like Action of Haemoglobin. Chemico-Legal Detection of Blood by Means of Peroxidase Reactions. Res- piratory Coloring Substances. Artificial Peroxidases. Doubt as to the Ferment Character of Peroxidases. Indophenoloxi- dases. Purin Oxidases. Aldehydases. Summary. XXIII. Catalases. Tissue Respiration 556 Catalases. Definition of Catalases. Demonstration. Deter- mination of Activity of Catalase Preparations. Physiological Significance of Catalases. Tissue Respiration. Cardinal and Accessory Respiration. Reducing Tissue Components. Oxygen Consumption in the Blood. Living and Dead Protein. Meth- ods of Study of the Respiration of Isolated Organs. Barcroft and Haldane Blood Gas Analysis. Cohnheim's Respiration Apparatus for Isolated Organs. Thunberg's Microrespirometer. Gas Interchange of Muscle. Oxygen Requirement of Nervous Tissue. Gas Interchange of Salivary Glands. Gas Exchange in the Liver and Kidney. Gas Exchange in the Intestine. An- oxybiotic Processes. CONTENTS xv XXIV. The Coloring Matter of the Blood. Blood Gases. Gas Inter- change in the Lungs. Physiology op Alpinism 579 Haemoglobin. Production of Haemoglobin Crystals. Variability of Haemoglobin. Hoemochrome and Crystallized Blood Coloring Matter. Individuality of Haemoglobin. Importance of Iron in the Blood Coloring Matter. Methaemoglobin. Blood Gases. Technic of Blood-Gas Analysis. Objective Haemoglobinometry and Spectrophotometry. Coefficients of Absorption, Invasion and Evasion. Tension Curves. Influence of Carbonic Acid Pressure. Influence of Temperature. Influence of the Salts in the Medium and Other Factors. Physical-Chemical Concep- tion of Oxygen Fixation by Haemoglobin. Carbonic Acid Com- bination in Blood. Process of Exchange Between the Blood Corpuscles and Serum. Gas Exchange in the Lung Mechanism of Gas Exchange. Secretion of Oxygen in the Swim-Bladder of Fish. Partial Pulmonary Exclusion. Cutaneous Respiration. Intestinal Respiration. Physiology of Alpinis-m. Sources of Observation Material. Influence of Climatic Factors. Increase of Blood Corpuscles. Changes in Cardiac Activity. Changes of Respiration. Acapnia. Loss in Alkalescence of the Blood. Increase of Energy Exchange. Nitrogen Retention. XXV. Fever 610 Total Exchange. Influence of Increased Muscular Activity. Increase of Reaction Velocity of Metabolic Processes with the Temperature. Frugality of Body in Chronic Febrile Conditions. Respiratory Quotient. Reduction Power of Tissues. Decreased Heat Elimination. Protein Destruction. Inhibition of Febrile Protein Destruction by Increasing Carbohydrate Food . Elimina- tion of Nitrogenous Metabolic End-Products. Acidosis and Fat Destruction. Relation of Carbohydrate Metabolism to Fever. Changes in the Constitution of Blood Plasma. Water Economy in I'ever. Swelling of Cellular Protoplasm. Chlorine Retention. Chemical and Physical Heat Regulation. Importance of Nerv- ous System in Regulation of Temperature. Fixation of Heat Regulation at a Higher Level. Increased Excitability of Heat Regulating Centres in Fever and its Reduction by Antipyretics. Pyrogenic Properties of Proteins and Protein Derivatives. Fever Following the Introduction of Corpuscular Elements into the Circulation. Hyperthermias Produced by Chemically Definite Substances. Salt- and Sugar-Fever. Significance of Fever. Epilogue. THE PHYSIOLOGICAL AND PATHOLOGICAL CHEMISTRY OF METABOLISM CHAPTER I INTRODUCTION TO THE STUDY OF METABOLISM. PROTEIN DIGESTION IN THE STOMACH INTRODUCTORY Every one knows full well with what feelings of joy and satisfaction the mountain climber after long and tiresome trudging at last reaches some hoped-for height and views the silent space around and about him. If fortune smile and the blue dome arch out before him without a cloud to its farthest reaches, how easy it is for the wanderer to lose himself in contemplation of the unending, shining distances, and to allow to dawn within him a shadowy presentment of the eternal and infinite. But days of such good luck are rare ; much more often the traveller on the heights must be satisfied if spiteful mists do not completely cheat him of the reward of his labor, and if some little part of the majesty he had hoped to look upon remain unfilched from view. It may well be that only one small part in all that mountain world stands forth in sharply outlined configuration to satisfy his gaze. All other regions seem covered by a vast cloak of vapor, with only the uncertain shadows of grosser outlines shown. In the far distances lie thick banks of clouds and rolling, steam- ing shapes of mist, sweeping out from the clefts, clinging in the valleys, and refusing to permit even a guess of what lies buried behind them. So, too, with us, after long and tiresome wandering, this mountain pass is attained; 1 from which we may hope to advance some little way, perchance, into the broad, mys- terious domain of the physiology of metabolism. In allur- 1 Referring to the series of lectures preceding the present text, in the volume entitled " Chemistry of the Tissues," in the original German edition. 1 2 INTRODUCTION TO STUDY OF METABOLISM ing change are unfolded splendid open stretches in full bright light, and dark cloaks of vapor with only here and there a ray breaking through ; thick, still banks of clouds, and rolling veils of mist through which the picture, before clear and transparent, becomes in the next moment dark and uncertain. We view with wonder and admiration the vast amount of labor which has been devoted to the study of metabolism since the days when Lavoisier first recognized the vital combustion processes and since, long after, Robert Meyer and Herman v. Helmholtz inflamed with their genius a torch destined to cast its beams of light into the darkness of all fact and error in the domain of vital phenomena. Under the dominance of the law of conservation of energy the newer physiology of nutrition, founded by Liebig, Pettenkof er, Voit and Pflüger, is seen to arise; modern chemistry at the task of uncovering the secrets of the intermediate metabolism; and pathologists in earnest endeavor to open to physiology the experiments on man which nature provides. But, on the other hand, how slow the steps of progress to one who never turns to observe the long path already traversed but keeps ever before him the goal constantly receding into the distance before his advance. In undertaking to present a clear outline of the problems which mainly occupy the attention of metabolic physiologists to-day it seems best to the author, in conformity with the plan followed in the preceding volume upon the Chemistry of the Tissues, which started with a discussion of the protein prob- lem, to begin this study of metabolism also with the proteins. The proteins and their catabolic products will be first con- sidered, tracing them from their ingestion into the stomach as food, in their course through the body until their deriva- tives disappear in the unknown processes of intermediate metabolism. In turn the carbohydrates and fats will be taken up in the same way ; in preparation for the considera- tion, finally, of the vital combustion processes, the most im- portant phases of metabolic physiology. It is no short and "MOCK MEAL" 3 easy path along which the student is invited to trudge with the author. Yet it is scarcely in harmony with the duty of a guide that at the very beginning of an ascent he should paint fearful pictures of its difficulties. It is very much better to take up the way confidently and without misgivings, and to postpone to convenience all matters of reflection. Without further introduction, therefore, the subject- matter of the lecture may be taken up : PROTEIN DIGESTION IN THE STOMACH In all the mammalia, it need scarcely be said, the food after mechanical preparation by mastication passes to a reservoir, the stomach, where its proteid constituents under- go at once the first phase of digestion under the influence of the peptic ferment of the stomach. "Mock Meal" and Pawlow's Ventriculus. — Investiga- tions bearing upon the secretion of the gastric digestive juice in subjects having operative or traumatic gastric fis- tulas date back to the first half of the past century. A sys- tematic experimental study of the problem of the secretory function of the gastric mucous membrane was first made possible when Pawlow la perfected a satisfactory technic of artificial production of gastric fistula. In his method the oesophagus is diverted in the neck, in a dog in which a gastric fistula has been produced, to the surface and the orifice is stitched into the skin wound; so that by the passage of a "mock meal" considerable quanti- ties of pure gastric juice without admixture of food sub- stances may be obtained from the fistula, as all the food given the hungry animal passes out of the upper part of the gullet to the exterior, while at the same time the nervous secretory stimulus ("psychic secretion") excited by the act of chewing operates without complicating factors. Pawlow contributed another important step by his modifi- cation of Haidenhain's method of making a ventriculus or a a J. P. Pawlow, Arbeit der Verdauungsdrüsen, Wiesbaden, 1898; and Nagel's Handb. d. Physiol., 2, 699-762, 1907. 4 GASTRIC DIGESTION OF PROTEINS * 'little stomach. ' ' By simply cutting out of the gastric wall a section, closing the wound in the stomach and stitching the blind sac constructed from the exsected patch into the ab- dominal wound, a "ventriculus" is formed, which during digestion secretes coincidently with the stomach itself. In the older method, however, the secretory nerves are divided and the secretory conditions are therefore by no means normal. Pawlow corrected this fault in his method of oper- ating, by freeing completely only the mucous membrane of the patch and sparing a pedicle of the serous and muscular coats in attachment with the stomach proper, so that the vagus fibres going to the mucosa may pass from the wall of the large stomach to that of the small stomach by route of the pedicle. He has been able to show that the vagus nerve actually conducts secretory impulses to the stomach by the following methods. If both vagi are excluded, either by section or by atropine, the usual result of the "mock meal" fails to appear; by stimulation of a peripheral vagus seg- ment gastric secretion can be induced, provided stimulation is not practiced immediately after the section but preferably after sufficient time has elapsed for the degeneration of the vagus cardiac fibres. Nervous Mechanism of Secretion. — Efficient secretory stimulus, passing from the cortex of the brain by way of the vagus nerves to the stomach, producing in case of the "mock meal" experiment a full complement of active gastric juice, fails after section of the vagi; but after a time a certain amount of gastric adaptation takes place whereby a continuous secretion is maintained sufficient for the digestive requirements. 2 Bickel has observed con- tinuous secretion of normal gastric juice in the nerveless stomach in a dog in which he cut all the extra gastric nerves of a gastric diverticulum. 3 3 P. Katschowsky (Pawlow's Laboratory), Pfliiger's Arch., 84, 6, 1901. 3 A. Bickel, Deutsche med. Wochenschr., 1909, 704. Literature on the neuro-secretory mechanism of gastric secretion : A. Bickel, Handb. d. Biochemie, 3', 58-63, 1910. ACTION OF PSYCHIC INFLUENCES 5 By a long series of painstaking and carefully conducted investigations, the majority of which were done in the Insti- tute of St. Petersburg and in the laboratory of A. Bickel in Berlin, the excito-secretoiy influence exerted upon the gas- tric secretion by a great variety of physiological and patho- logical factors has been determined, including foodstuffs, condiments, mineral waters, medicinal substances, and the like. 4 These studies have been very materially supplemented by observations upon human beings with gastric fistulas, 5 in whom occasionally the same conditions have existed as obtain in the experimental "mock meal" procedures. For ex- ample, in the case of a girl who had had a gastric fistula produced after a corrosive lesion of the oesophagus, an cesophagotomy was later performed, and by means of a rub- ber tube direct communication established between the end of the upper oesophageal segment and the gastric fistula. Well-masticated morsels were transmitted with considerable force by the muscles of the gullet along this artificial oesophagus into the stomach ; while by removal of the com- municating tube the precise arrangement of a regular "mock-meal" experiment was obtained. Action of Psychic Influences. — In endeavoring to come to some definite understanding from the various studies upon animals and human beings as to the factors by which the secretion of the gastric juice is most actively affected, we can- not fail to recognize the dominating agency of psychic influ- ence. We know to-day that under appropriate conditions the mere idea of a toothsome meal, and to a greater degree the actual mastication of such food, not only ' ' make the mouth water" but also make the juices of the stomach flow. An as- sociative gastric-juice production has been clearly proved to 4 Literature: 0. Cohnheim, Nagel's Handb. d. Physiol., 2, 534-542, 1907. A. Bickel, Handb. d. Biochemie, 3', 66-70, 1910. * Observations of F. A. Hornborg, F. Umber, H. Bogen, A. Bickel, Sasaki, H. Kaznelson, Pflüger's Archiv., 118, 327, 1907; R. S. Lavenson, Arch. Int. Med., k, 271, 1909; Cf. O. Cohnheim, Die Physiologie d. Verd. u. Ernährung, p. 57, 1908. 6 GASTRIC DIGESTION OF PROTEINS exist in the case of a child with gastric fistula. Each time the little patient received food a certain note was sounded on a child 's trumpet ; and after a time, during which this practice was continued, the sound of the trumpet alone, without the actual food, sufficed to cause a flow of gastric secretion. 6 That anger and agitation disturb the appetite is a comparatively common experience of almost every one of us. But Bickel has produced experimental evidence in the same line, by showing that a dog whose equanimity was banished by the irritating sight of a cat not only manifested loss of appetite from anger, but in addition suffered actual cessation of gastric secretion during his period of indignation. 7 The normal taste perceptions are, however, not invariable prece- dents of actual gastric secretion; as Coronedi observed in the course of a "mock meal" experiment the production of an entirely normal gastric juice after having completely anaesthetized the tongue of the experiment animal against sense-perception by painting it with cocaine solution. 8 Excito-Secretory Stimuli. — Mechanical irritants have lit- tle effect in inducing secretion of the gastric juice. However, although Pawlow has denied their influence completely, Arthur Schiff has been able to present evidence that chronic irritation, as from sand particles and similar substances in fluid suspension, may be effective, at least in the sense of in- creasing the secretory flow. 9 In the same connection may be mentioned the fact that in dogs with the pylorus con- stricted by a silver band there occurs a continuous gastric secretion. 10 In comparing various foodstuffs, the most active secre- tory flow is noted after feeding meat. It has been shown, too, that meat-extractives are particularly influential; but e H. Bogen (Kinderklinik, Heidelberg), Pfltiger's Arch., 117, 150, 1907. 7 A. Bickel, Deutsche med. Wochenschr., 1905, 1829. *G. Coronedi and F. Delitala, Arch, di Fisiol (Festschr. f. Fano), 7, 17; Centralbl. f. d. Ges. Biol., 10, No. 1915, 1910. »A. Schiff (Inst. R. Paltauf), Zeitschr. f. klin. Med., 61, 220, 1907. 10 N. B. Foster and A. V. S. Lambert, Proc. Soc. Exper. Biol., 5, 109, 1908. GASTRIC SECRETIN 7 it is not known to what particular component of the extract this special secretion-stimulation is due. The flesh of fish seems to be more effective than other meats. 11 Extracts of yeast are much less active than meat-extract. 12 The latter shows marked influence, too, if introduced subcutaneously or per rectum. It is scarcely practicable here to enter into detailed mention of the action of the great mass of bitter stuffs, alkaloids, alcohol, mineral waters and neutral salts upon gastric secretion 13 ; but in actual practice all these sub- stances are of medicinal interest in the therapy of hyper- acidity and hypersecretion. It is but fair to remark, how- ever, that in this connection we are not upon safe ground, particularly as many of the affections which clinicians have been in the habit of classifying under the terms just stated, should, perhaps, be classed as disturbances of gastric motil- ity, hyperesthesia of the gastric mucous membrane and other comparable conditions. 14 While not assuming from a theoretical standpoint to enter into a full discussion of this subject, the author is unwilling to pass over a group of other factors of sig- nificant physiological character, of importance in stimulat- ing secretion of the gastric juice. An attempt has been made to establish a relation between the salivary glands and gastric secretion, from the circumstance that total extirpa- ion of the former in the dog has seemingly occasioned a considerable diminution of the gastric secretion 15 ; but the correctness of this assertion has been questioned. 16 Gastric Secretin. — It has been observed that when neu- tralized gastric juice or the product of acid-extraction of the gastric and intestinal mucous membranes and of many other 11 Cf. B, Lönnquist, Skand. Arch. f. Physiol., 18, 194, 1906. W. N. Boldy- reff, Arch. f. Verdauungskr., 15, 268, 1909. 12 W. Hoffmann and M. Wintgen, Arch. f. Hygiene, 61, 187, 1907. "Literature: A. Bickel, Handb. d. Biochem., 3', 66-69, 1910. 14 Cf. V. Rubow, Arch. f. Verdauungskr., 12, 1, 1906; R. Kaufmann, Zeitschr. f. klin. Med., 57, 491, 1905. a Hemmeter, Biochem. Zeitschr., 11, 238, 1908. "A. S. Löwenhart and D. R. Hooker, Proc. Soc. Exper. Biol., 5, 114, 1908. 8 GASTRIC DIGESTION OF PROTEINS tissues is introduced subcutaneously or intravenously, secre- tion of gastric juice ensues. 17 Bayliss and Starling are there- fore disposed to accept the existence of a ' * gastric secretin. ' ' According to this view the normal secretory activity of the gastric mucous membrane is to be referred to two factors : the more important role is to be ascribed, of course, to the nervous stimuli transmitted by the vagus nerves ; but a sec- ond influence, determining the continuation of secretion long after the influence of the first has ceased, is held to be an agency of chemical nature. This is believed to be a specific substance generated in the pyloric mucous membrane under the influence of acid, which, passing into the blood, is returned to the mucous membrane of the stomach as a "hormone" or chemical messenger, stimulating it to renewed secretory activity. 18 Discussion of the hypothesis is postponed to the following lecture in connection with the subject of the pan- creatic secretin. Inhibition of Secretion. — In addition to those influences which induce secretion of gastric juice, others are known which act to inhibit it, an example being manifested in the failure to secrete following the introduction of fatty foods into the stomach. Pawlow has shown that the inhibition in such case arises rather from the duodenum than from the stomach. Apparently alkalies also act to inhibit the secre- tion at times by an influence arising from the intestine. Attention has recently been called by clinicians to the absence of free hydrochloric acid in the gastric secretion in cases of disease of the gall-bladder, and to the value of this phenomenon in diagnosis. 19 Origin of Free HCl in Mucous Membrane of Stomach. — This brings us again to the old problem of the nature of * Edkins, Jour, of Physiol., 34, 133, 1906. " E. H. Starling, Lectures on Recent Advances in the Physiology of Diges- tion, p. 75, et seq., London, 1906; W. M. Bayliss and E. H. Starling, Ergebn. d. Physiol., 5, 676-677, 1906. 19 H. Hohlweg (Voit's Clinic at Giessen), Deutsche Arch. f. klin. Med., 108, 255, 1912. ORIGIN OF FREE HCl 9 the particular process by which the gastric mucosa is enabled to separate a free mineral acid as a secretory product. There is no occasion to weary the reader with a statement of the older attempts to explain the phenomenon. Neither the idea of mass-action of carbonic acid, nor that of alkali-binding by lecithin-albumen, nor that of a supposed impermeability of the gastricmucous membrane to chlorine ions has withstood criticism 20 ; and it has been impossible to find in the mucous membrane of the stomach any organic compounds of chlorine from the dissociation of which hydrochloric acid could by any possibility be produced. 21 It is entirely reasonable to regard the sodium chloride of the ingested food as the source of the gastric hydrochloric acid. Rosemann has established by careful investigation the fact that it is impossible to pro- duce any important reduction of the chlorine stored in the economy by starvation and the use of food poor in chlorine, because the system is able to protect itself against chlorine impoverishment by lowering the excretion of chlorine in the urine to a minimum. It is possible, of course, to withdraw considerable amounts of chlorine from the body by "mock feeding" because of the hydrochloric acid secretion thus in- duced. In the end secretion of fluid ceases, and that, too, at a time when the general body still contains a notable amount of chlorine. Apparently only about one-fifth of the total chlorine content of the body can be discharged in the gastric secretion. 22 From studies conducted upon a professional female starvationist it has been determined that even after a twenty-four day fast the stomach will respond to the stimulus of a test-breakfast by production of a fluid which 80 Cf. L. v. Rhorer, Pflüger's Arch., 150, 416, 1905. * H. Dauwe, Arch. f. Verdauungskr., 11, 137, 1905. a H. Rosemann (Münster), Pflüger's Arch., 11^2, 208, 1911; cf. also refer- ences of J. Wohlgemuth, Exp. Untersuchungen über den Einfluss des Kochsalzes auf den Chlorgehalt des Magensaftes, Berlin, Hirschwald, 1906; and also the older investigations of Nencki, Külz, A. Kahn and others. 10 GASTRIC DIGESTION OF PROTEINS while showing decided lowering of the hydrochloric acid is nevertheless entirely able to digest. 23 If free hydrochloric acid is to be derived from sodium chloride, the latter must react with water in conformity with the equation: NaCl + H 2 = NaOH + HCl. For each molecule of hydrochloric acid thus separated a molecule of sodium hydroxide must remain in the organism. This may explain why at the height of the acid secretion the sodium chloride excretion in the urine is diminished and why in the ensuing period of resorption of the strongly acid contents of the stomach more ammonia fails to be synthesized into urea, being utilized to neutralize the free acid. 24 We may conceive, too, why the body of an animal in the state of chlorine starvation reacts to sodium chloride administration by a notable increase of alkalinity of the urine (as pointed out by the author's lamented friend, Leo Schwarz, in Hof- meister 's laboratory 25 ). The fact that the hydrochloric acid of the gastric juice can be replaced by hydrobromic acid up to a certain degree indicates that sodium bromide acts in a comparable manner. Physicochemical Explanations. — Attempts have been fre- quently made to apply the modern ideas of physical chem- istry to the problem of the production of HCl in the gastric juice. When the ion theory began slowly to penetrate into the minds of biologists it came to be recognized that often an explanation may be realized by expressing a problem in the terms of the theory of ions. As a matter of fact, as you know, if to-day a problem is laid before the great public, no matter how learnedly, in technical phrases, the real meaning is no more grasped than if it had been told in the Spanish or Russian language. Unfortunately here and there we can 23 L. Riitimeyer (Basel), Zentralbl. f. innere Med., 1909, 233. ^A. Müller and P. S'axl (I. Med. Clinic, Vienna), Zeitschr. f. klin. Med., 56, 546, 1905; A. Loeb (Med. Clinic, S'trassburg ) , ibid., 56, 1905; S. A. Gam- meltoft (Copenhagen), Zeitschr. f. physiol. Chemie, 75, 57, 1911. 25 L. Schwarz (Physiol. Chem. Instit., Strassburg), Hofmeister's Beiträge, 5, 56, 1903; cf., ibid., also the work of Falck, M. Gruber, and Rosell. ACID-PRODUCTION BY MARINE SNAILS 11 still find in modern science (especially in medicine) some lingering residuum of the painstaking endeavors of the mas- ters of the Middle Ages to impress properly the audience with the fact of the extreme difficulty of understanding their very learned disquisitions. However, a physicochemical explanation in point is to be found in the proposition which Daneel predicates upon the principle of ion activity: If it be conceived that free organic acids are produced in the gastric mucous membrane (and we have every reason for assuming that the production of free lactic acid is a function common to cells in general, although the acid is quickly neutralized by the alkalies of the circulating fluids), we may represent by the symbol R an organic acid radical; and the dissociation of sodium chloride and of acid would follow the formulas : NaCl = Na' + Cr HR =H' + R'. Of the two cations H' is more active than Na' and of the anions CI' more active than R' ; wherefore from a mixture of sodium chloride and organic acid it is possible for HCl to form by diffusion, as if the stronger mineral acid were driven out of its salt by the much weaker organic acid. It would, of course, not be difficult for any physical chemist to raise exception to this method of explanation. The author, however, has a constant feeling that more attention should be given to the matter; but further progress in the chemistry of colloids, particularly in the obscure field of the phenomena of adsorption, must doubtless be attained before the subject can be further clarified. Acid-production by Marine Snails. — In this place it is possible only in the briefest manner to remind the reader that the gastric production of acid among mam tu als is by no means an isolated phenomenon of this sort in nature. When Johannes Müller visited Messina in 1854 he saw with astonishment that a streak of fluid from the proboscis of the 12 GASTRIC DIGESTION OF PROTEINS large "partridge-shell" snail, Dolium galea, on coming in contact with the marble tiles with which the room was paved, caused an effervescence just as if some strong acid had been dropped upon the tile. Since then we have come to know that a number of marine snails secrete an acid salivary fluid, and that that of Dolium contains appreciable amounts of free sulphuric acid. 26 Determination of Acidity of Gastric Juice. — In any observation relative to acid secretion in the gastric juice the quantitative determination of the free hydrochloric acid is naturally a matter of much importance; some of the more definite advancements of method deserving brief mention at this time. A number of new features have been added to the old color reactions for the recognition of free hydrochloric acid, as the employment in modification of Günzberg's reagent of a mixture of p-oxybenzaldehyde with phloroglucin or of a mixture of dimethylamidobenzaldehyde with indol. It is of more importance, however, that we have come to recognize that estimation of the true acidity of the gastric juice, that is, its hydrogen ion concentration, is impossible by simple titration. For this we need either measurement of the electromotor power of the actual hydrogen-concentration chains 27 or the indicator method. In Müller 's method gastric juice is mixed with a solution of tropaeolin ; the fluid then assumes a color, which ranges ac- cording to the degree of acidity between a reddish brown and yellow, and by comparison with a color scale the percentage presence of "free hydrochloric acid" can be directly de- termined. 28 The method of Dreser 29 is based on quite a 26 Literature upon Acid Secretion in Gastropoda : O. v. Fürth, Ver gleichende chemische Physiologie der niederen Tiere, pp. 208-215, Jena, 1903 27 C. Foft, C. R. Soc. de Biol., 1905, I, 865; II, 2; F. Tangl, Pflüger's Arch. 115, 64, 1906. L. Michaelis and H. Davidsohn, Zeitschr. f. exper. Pathol. 8 398, 1910; J. Christiansen (Copenhagen), Deutsche Arch. f. klin. Med., 102 103, 1911. 28 A. Müller (Clinic of v. Noorden, Vienna), Med. Klinik, 1909, 1438. 28 H. Dreser, Hofmeister's Beitr., 8, 285, 1906. BINDING OF THE HYDROCHLORIC ACID 13 different principle. An excess of relatively insoluble barium oxalate is added to the gastric juice as "ground substance" and the quantity of oxalic acid thrown into solution by the hydrochloric acid is estimated by titration with potassium permanganate. In another test 30 the amount of iodine freed from a mixture of potassium iodide and potassium iodate in the presence of hydrochloric acid is determined by titration and used as measure of the latter. The test proposed by Holmgren 31 is particularly original; it is based upon the adsorption of acids in capillary media. If an acid solution is allowed to drop from a pipette upon a filter paper spread out horizontally it will be observed that, as the drop spreads out circularly, only the central part of the moist circle shows an acid reaction, the peripheral part containing nothing but water. The acid moieties are thus more freely adsorbed by the filter paper than the water molecules. And it has been shown that the more concentrated the acid the narrower the acid-free ring at the margin; and that the surface of the central circular acid area and that of the peripheral acid-free zone vary in relation to each other, according to the acidity of the solution employed. The outcome is strikingly regu- lar, and the results may be obtained almost at a glance if the paper used has been colored with litmus ; and the test, car- ried out in a very few moments, accords a fairly delicate esti- mate for practical purposes of the acidity of a sample of gastric juice. A very small quantity of the latter, about one-tenth of a cubic centimeter, serves for the determination. Binding of the Hydrochloric Acid by Protein Bodies. — There is no lack of methods, it is true, for the determina- tion of the free hydrochloric acid in the gastric juice ; but the question then comes up as to what can be the particular value of them at best. For it must be confessed that the importance of determining free hydrochloric acid, especially 30 M. Wegrumba (Berne), Internat. Beitr. z. Pathol, u. Ther. d. Ernäh- rungsstörungen, 3, 53, 1911. 81 J. Holmgren (Stockholm), Deutsche med. Wochenschr., 1911, 247. 14 GASTRIC DIGESTION OF PROTEINS for clinical purposes, has been very much overestimated. The actual facts may from a practical standpoint be stated without hesitation as follows : The free hydrochloric acid of the gastric juice in the presence of albuminous food is quickly linked either completely or partly to the proteins or their cleavage products. It should be kept in mind that proteins are such weak bases that their salts are open to extensive hydrolytic dissociation 32 — so much so that in dissolving them a part only of a given protein-salt remains as such, the remainder reverting at once to protein and free hydrochloric acid. The old belief was that the free acid alone had any important bearing upon the digestion, and it was for this reason that so much stress was laid upon its estimation. 33 It was doubted, for example, because the measurement of the concentration of hydrogen-ions in the infant's stomach showed only very low tension, whether the pepsin here could have as its chief function a digestive ac- tivity or whether it would not much more likely act as a lab ferment. 34 It seems important that, as Julius Schütz demonstrated, the presence of H-ions is not absolutely necessary for peptic digestion; the process begins at once in the presence of no more than a very small amount of combined acid (hydrochloric acid linked to albumin). Schütz believes that the principal purpose of the excessive (i.e., "free") hydrochloric acid is probably to protect the native proteins from loss of the hydrochloric acid linked to them which the proteid catabolic products arising in the course of digestion endeavor to abstract; that therefore it is no more than a reserve stock. From this point of view it is not the relative concentration but the absolute quantity of free acid present which should stand as a measure of the 33 Literature: O. Cohnheim, Nagel's Handb. d. Physiol., 2, 543-547, 1907. 8S Ci. A. Müller (I. Med. Clinic, Vienna), Deutsche Arch. f. klin. Med., 94, 27, 1908. 34 H. Davidsohn, Zeitschr. f. Kinderheilk., 2, 420, 1911. PROTEIN DIGESTION IN THE STOMACH 15 intensity of the digestive process. 35 Then, too, there exist very suggestive observations indicative of the impossibility of obtaining by distillation free hydrochloric acid from gas- tric juice during active digestion. 36 In contradistinction to this idea there are, of course, other authors who insist that the presence of free hydrochloric acid is absolutely essential for thorough utilization of the food. 37 Extent of Protein Digestion in the Stomach. — Let us now turn to the question as to the extent to which pro- tein digestion actually takes place in the stomach and the catabolic products which are normally produced there. This subject has been so well worked out that the actual facts of the matter can be stated very briefly, even though the litera- ture thereupon is extensive. 38 This is due primarily to the labors of Edgard Zunz, of Brussels, 39 conducted by him with much success through a long series of years, and also to the united work of London and Abderhalden 40 and their numer- ous collaborators, their studies combining happily the most advanced ideas of a finished fistula-technique with those of the chemistry of proteins. "The coagulated proteins of meat," to quote E. Zunz, writing about ten years ago, 41 "are in turn dissolved in the 35 J. Schütz, Wiener med. Woehenschr., 1906, Nos. 41 and 42; Biochem. Zeitschr., 22, 33, 1909; Arch. f. Verdauungskr., 11, 11, 1911; note Literature in latter article; cf., also, H. Jastrowitz (Siegfried's Lab.), Biochem. Zeitschr., 2, 157, 1906. 30 F. Landolph, Nouvelles etudes chimiques sur le sue gastrique : Buenos Aires, 1911; Zentralbl. f. Physiol., 25, 539, 1911. 37 Cf. G. Ewald (Med. Clinic, Erlangen), Deutsche Arch. f. klin. Med., 106, 498, 1912. 88 Literature upon the extent of gastric digestion: E. Zunz, Ergebn. d. Physiol., 5, 622-663, 1906; E. S. London, Handb. d. Biochemie, 3", 68-80, 1909. 39 E. Zunz, 1. c. and Bulletin de la Soci£te roy. des Sciences med. et natur. de Bruxelles, 1910, No. 3; Memoires couronnes et autres memoires publies par lAcad. roy. de med. de Belgique, 19, 3, 1906; 20, 1, 1908; Bull, de lAcad. roy. de med. de Belgique, 2Jf, 241, 1910; abstracted in Jahresb. f. Tierchem., 1^0, 371. 40 E. Abderhalden and E. S. London, together with K. Kautsch, L. Baumann, O. Prym, K. v. Körösy, C. Vögtlin, Zeitschr. f. physiol. Chem., 48, 549, 1906; 51, 383, 1907; 53, 147, 343, 1907. 41 E. Zunz, Hofmeister's Beitr., 3, 339, 1902. 16 GASTRIC DIGESTION OF PROTEINS stomach by the gastric juice, in which process there arise a very small proportion of acid-albumin, a large quantity of albumoses and a smaller quantity of more advanced diges- tive products (peptone, peptoids, and perhaps some of the crystallizable end-products). The portion which has been dissolved passes for the most part into the small intestine, where it rapidly undergoes further dissociation and resorp- tion. A small residue is resorbed directly in the stomach ; the advanced products of digestion are next to be absorbed, while albumoses are taken up with difficulty." The author has in a previous volume (Vol. I of this series, p. 77, et seq., The Chemistry of the Tissues) taken occasion to indicate the change of view which has arisen within the past decade in re- spect to the albumoses, which in strict parlance are no longer accepted. The schematic tables of the sequence of the various types of ' ' albumoses ' ' and ' ' peptones ' ' in the course of gastric digestion, on which so much time and labor used to be expended, have today become practically meaningless. But it is important, as established by Abderhalden and Lon- don,, that cleavage of aminoacids from the protein molecule scarcely occurs at all in the stomach, as, too, that synthetic Polypeptids supplied are not appreciably affected. 42 The velocity of solution depends upon the nature of the protein (for instance, gelatine is " digested" much more rapidly than serum-albumin or egg-albumin). The dissolved pro- teins of the gastric contents correspond for the most part to the old definition of ' ' albumoses. ' ' Another important mat- ter is the rapidity of passage of the dissolved material in the stomach into the intestine. It has been long known that the degree of coagulation of the proteins is here an impor- tant matter and that it makes decided difference whether they are ingested raw or cooked. There are, moreover, other items not well understood. For example, while raw egg-albumin has left a dog's stomach, for the most part 42 E. Abderhalden and E. S. London, 1. c. PROTEIN DIGESTION IN THE STOMACH 17 changed, within but little more than an hour, raw meat is retained for considerably longer time. 43 The resorption of the products of protein cleavage in the stomach is always of very minor importance. Meat and albumin leave the stomach of the dog, according to London, without having undergone any notable nitrogenous resorp- tion. Moreover, the products of protein catabolism, which are readily absorbed in the intestine, can be quantitatively recovered if in the stomach even for a number of hours. (It is true, however, that Salaskin, in agreement with the older views, insists upon the actual absorption of protein sub- stances in the stomach.) 44 In this connection the reduced ability of proteins which have been acted upon by pepsin and hydrochloric acid to withstand the subsequent action of trypsin, as pointed out by Carl Oppenheimer and Aron 45 and by Emil Fischer and Abderhalden 46 has important physiological bearing. As the gastric function in protein digestion therefore is in a general way a preparatoiy one it cannot be regarded as a very wonderful thing that it has been found possible to perform complete gastrectomy in animals and in man, since the well-known experiments of Karl Ludwig and Ogata and the actual total extirpation of the human stomach by Czerny. 47 As to the extent of resorption, opinions vary widely. While London, as above stated, regards it as of little conse- quence, Scheunert 48 accepts for the stomach a fixed power of absorption as proved (basing his opinion upon the observa- 18 E. S. London, with W. Polowzowa, A. Th. Sulima, C. Schwarz, Zeitschr. f. physiol. Chemie., 46, 209, 1905; 49, 328, 1906; 68, 378, 1910. **S. Salaskin, Zeitschr. f. physiol. Chem., 51, 167, 1907. " C. Oppenheimer and H. Aron, Hofmeister's Beitr., 4, 279, 1903. " E. Fischer and E. Abderhalden, Zeitschr. f. physiol. Chem., 40, 215, 1903. *' M. Ogata, G. Carvallo and V. Pachon, Langenbuch, C. Schlatter ; also A. Carrel, G. M. Meyer and P. A. Levene, Amer. Jour, of Physiol., 26, 369, 1910; E. S. London and W. F. Dagaew, Zeitschr. f. physiol. Chem., 14, 330, 1911. "A. Scheunert, Zeitschr. f. physiol. Chem., 51, 519, 1907. 2 18 GASTRIC DIGESTION OF PROTEINS tions of Lang and Tobler, as well as his own, upon horses and dogs). Comparative Physiological Considerations. — Our view of physiological processes of any sort is bound to be un- duly warped and limited if we confine ourselves to human beings and the ordinary laboratory experiment ani- mals. Nothing less than a consideration of all classes of life forms can assure a thoroughly scientific basis. William Biedermann 49 by the monumental work in which he has collected and critically dealt with the whole mass of material bearing on digestion from the standpoint of comparative physiology, has certainly earned a claim to general gratitude from all who are concerned with biological studies. The author feels that at least a few brief considerations should be given to this side of the subject, restricting himself, how- ever, to the vertebrates because he has elsewhere dealt with the chemistry of digestion in invertebrates. 50 In the first place, among fishes 51 there occur forms with- out stomachs (among others, amphioxus, the cyclostomata and cyprinoids). In the cyprinoid fishes the opening of the gall duct lies just back of the oesophageal opening into the intestine; and, of course, it is impossible that in such case there could be the least trace of gastric digestion in its ordinary meaning. Most fishes, however, possess a stom- ach, the glands of which secrete an enzyme capable of digest- ing protein substance in an acid reaction. The most exact work in this connection has been done upon the selachians. There is divergence of opinion as to the existence of free hydrochloric acid in the gastric juice of sharks (recently 49 W. Biedermann, Handb. d. vergl. Physiol., H. Winterstein, 2' (Die Aufnahme, Verarbeitung und Assimilation der Nahrung, pp. 1563, Jena, 1911). 60 O. v. Fürth, Vergleichende chemische Physiologie der niederen Tiere, pp. 140-330, Jena, 1903. 61 Literature upon gastric digestion in fish : A. Scheunert, Handb. d. Biochem., 3", 163-167, 1909; W. Biedermann, 1. c, pp. 1088-1106; cf. also D. D. Van Slyke and G. F. White, Journ. of Biol. Chem., 9, 209, 1911. PHYSIOLOGICAL CONSIDERATIONS 19 discussed by E. Weinland and by M. van Herwerden) 52 but for that matter the entire question is of lessened importance, since we have come to recognize the positive influence of com- bined hydrochloric acid in peptic digestion (v. sup.). Be- sides, the pepsin of fishes apparently is not entirely identical with that of the mammalia. Gastric digestion in amphibia, reptiles and birds 53 cor- responds doubtless with the peptic type. In graminivorous birds, whose food is first acted upon in a crop, the peptic catabolism of protein may begin even in this organ. From the point of systematic investigation of the com- parative physiology of digestion in mammals 54 Ellenberger and Scheunert, scientists in the Veterinary High School in Dresden, have rendered most important service. In the ruminants, with three complex proventricles preceding the glandular stomach, it goes without saying that complicated processes of digestion are met. The hamster with its two- chambered stomach presents interesting features, the first lined with a cutaneous type of mucous membrane and con- nected by a very narrow opening with the glandular stomach proper, thus occupying an intermediate position between the many-chambered stomach of the ruminants and the single- chambered stomach of the solipeds, the hog and other mammals. The digestion of the food proteins by the gastric juice takes place only in the gland-bearing stomach, while 52 M. van Herwerden (Physiol. Institut., Utrecht), Zeitschr. f. physiol. Chem., 56, 453, 1908; E. Weinland, Zeitschr. f. Biol., 55, 58, 1911. "Literature upon gastric digestion in Amphibia, Reptiles and Birds: A. Scheunert, Handb. d. Biochem., 3", 168-170, 1909; W. Biedermann, 1. c, pp. 1272-1281. w Literature upon the comparative physiology of gastric digestion in Mammalia: A. Scheunert, Handb. d. Biochem., 3", 153-162, 1909; W. Bieder- mann, 1. c, pp. 1299-1311; W. Ellenberger and A. Scheunert, Lehrb. d. vergl. Physiol., Berlin, P. Parey, 1910; A. Scheunert, Vergleichende Studien über den Eiweiszabbau im Magen, O. Wallach, Festchrift, Göttingen, 1909; and Pflüger's Arch., 109, 145, 1905, and 139, 131, 1911; A. Scheunert and E. Rosenfeld, Deutsche tierärztl. Wochenschr., 11, No. 25; A. Scheunert. and W. Grimmer, Zeitschr. f. physiol. Chem., 58, 27, 1906; E. Rosenfeld, Inaug. Diss., Leipzig, 1908; A. Schattke, Inaug. Diss., Dresden, 1909; F. Bengen and G. Haane, Pflüger's Arch., 106, 267, 286, 1905. 20 GASTRIC DIGESTION OF PROTEINS in the proventricle processes of bacterial decomposition of the proteins may be in play. However, even in animals with simple stomachs the details of the process may show vari- ations. For example, in the dog, as above mentioned, the proteid catabolic products arising in the stomach consist for the most part of albumoses ; while in the horse the quantity of albumoses is never marked enough to permit their pre- dominance over syntonins, peptones and abiuret products. At this point we may take up at least briefly a considera- tion of the digestive ferment of the stomach, pepsin.™ Efforts to Isolate Pepsin. — A great deal of effort has been expended in attempts to isolate pepsin, 56 and an apparently albumin-free ferment has a number of times been obtained, although usually it is thrown down along with other precipi- tates of extremely varied types. Thus in Hofmeister 's labo- ratory 57 a pepsin, "albumin-free" in the ordinary sense, has been produced by expression by aBuchner press from gastric mucous membrane ground up with infusorial earth, nitration of the fluid through a Chamberland filter, subsequent dialysa- tion (Brücke's method), and displaced with an alcohol-ether solution of Cholesterin. The pepsin adheres to the separat- ing Cholesterin precipitate. By suspending the latter in water and removing the Cholesterin with ether, there re- mains a clear fluid with active digestive power failing to show protein reaction or lab activity. These occasionally recurring efforts to produce "analytically pure" pepsin are growing more and more infrequent, as it is gradually being appreciated that at best it is a fruitless labor. Even if in the end, after much care and effort, a ferment is obtained so thor- oughly isolated that a protein reaction can no longer be 66 Literature upon pepsin : A. Cohnheim, Nagel' s Handb. d. Physiol., 2, 548-552, 1907; F. Samuely, Handb. d. Biochem., 1, 546, 1909; A. Bickel, ibid., 8\ 100, 1910; C. Oppenheimer, Fermente, III. Aufl., 256-280, 1910; W. Bieder- mann, 2, I. Hälfte/ 1257-1264, 1911. 68 Recent experiments of Sundberg, Sjöquist, Mrs. Schoumow-Simonowsky, Friedenthal, Pekelharing, Schrumpf and others. " P. S'cbrurnpf, Hofmeister's Beitr,, 6, 396, 1905. METHODS FOR ESTIMATION OF PEPSIN 21 obtained there is bound to remain the objection that this means nothing more than that enzyme action may continue in degrees of dilution in which the most delicate reactions fail to show protein. It is utterly impossible for any man at the present time to say whether the ferments are of a proteid nature or not. When a French physiologist gave it as his opinion that ferments are in no sense material, but energy alone, his statement called forth vehement opposi- tion. To-day, however, when a slow but sure change is taking place in our fundamental ideas about atoms, and the very basis of our older conception of nature, the duality of matter and force, is decidedly wavering, such heretical theories would scarcely excite anything like the same indignation. Methods for Estimation of Pepsin. — Numerous methods have been applied to the quantitative determination of pepsin. Grützner estimates the amount of coloring mat- ter passing into solution in the digestion of fibrin stained with carmine, using a wedge-colorimeter. 58 In Mett's method the length of a column of coagulated albumin enclosed in a fine glass tube is measured after a fixed period of digestion. Hammerschlag estimates by precipitation with Esbach's reagent the amount of undigested protein remaining after digestion of a known albumin solution for a certain fixed time. Spriggs tries to draw a conclusion from the loss of viscosity of a protein solution as to the progress of its digestion. Vollhard begins with a known solution of casein ; throws out the undigested casein by sodium sulphate, and estimates the amount of digested protein in the filtrate by its alkali-binding power determinable by titration : the more advanced the digestion the more potassium or sodium hydrate required. E. Fuld arranges a series of small beakers each containing the same quantity of a hydrochloric acid solution of a pure protein, edestin, adding graduated quantities of the solution whose peptic quantity is to be 88 P. v. Grützner (Tübingen), Pflüger's Arch., IM, 545, 1912. 22 GASTRIC DIGESTION OF PROTEINS determined; after appropriate time for digestion sodium chloride is added to all of the samples, and the result esti- mated by the degree of turbidity ensuing (absent in fully digested specimens). M. Jacoby and Solms substitute ricin for edestin; Gross casein; and Eose protein (globulin) of garden-pea in modification of the latter method. 59 Law of Pepsin Fermentation. — Based upon these methods it has been thought possible to establish a " fer- mentation law" for the activity of pepsin. The Schiitz- Borissow rule, making the effectiveness of the pepsin proportionate to the fourth root of its quantity, has at- tained considerable popularity and has been very much discussed. 60 Without going into the details of ferment activity it may be stated that P. v. Grützner 61 has concluded from his careful and critical studies in reference to pepsin and trypsin ' ' that there is no uniform law prevailing during the entire course of a process. ... In the processes of digestive fermentation at the beginning one rule prevails, which is not the same for the intermediate stage or for the terminal part. Moreover, as stated, the absolute and rela- tive quantity of a ferment will cause variation of the law and it is impossible to speak of a law in the usual meaning of the term for any one ferment. ' ' Propepsin. — Pepsin does not exist in completed state in the glands of the gastric mucosa, as shown by the studies of Ebstein and Grützner and those of Langley, but in the form of a proferment which (in contrast to pepsin, which is very sensitive to alkalies) is resistant to alkali, and immediately changes into pepsin when brought in contact with hydro- 58 Cf. critique of methods, P. v. Grützner (Festschr. f. G. Fano), Arch, di Fisiol., 7, 223, 1909; E. Henrotin, Ann. de la Soc. roy. des Sciences mod. et natur. de Bruxelles, 18, fasc, 2, 1908; W. C. Rose, Arclr. of Intern. Med., 5, 459, 1910; A. Fubini, Gaz. Osped., 30, 409, 1909; P. W. Cobb (Cleveland), Amer. Journ. of Physiol., 13, 448, 1905. 60 Cf. J. Schütz (Laborat. Hofmeister), Zeitschr. f. physiol. Chem., 30, 1, 1900; Reichel, Wiener klin. Wochenschr., 1908, No. 30. 64 P. v. Grützner, Pfliiger's Arch., l.' f l, 115, 1911. RESISTANCE OF STOMACH TO AUTODIGESTION 23 chloric acid. Glässner 62 has succeeded in separating pro- pepsin from labproferment by precipitation with uranyl acetate. Psendopepsin. — The existence of a pseudo pepsin, ob- tained from the pyloric mucous membrane by the investi- gator mentioned, said to act under weakly alkaline reaction and to split protein with production of tryptophane, is a matter of considerable doubt; as in the first place it can scarcely be distinguished from autolytic tissue enzymes and, as is well known, the entrance of trypsin into the pylorus is not an infrequent occurrence. 63 Passage of Pepsin into the Intestinal Canal. — Abder- halden has made interesting observations showing the marked readiness with which pepsin is taken up by elastin and similar substances. The pepsin may be practi- cally all removed from the gastric juice by means of elastin. Protected within such albuminoids the enzyme may be car- ried into the intestine and there complete its action. Con- siderable amounts of active pepsin have been detected in the upper portion of the intestine by the elastin method ; and apparently peptic digestion is not limited entirely to the stomach, but plays an important role in the intestine as well. 64 Resistance of Stomach to Autodigestion. — The physi- ology of gastric digestion includes another problem which has long stimulated investigation in a marked degree. This concerns the method of protection of the digestive organs against self-digestion. Every thoughtful person who has ever noticed the rapidity with which a bit of albumin is digested by active gastric juice, has necessarily asked him- °K. Glässner, Hofmeister's Beitr., 1, 24, 1901. "* Cf. the publications of F. Klug, A. Scheunert and Grimmer, Pekelharing (cited in Handb. d. Biochem., 1, 551, 1909) and F. Reach (Hofmeister's Beitr., h, 143, 1903). ** E. Abderhalden in association with F. W. Strauch, F. Wachsmuth, O. Meyer and K. Kiesewetter, Zeitschr. f. physiol. Chem., 71, 314, 339, 1911; H, 67, 411, 1911. 24 GASTRIC DIGESTION OF PROTEINS self how it is that the mucosa of the living human and animal stomach is able to withstand the peptic action of the gastric juice. As early as in the eighteenth century Hunter was searching for an answer to this question ; and ever since the great Claude Bernard interested himself in it it has never been dropped from the list of the day's work awaiting the physiologist. 65 The author has in an earlier volume taken up the discus- sion of anti-ferments (in the first volume of this series, p. 559, Chemistry of the Tissues). Following A. Danilewski's demonstration of the existence of an antipepsin in the lining of the stomach and that of Ernst Weinland 66 proving the resistance of parasitic worms to the gastric and intestinal ferments from this standpoint, numerous investigations have been made upon the subject. A diffusible agent has been found in the gastric juice which withstands heating, is re- sistant to acids and alkalies and is capable of inhibiting peptic activity 67 ; but it should be remembered that blood serum freed of diffusible material by dialysis may also show antipeptic activities. 68 0. Schwarz, working in Hofmeister 's laboratory, found after heating pepsin solutions to above 60° C. a substance capable of inhibiting peptic activity which apparently had been in the solutions before subjection to heat and which was not formed directly from the pepsin itself. 69 Whether any of these contributions, however, have a physiological bearing seems decidedly questionable. For example, knowing that pepsin can be easily separated from a solution by animal charcoal one might hesitate to 65 Literature upon the protection of the stomach and intestine from auto- digestion: CI. Fermi (S'assari), Centralbl. f. Bakteriol., 56, 55, 1910, and Arch, di farmacol. sperim., 10, 449, 1911; cf., Centralb. f. d. ges. Biol., 13, No. 369, 1912; C. Oppenheimer, Fermente, 3d ed., 270-271, 1910. 66 E. Weinland (Physiol. Inst., Munich), Zeitschr. f. Biol., U, 1, 45, 1902. " L. Blum and E. Fuld, Zeitschr. f . klin. Med., 58, 5-6, 1906. M M. Jacoby, Biochem. Zeitschr., 2, 247, 1906. 89 O. Schwarz (Physiol. Chem. Institut., Strassburg), Hofmeister's Beitr., 6, 524, 1905. AUTODIGESTION 25 think that the prevention of the action of a gastric juice after addition of gastric epithelial cells is due solely to "anti- pepsin." 70 The following experiment by E. S. May in the laboratory of Rene du Bois-Reyniond 71 is instructive. The serosa and mucosa of a dog's stomach were separately prepared and spread out over glass plates, and placed in gastric juice. After eighteen hours the mucosa was not changed ; but the serosa was markedly acted upon, at one place with complete penetration. The question has been approached from another stand- point, too, by introducing living tissue into the opened stomach of an animal. Although these experiments have not been productive of uniform results (a living spleen with its blood vessels attached has been digested quite rapidly), we must accept the fact that, for example, the foot of a live frog introduced into the stomach of another frog, or perhaps a loop of living intestine placed into an incised stomach, may remain intact for many hours. 72 On the other hand, very active solutions of trypsin have been known to digest living tissue (tails of rats and mice). 73 In conclusion mention may be made of such observations as those of Claudio Fermi showing that many aquatic ani- mals (protozoa, worms, crustaceans, insects) may live with- out the least harm in trypsin-solutions. 74 A solution of trypsin, capable of rapidly digesting a large lump of coagulated albumin, has been found to be without any effect upon the tiny mass of protoplasm of an infusorian, unpro- 10 R. duBois-Reyniond, Berl. physiol. Ges., July 21, 1911, Centralbl. f. Physiol., 25, 774, 1911. T1 1. c. " Neumann, Centralbl. f . allg. Pathol., 18, 1, 1907 ; Kathe, Berliner klin. Wochenschr., 1908, 2135; Katzenstein, ibid., 1749; G. Hotz, Mitt. a. d. Grenzgebieten d. Med. u. Chir., 21, 143, 1909; R. du Bois-Reymond, 1. c. "L. Kirchheim (Labor. M. Cremer, Cologne), Arch. f. exper. Pathol., 26, 352, 1911. 71 CI. Fermi, 1. c. 26 GASTRIC DIGESTION OF PROTEINS tected by any tegumentary covering, after as much as a month's exposure. The Italian author just mentioned has concluded therefore that the living cell protects itself against the digestive enzymes of the stomach, intestine, and pan- creas, not by antienzymes, nor by protective coverings, nor yet by any special sort of impermeability, but much more likely by the resistive ability of the whole living cell itself. The simple reply to the question why the living cell is not attacked is therefore this: "Because it is alive." With such an answer the problem has circled back to exactly the same point where Hunter started one hundred and forty years ago. It is to be hoped that perhaps the next century and a half will really accomplish something in the way of progress. Origin of the Round Ulcer of the Stomach. — In a limited way the question as to the mode of origin of the round ulcer of the stomach is related with that of gas- tric autodigestion. Time after time explanations for this lesion have proposed some local circulatory disturbance in a given area of the mucosa (from a spasm of the vessels, thrombosis or hemorrhage) with autolysis of the portion involved. 75 We have been able repeatedly, too, to produce gastric ulcers experimentally in animals, as by injection of diphtheria toxin, 76 gastrotoxic serum, 77 by feeding bouillon cultures of bacterium coli, 78 by repeated serum injections 79 (as one of the features of anaphylaxis), as well as by the poison of the Gila monster (Heloderma suspectum). 80 The last mentioned experiments (conducted under Leo Loeb) 75 Cf. the extensive material of R. Beneke, Verb. d. Deutsche pathol. Ges., Kiel, 1908, 284. '"Rosenau and Anderson, Journ. Infect. Diseases, 4, 1, 1907. 77 M. J. Bolton, Proc. Roy. Soc, 1905-06, Series B, 77 ; 1909, Series B, 79, cited by Rehfuss, v. infra. T. B. Turck, Journ. Amer. Med. Assoc, 1906, 1753, cited by Rosenau and Anderson, v. sup. 79 Gay and Southard, Journ. of Med. Research, 190S, cited by Rehfuss, v. infra. 80 M. E. Rehfuss, University of Pennsylvania Medical Bulletin, 22, 105, 1909. CHYMOSIN 27 suggest that ' the hemorrhages in the mucosa as well as thromboses may not be primary lesions in the production of ulcers, but rather secondary to and produced by the ulceration. Chymosin. — A short review of the problem of the lab- coagulation will serve to conclude the present lecture. It may be confidently asserted that the question of the rennet-ferment or chymosin is the oldest physiological- chemical problem which has concerned mankind. For, many, many thousands of years ago, when humanity was not interested in observation of nature and in contemplations about natural philosophy, the nomadic herdmen were well acquainted with the use of the gastric mucous membrane in curdling milk. A mere glance over the extremely extensive literature dealing with the lab process gives one the impression of an impenetrable thicket of apparently diametrically opposed observations and theories, just as in the study of blood coagulation. Starting with the observations of Hammersten many attempts have been made to outline the lab-process as a double-phased one in which the bulk of the casein is first changed into paracasein, which upon the addition of a suf- ficient amount of calcium salts is thrown down as the almost insoluble calcium paracaseinate or cheese, a small part of the protein remaining, however, in solution as an albumose- like whey-albumin from a coincident splitting process. This simple formulation has not entirely harmonized, however, with all the experimental findings of a large group of investi- gators who have been interested in the subject, relating to ferment-kinetics, the physical-chemical characteristics of milk, the inhibition and stimulation of the lab-process by various agents, to parachymosin and prochymosin, the part played by calcium salts, antiferments, and other comparable 28 GASTRIC DIGESTION OF PROTEINS factors. 81 The many contradictions are not to be wondered at when we remember that the lab-coagulation is really a complex process. Ivar Bang concludes his exhaustive in- vestigations upon the subject with the opinion that the calcium salts of the milk are distributed among the organic and inorganic acids, the lactalbumin, lactoglobulin and casein; that the casein by virtue of its acid nature combines with the whole group of milk bases; that there is not one paracasein alone but that there are formed, long before coagulation is apparent, a number of different paracaseins with variable but increasing affinity for calcium phosphate, and that at a certain stage these compounds separate from solution and coagulation takes place. 82 Ultramicroscopic Studies of Lab-process. — Kreidl and Neumann 83 have been able to explain very satisfactorily by direct ultramicroscopic observation a number of features (especially that involved in the differences be- tween cow's milk and human milk) which have more than once been regarded as due to variation in the degree of solu- tion of the casein in the milk. In the milk of different species of animals there arevisible minute bodies (lactokonids), pos- sibly identical with suspended casein or caseinated calcium. "It has been shown by ultramicroscopic study of milk of various animals that in all kinds of milk except human there may be found in addition to the fat globules great num- bers of a second corpuscular element. The plasmatic space 81 M. Arthus, J. Bang, G. Becker, L. Blum, E. Fuld, M. van Herwerden, S. G. Hedin, H. Köttlitz, S. Löwenhart, E. Laqueur, L. Morgenroth, L. Pinkus- sohn, E. Petry, C. Pages, H. Reichel, K. Spiro, W. Sawjalow, B. Slowzoff, S. Schmidt-Nielsen, G. Warneken, J. Wohlgemuth and many others. Literature upon the Lab-process and the Proteins of Milk: E. Fuld, Ergebn. d. Physiol., 1, 408-504, 1902; R. W. Raudnitz, ibid., 2, 193-251, 1903; E. Laqueur, Biochem. Centralbl., 4, 318, 1905; F. Samuely, Handb. d. Biochem., 1, 567-570, 1909; C. Oppenheimer, Fermente, 3d ed., 286-312, 1909; A. Schlossmann and S. Engel, Handb. d. Biochem., 3', 405-432, 1910. 82 1. Bang (Lund), Skandin. Arch. f. Physiol., 25, 105, 1911. 83 A. Kreidl and A. Neumann (Physiol. Instit., Univ. of Vienna), Sitzungs- bericht d. Wiener Akad. Mathem-Naturwiss., kl., 117'", March, 1908; cf. also Centralbl. f. Physiol., 22, 133, 1908, Pfliiger's Arch., 128, 523, 1908. BACTERIAL INVOLVEMENT IN LAB-PROCESS 29 between the fat globules is full of very minute particles in active molecular movement, often in such great numbers that nothing of the otherwise dark looking plasma is to be seen, and the whole field of vision is apparently filled by a dancing mass in which the fat globules lie embedded. . . . Fresh human milk may be distinguished from this picture at the first glance. In the human milk the plasmatic field looks black. . . . If a preparation be made of a milk with lab- ferment added it will be seen that at first the particles collect into minute groups recognizable as composed of the lacto- konids ; the smaller aggregates then unite into larger ones, the latter entangling the fat globules and finally sinking to the bottom of the container. . . . Coincidently in the tube from which the preparation was taken coagulation can be grossly seen." From this one would picture the lab coagulation as a process characterized by the gradual clumping of suspended colloidal particles, the individual phases of which are con- stantly intermingling and therefore beyond any chance of schematic arrangement in chemistry. Here, just as in case of the process of blood coagulation, one cannot but feel that too much importance has been assigned to the details of a separation process which is an expression of disturbance of equilibrium, and that too at tremendous expense of effort and ingenuity. An architect who is seeking information about the collapse of a building is, of course, particularly anxious to discover the cause of the collapse. But he would scarcely waste any great amount of energy in finding out in detail whether at the moment of the fall a certain gable or a certain arch had given way a little earlier or later or a little to one side or to the other. Bacterial Involvement in Lab-process. — Recent observa- tions have been made by Kreidl and Lenk 84 which deserve "A. Kreidl and E. Lenk, Biocliem. Zeitschr., 36, 357, 1911. 30 GASTRIC DIGESTION OF PROTEINS notice, bearing upon the participation of a hitherto neglected factor, that of bacterial infection, in the process of lab- coagulation. They point out that sterile milk will not coagulate in sterile vessels if treated with sterile rennin; but merely touching the milk with the non-sterile finger or the addition of a few drops of ordinary milk will cause curding. Much thought has been devoted to the formulation of an acceptable conception of the physiological purpose of the lab-process. That an eventual change of the milk intro- duced into the stomach into a firm curd, so that this can pass into the intestine only bit by bit, gradually, thus protecting the intestine against being flooded with food-proteins, and that this in many animals is of importance in the nourish- ment of the young, — all this goes without question. There is reason to question, however, whether the greatest value of the rennin, which is found widely distributed in the digestive organs of vertebrates, many invertebrates and, too, in the juices of many plants, 85 should not be sought for in an alto- gether different direction. Plastins. — Observations of A. Danilewski and his numer- ous students 86 that rennin (and also extracts of intestine and of pancreas) gives rise to precipitation in solutions of products of peptic digestion suggest that these precipitates may be considered as an expression of a synthetic fermenta- tion, possibly directed toward the regeneration of the split protein. But the attractive prospect that in plastins we are seeing intermediate products of a fermentative protein synthesis, has unfortunately all too soon met the fate of the most of the brilliant expectations of this world. Question of the Identity of Pepsin and Rennin. — Obser- vations of this kind (mainly from Pawlow and his 85 W. Biedermann, Handb. d. vergleich. Physiol., 2', 1289, 1911. ""Kurajeff, Lawrow, Lukomnik, Nürnberg, Okunew, Salaskin, Sawjalow, Schapirow and others. Cf. also R. 0. Herzog, Zeitschr. f. physiol. Chem., 39, 305, 1903; H. Bayer (Hofmeister's Laborat.), Hofmeister's Beitr., If, 554, 1903. INDENTITY OF PEPSIN AND RENNIN 31 school) have developed the idea that pepsin and rennin are fundamentally identical ; that the lab-action is nothing else than the reversed synthetizing manifestation of the same ferment which normally behaves as a peptic protein cleav- age-enzyme. This view undoubtedly is an attractive one, and meets our demand for simplification of our concepts of complicated natural phenomena. The great interest mani- fested in this view is probably explicable on this basis. An unusually large number of comprehensive researches have been devoted within recent years to this question of the identity of pepsin and lab-ferment. Pawlow's many associ- ates 87 are constantly directing attention to the distinct parallelism between the action of pepsin and lab. They have been unable to separate the two ferments either by dif- fusion, 88 filtration 89 or by electric conduction. 90 Hammer- sten and very many other authors, 91 on the other hand, have unquestionably produced in different ways solutions of pepsin which no longer coagulate milk, and, too, lab solutions which no longer manifest any peptic ability. An American writer 92 has demonstrated that on passing an electric cur- rent under certain conditions through a fluid containing both rennin and pepsin the latter will be completely destroyed while the lab remains unchanged. The physiologist Duc- w Cf. J. W. A. Gewin (Physiol. Instit., Utrecht.), Zeitschr. f. physiol. Chem., 54, 32, 1907; W. Sawitsch, ibid., 55, 84, 1908; W. van Dam, ibid., 64, 316, 1910; Th. J. Migay and W. Sawitsch, ibid., 63, 405, 1909; W. Sawitsch, ibid., 68, 13, 1910. 88 R. O. Herzog (Karlsruhe), Zeitschr. f. physiol. Chem., 60, 306, 1909. M C. Funk and A. Niemann, Zeitschr. f. physiol. Chem., 68, 263, 1910. 90 C. A. Pekelharing and W. E. Ringer, Zeitschr. f. physiol. Chem., 7.5, 282, 1911. 91 O. Hammersten, Zeitschr. f. physiol. Chem., 56, 18, 1908; 68, 119, 1910; 74, 142, 1911; S. Schmidt-Nielsen ( Hammersten's Laboratory), ibid., 68, 92, 1906; A. Rakoczy, ibid., 68, 421, 1910; J. F. B. van Hasselt, ibid., 10, 171, 1910; A. E. Porter, Journ. of Physiol., 42, 389, 1911; L. Blum and W. Böhme, Hofmeister's Beitr., 9, 74, 1906; A. E. Taylor, Journ. of Biol. Chem., 5, 399, 1909: A. Rakoczy (Kiew), Zeitschr. f. physiol. Chem., 68, 421, 1910. 92 W. E. Bürge (Johns Hopkins Univ.), Amer. Journ. of Physiol., 29, 330, 1912. 32 GASTRIC DIGESTION OF PROTEINS ceschi, 93 working in Argentina, has shown that pepsin but not chymosin is to be found in the stomach of certain mar- supials (Didelphis) . In view of positive evidence like this, indicating the duality of pepsin and rennin, the frequent failure to establish such differentiation cannot, in the opinion of the author, be held as contradictory ; positive results are of more significance, it seems to the author, than negative ones in such a matter. Identity between pepsin and rennin for the present at least certainly does not seem to be proved. As for the frequently expressed suggestion that the two ferments may not be precisely identical but different ' ' sides ' ' of one single ferment, slightly different sidechains of one " giant enzyme molecule," the author confesses his complete ignorance, not knowing how to proceed in attempting to make an exact differentiation between different enzymes and " different sidechains of some one ferment." To use the expression of a distinguished jurist, the author is not enough of an expert in this matter to see even darkly. 83 V. Ducceschi (Cordova), Arch, di Fisiol., 5, 413, 1908. CHAPTER II THE PROTEOLYTIC PANCREATIC FERMENT Transfer of the Food from the Stomach into the Intes- tine. — The previous chapter having been devoted to the process of protein digestion in the stomach, the subject of the method of transfer of the food from the stomach into the intestine may with propriety be next considered. This process has been approached experimentally in a variety of ways, as by the production of gastric and duo- denal fistulas, by rontgenoscopy of the stomach after intro- ducing bismuth nitrate into the food ingested, by tube analysis of the gastric contents, etc. Hofmeister and Schütz studied by direct observation the movements of a living exsected stomach kept in a moist chamber ; P. v. Grützner examined frozen sections of stomachs of animals killed in different stages of digestion ; Scheunert followed the move- ments and partition of the gastric contents by the special means of administering colored food, etc. Although it is im- possible to go at length into the subject the attention of the reader should be directed at least to the importance of chem- ical regulation of the reflex pylorus closure from the intes- tine; our knowledge of which is mainly due to the recent studies of Pawlow, Moritz, Cannon, London, Cohnheim, Tob- ler and others. 1 We know from observation that a dog with a duodenal fistula discharges the water through the fistula 1 Literature upon the Passage of the Food from the Stomach into the Intestine: 0. Cohnheim, Nagel's Handb. d. Physiol., 2, 560-568, 1907; Physiol, d. Verd. u. Ernähr., pp. 13-26, 1908; E. S. London, Handb. d. Biochem., 8" 68- 73, 1909; 0. Cohnheim and F. Marchand, Zeitschr. f. Physiol. Chem., 63, 41, 1909; F. Best and 0. Cohnheim, ibid., 69, 113, 1910; Sitzungsber. d. Heidelber- ger Akadamie, 1910; W. B. Cannon (Harvard Medical School), Amer. Jour, of Physiol., 20, 283, 1900; cf. also F. Meyer (Kissingen), Zeitschr. f. physiol. Chem., 11, 466, 1911; M. Kirschner and E. Mangold (Greifswald), Mitteil. a. d. Grenzgebieten d. Medizin u. Chir., 23, 446, 1911; R. Kaufmann and R. Kienböck, Med. Klinik, 1911, 1150. 3 33 34 PROTEOLYTIC PANCREATIC FERMENT about as it is drunk, quite naturally suggesting comparison with Baron Münchhausen's well known horse, with all its drinking merely pouring out the water from the cut which has severed away the posterior half of its body. But if, instead of water, acid gastric contents come in contact with the duodenal mucous membrane, there at once occurs a reflex closure of the pylorus. In this way the intestine is protected from further flooding with acid gastric material. At the same time entrance of alkaline digestive fluids (the bile, pancreatic juice and intestinal juice) into the duodenum is brought about by the acid material, these juices neutralizing the acid. As soon as the pylorus opens again another charge of acid is expelled into the duodenum ; and the same perform- ance automatically repeats itself. According to Cannon reflex relaxation is induced, directly by contact of the acid chyme upon the pars pylorica. Besides the influence of acid from the intestine, closure of the plorus may be caused by fat; and according to the studies of 0. Cohn- heim and his associates it is not a matter of consequence where the oil or acid is placed in the small intestine. From the fact that cocainizing the intestine prevents the contrac- tion the reflex nature of the act cannot be in doubt. There are, too, in all probability a number of other factors involved in the regular discharge of the gastric contents besides the hydrochloric acid and the fat of the chyme, especially the consistence of the food. Passage of the Intestinal Contents into the Stomach. — In contrast, a matter of physiological interest, first brought to attention within the past few years, is that of the entrance of intestinal contents into the stomach, observed by Pawlow and Boldyreff. After the introduction of fatty foods particu- larly, but sometimes also after fasting, and, too, when there is an especially high degree of gastric acidity, it may happen that a mixture of intestinal secretion, pancreatic juice and bile regurgitates into the stomach to such an extent that the hydrochloric acid of the stomach may be neutralized and PANCREATIC FISTULAS 35 peptic digestion interfered with. Abderhalden and his as- sociates have shown that the splitting of a dipeptid, glycyl- 1-tyrosin, which is not produced normally in the stomach, can in these cases be detected in the gastric juice by polarimetry (after neutralizing the acidity of the gastric juice by sodium bicarbonate). It has been proposed to base a method for obtaining the human pancreatic juice for diagnostic pur- poses upon this fact. In application an "oil test-break- fast," consisting of about 200 ccm. of olive oil, containing free oleic acid, is introduced by a stomach-tube after neu- tralization of the gastric acidity by alkali. The gastric con- tent withdrawn after a half hour is readily freed from the oil and often is found stained greenish by bile and may show the presence of trypsin. Whether the method, for which great expectations were entertained, will prove of actual value in diagnosis must for the present be held in judgment. 2 Pancreatic Fistulas. — As is well known, the chyme having passed into the intestine becomes mixed with the secretion of the pancreatic gland, to the secretory process of which organ attention should next be called. Although Claude Bernard had introduced canulas into the excretory duct of the pan- creas and had endeavored to thus study the method of secre- tion of this important organ, for a long time progress was not satisfactory, simply because there was too great a departure from the normal physiological conditions arising from the severity of the operative procedure. Very often after such a ' ' temporary" fistula has been made, even if the experimental animal be in the height of digestion, but little or no secre- tion can be obtained. Here again the incomparable Eussian physiologist, Pawlow, 3 was the first to overcome 2 Literature upon the Passage of Pancreatic Juice and Intestinal Fluid into the Stomach : Important monographs by W. Boldyreff, Ergebn. d. Physiol., 11, 127-213, 1911; cf. also W. Boldyreff, Pflüger's Arch., 121, 13, 1908; E. Abder- halden and F. Medigreceanu, Zeitschr. f. physiol. Chem., 57, 317, 1908; E. Abderhalden and Schittenhelm, ibid., 59, 230, 1909; J. Lewinski (Minkowski's Clinic, Greifswald), Deutsche med. Wochenschr., 1908, 1582. 3 Literature upon Production of Pancreatic Fistulas : J. Pawlow, Ergebo, d. Physiol., 1, 266-272, 1902; Nagel's Handb. d. Physiol., 2, 728-742, 1907. 36 PROTEOLYTIC PANCREATIC FERMENT the special technical difficulties. He proceeded to bring a patch of the duodenal wall surrounding the orifice of the excretory duct to the skin surface and stitched it into the cutaneous margins of the wound. In this way he obtained a permanent fistula and the advantage of continuous observa- tion of the glandular activity at his convenience. Even with this advance in technique the results obtained were not thoroughly satisfactory because of the activation of the secretory fluid obtained from the fistula by the enterokinase (v. infra.) of the mucous membrane surrounding it. Only by careful removal of this mucous membrane which had been healed into the cutaneous wound and by isolating the orifice of the duct by stitching it to the margins of the wound can there be obtained sufficient approach to physiological conditions to insure that really normal pancreatic fluid will be obtained. Such conditions being established it is possible to de- termine the influences by which the pancreas is normally stimulated. Undoubtedly the most important factor in this connection is the entrance of the acid content of the stomach into the duodenum. The hydrochloric acid is, it is safe to say, the most effective stimulant to pancreatic secretion ; the influence of the fatty substances of the food is apparently more or less questionable 4 ; but a psychic agency must in all likelihood be recognized. As to the method by which the secretion of the gland is set in operation, doubtless both nervous and chemical mechanism must be held possible. Secretin. — Bayliss and Starling were able to demonstrate that the introduction of acid into an intestinal loop will maintain the pancreas in activity even after section of both vagi and the splanchnic nerves, and after extirpation of the solar plexus. After exclusion of all nervous communication between the intestinal loop and the rest of the body the flow of pancreatic juice is quite as free as if the nerves were in- *0. Cohnheim and Ph. Klee, Zeitschr. f. physiol. Chem., 78, 464, 1912. (Soaps apparently have more influence than do the neutral oils.) SECRETIN 37 tact. From this fact the authors named conclude "it was clear that the message from the isolated loop was carried by way of the blood to the pancreas and that it must be some new type of chemical substance which originated in the intestinal mucous membrane under the influence of the acid. In confirmation of this conclusion the mucous membrane of the upper portion of the small intestine may be scraped off, mixed with 0.4 per cent. HCl and the mixture filtered, when it will be found that injection of this filtrate directly into the circulation will induce a special flow of pancreatic fluid. To this new substance produced under the influence of acid in the intestinal cells we have applied the name 'secretin.' " 5 The work of the authors quoted has been amply confirmed in many quarters; and the chain of proof has been in a sense completed by Wertheimer's discovery of secretin in the blood of an isolated intestinal loop into which acid had been introduced. The well considered and masterly studies of Bayliss and Starling should be regarded as truly classical. In this connection Swale Vincent 6 in a critical review of the whole question of internal secretion regards the action of secretin (after the glycogenetic function of the liver) as the best-attested example of an "internal secretion" and in strict sense more definitely established than the internal secretions of the thyroid gland and the adrenal. And yet the conditions of pancreatic secretion remain so confused that the question of the role and significance of secretin is in the author's opinion still far from final solution. In the first place it should be remembered that, as shown by Wertheimer and Fleig, the flow of pancreatic secretion may be induced by the introduction of acid into an intestinal loop even if the blood of the loop is completely cut out from 8 Literature upon Secretin : W. M. Bayliss and E. H. Starling, Ergebn. d. Physiol., 5, 670-676, 1906; E. H. Starling, Lectures on Recent Advances in the Physiology of Digestion, London, 1906; J. Pawlow, Nagel's Handb. d. Physiol., 2, 734-742, 1907; S. Rosenberg, ibid., 8', 141-146, 1910; C. Oppen- heimer, Fermente, 3d ed., 193-194, 1910. "Swale Vincent, Ergebn. d. Physiol., 9, 496-500, 1910. 38 PROTEOLYTIC PANCREATIC FERMENT the general circulation and fails to pass to the pancreas. A number of authors, especially those of the Pawlow school, are disposed to assume that the pancreatic secretion is gov- erned by a dual mechanism, partly nervous and partly humoral. 7 Secretin seems to be a thermostabile substance, soluble in alcohol, not specific for any particular kind of animal, but apparently identical in all vertebrates. The belief that it is formed from a "prosecretin" when acid comes in contact with the intestinal mucosa is improbable, as it can be shown 8 that solutions having secretin-like influence may be obtained by treating the mucous membrane of the intestine with sodium chloride solution, soaps, alcohol, peptone, chloral hydrate or even with hot water ; the usual employ- ment of mineral acids is readily explicable from the fact that such an agent prevents the destruction or masking of the secretin action by other agents contained in the extracts of intestinal mucous membrane. Fleig's assumption of the necessity of recognizing different kinds of secretin ("sapo- krinin," "ethylokrinin," etc.), according to the method of derivation, is probably not justified. 9 Secretin and Cholin and the Vasodilations. — From an investigation which the author's friend, Carl Schwarz, 10 and the author conducted, it was found that cholin exists in the secretin prepared according to the method of Bayliss and Starling. The influence of such an extract is unquestionably to be partly attributed to this base, 7 Cf . A. Bylina (Institut, exp. Med., St. Petersburg), Pfliiger's Arch., 142, 531, 1911. 8 Delezennes, and Pozerski, Fleig, Camus, Falloise, Gley and others. * While C. Delezennes and E. Pozerski, Journ. de Physiol., 1%, 521, 540, 1912, in their most recent publications insist upon the non-existence of a " pro- secretin," W. Stepp (Univ. College, London: Journ. of Physiol., 43, 441, 1912) holds that secretin practically always is to be found in the intestinal mucous membrane as prosecretin, free secretin being found only occasionally in small quantities; cf. also S. Lalou, Journ. de Physiol., U h 241, 465, 530, 1912; E. Gley, ibid., 507. 10 0. v. Fürth and C. Schwarz, Pfliiger's Arch., 12^, 427, 1908; cf. Litera- ture thereto appended. SECRETIN AND CHOLIN 39 the physiological influence and significance of which have elsewhere (Vol. I of this series, Chemistry of the Tissues, pp. 185-190) been detailed. Secretin cannot, however, be identified as cholin, the activities of the two sub- stances being in no wise parallel ; the secretory effect of cholin (but not of secretin) being completely eliminated by atropin. "Secretin" is, however, apparently not a simple substance, but is perhaps a mixture of a number of agents capable of exciting secretory activity, in the group of which cholin doubtless is included. Keeping in mind the fact that cholin is an exceedingly labile substance, subject to marked changes from comparatively simple disturbances (as seen, for in- stance, in its transformation into neurin, muscarin and acetylcholin) and to functional exaggerations (cf. Vol. I, p. 189, of this series, The Chemistry of the Tissues), it is not easy to get rid of the idea that perhaps secretin is nothing more than a mixture of cholin and of its transformation products. Of course this is no more than an unproved con- jecture, which it is true would serve to bring secretin and the "vasodilatins" (probably closely related to cholin) under a common heading. As previously indicated, cholin is one of the general tissue constituents ; and from the investigations of Popiel- ski n and his students " vasodilatins' ' can doubtless be ex- tracted from practically all tissues. These are powerful substances capable, when injected intravenously with proper precautions, of inducing fall of blood-pressure, secretion of saliva, of gastric, intestinal and pancreatic fluids, increase of peristalsis, spasms, loss of haemic coagulability and in- creased flow of the lymph. Popielski believes that a relative anaemia, dependent upon the lowered blood-pressure, and consecutive irritation of the nervous centres are fundamen- 11 L. Popielski (Lemberg), Centralbl. f. Physiol., 16, 505, 1902; 19, 801, 1906; Pflüger's Arch., 120, 451, 1907; 121, 239, 1908; 126, 483, 1909; 128, 191, 223, 1909. dO PROTEOLYTIC PANCREATIC FERMENT tal to the symptom-complex ; he does not accept the physio- logical importance of secretin for normal production of the pancreatic secretion, and, while willingly acknowledging the existence of a nervous mechanism as operative in the process, does not recognize a humoral factor. However, it must be insisted upon that, from the studies of Bayliss and Starling, the substance in intestinal extracts which depresses the blood pressure is not identical with secretin, and that secretin-containing solutions can be prepared which are apparently free from the former. It must be acknowledged that a clear insight into the matter has by no means been attained. The attendant difficulties may be the more readily appre- hended if it is recalled that the nervous mechanism con- cerned in the pancreatic secretory process is a complex one and is really very little understood. We know that the pan- creas receives its nervous impulses both from the vagus and the splanchnic ; both nerves furnish fibres to the blood ves- sels of the gland, and from stimulation of either according to circumstances there may be induced either increase or in- hibition of the secretion. C. Schwarz 12 was able to show that cholin can influence the pancreatic secretion in one way or the other according to the quantity injected, on the one hand inhibiting the flow through its excitation of the vagus centre, on the other inducing activity by stimulation of peripheral autonomous secretory nerves. It may therefore be appreciated without further discussion that it is difficult if not impossible to come to a satisfactory conclusion as to the identity or differentiation of any of the active con- stituents of organic extracts from simple comparison of their influences upon the pancreatic secretion and from other physiological factors. Until we are able to deal with chem- ically definite substances, it is to be feared we will be unable to escape from such contradictions. 12 C. Schwarz (Vienna), Centralbl. f. Physiol., 23, 337, 1909. ENTEROKINASE 41 Mention should be made also of the fact that acid exhibi- tion is capable of stimulating reflexly the pancreatic secre- tion not only from the duodenum but also, as discovered by Popielski, 13 and by London and C. Schwarz, 14 from a large part of the small intestine. London and Schwarz determined the fact that besides the duodenum, the whole of the jejunum and the upper portion of the ileum, but not the middle part of the ileum, manifest this ability, which therefore resides in about two-thirds of the entire length of the intestine. Besides secretin and cholin, pilocarpin is capable of stimulating the secretory activity of the pancreas if intro- duced into the blood; albumoses and other similar sub- stances may act in the same way. 15 Enterohinase. — Passing to consideration of the pancre- atic secretion itself, Pawlow is to be credited with the impor- tant discovery that trypsin is not secreted in a completed form. The secretion as it passes out of the duct of the gland contains rather a trypsin-zymogen, which is activated by con- tact with an agent arising from the intestinal mucosa, entero- hinase, and is transformed thereby into true trypsin. This puzzling agent, which is also doubtless of importance in acti- vating the human pancreatic secretion, 16 has been the object of intensive study for the past few years. A group of eminent French authors 17 and a number of other investigators 18 have endeavored to determine the real nature of enterokinase ; but opinions arrived at are at wide variance. 19 The belief that the substance is a ferment has met with considerable oppo- 13 L. Popielski, Zeitschr. f. physiol. Chem., 11, 186, 1911. 14 E. S. London and C. Schwarz, Zeitschr. f. physiol. Chem., 68, 346, 1910. 15 E. Gley, Journ. de Physiol., U h 507, 1912. 18 Observations of A. Ellinger, M. Kohn, K. Glässner and J. Wohlgemuth. 17 Dastre, Camus, Delezennes, Frouin, Gley and associates. 18 Bayliss and Starling, O. Cohnheim, Ciaccio, C. Foä, Hamburger and Hekma, Vernon, E. Zunz. 19 Literature upon Enterokinase: Th. Brugsch, Handb. d. Biochem., 3', 115- 116, 1910; S. Rosenberg, ibid., 3', 127-136, 1910; C. Oppenheimer, Fermente, 3d ed., 208-215, 1910; W. Biedermann, Winterstein's Handb. d. vergleich. Physiol., 2', 1409-1415, 1911. 42 PROTEOLYTIC PANCREATIC FERMENT sition. A number of authors would relate the adsorptive readiness of enterokinase (as in its ready fixation by fibrin) with its action, and believe that, in the same way as in haemolysis complement is fixed to a red blood cell by the intermediation of an amboceptor, trypsinogen is fixed to the protein molecule as complement by the binding power of enterokinase (as amboceptor) . Enterokinase is a thermola- bile substance; but 0. Cohnheim's observation that it is soluble in 90 per cent, alcohol is clearly not in harmony with the idea that it is of the nature of an enzyme. It is scarcely believed any longer that enterokinase is derived from leucocytes ; it is apparently directly produced from the cells of the intestinal mucous membrane, although there seems to be some active material of similar character also present in leucocytes at times. Activation of Trypsinogen by Calcium Salts and Similar Substances. — Besides enterokinase a considerable group of other agents is known to be capable of activating trypsino- gen. Calcium salts particularly have been the object of care- ful investigation by Delezenne in their marked relations with trypsinogen. Activation of an inactive dyalized pancreatic juice, after the latter, mixed with a salt of calcium, has been kept in an incubator for a number of hours, may result sud- denly, as if explosively ; if paraffined tubes have been used activation by calcium salts may be postponed for some days. 20 With special precautions activation of trypsinogen may be induced by quite a variety of colloids (as by toluidin blue) . It is therefore scarcely remarkable that the expressed juices from various organs, milk and similar substances, have been found capable of inducing the same result. Even bacteria can convert trypsinogen into trypsin ; the author has in an earlier volume (cf. Vol. I of this series, Chemistry of the 20 Cf . also J. Wohlgemuth ( Experimental Division of the Pathol. Institut., Berlin), Observations upon Human Pancreatic Fluid, Biochem. Zeitschr., 39, 302, 1912. INDIVIDUALITY OF TRYPSIN 43 Tissues, p. 501) discussed their probable relation with the genesis of the once celebrated freighting -theory (in accord- ance with which the pancreas was freighted with digestive ferment from the spleen). Even trypsin itself seems to belong to the group of acti- vators of trypsinogen ; at any rate when the least amount of trypsin is introduced into an inactive solution of the ferment it is spontaneously activated. Individuality of Trypsin. — Question has arisen whether the digestive pancreatic ferment is chemically one single sub- stance. L. Pollack has asserted that besides the trypsin there is a mucin digesting ferment, "glutinase," in the pan- creatic juice ; Vernon has accepted the existence of a peptone- splitting ferment {"pancreatic erepsin"). Other investiga- tions rather favor the idea of the individuality of the protein- digestive ferment of the pancreas. For example, the expla- nation of the observed fact that trypsin, first acidulated with hydrochloric acid and then neutralized, will freely digest gelatin but no other proteins is to be found in the fact that in highly alkaline conditions it digests all proteins but on addi- tion of acid digests only gelatine; if, thereafter, alkali be added the normal digestive properties recur. 21 In a recent study K. Glässner and A. Stauber have again asserted the existence of an erepsin in the pancreatic fluid along with trypsin. 22 Doubtless the activity of trypsin is influenced very largely by the ions present in the solution. A dialysed solu- tion of trypsin, for instance, shows diminished activity ; but 21 H. M. Vernon, Jour, of Physiol., 30, 330, 1903; K. Mays (Laboratory of A. Kossel), Zeitschr. f. physiol. Chem., 38, 502, 1903; J t 9, 124, 188, 1906; W. M. Bayliss and E. H. Starling, Journ. of Physiol., 30, 61, 1903; L. Pollack, Hofmeister's Beitr., 6, 95, 1905; Arch. f. Verdauungskr., 11, 362, 1905; M. Ehrenreich, Arch. f. Verdauungskr., 11, 261, 364, 1905; A. Ascoli and B. Neppi, Zeitschr. f. physiol. Chem., 56, 135, 1908; G. Schaeffer and E. F. Terroine, Journ. de Physiol., 12, 884, 906, 1910. n K. Glässner and A. Stauber ( E. Freund's Lab. ) , Biochem. Zeitschr., 25, 204, 1910. 44 PROTEOLYTIC PANCREATIC FERMENT it is interesting to note the return of its efficiency as soon as the normal salts of pancreatic fluid are added. 23 Only the briefest comment can here be made upon the extensive literature dealing with the influence of variations of alkalin- ity, temperature, neutral salts, poisons of various kinds, * * antif erments ' ' and adsorbing media upon the activity of trypsin; the author must refer for fuller details of these subjects to Carl Oppenheimer 's valuable work on ferments. 24 Conversion of Trypsin into Zymoid. — The observations of Bayliss are presumably of general interest as they may perhaps bring us a step nearer to comprehension of the mystery of ferment activities. By determination of con- ductivity Bayliss discovered that trypsin inactivated by standing does not lose its power of fixation of protein. He compares the inactivation of trypsin to Ehrlich 's conception of the change of a toxine into a toxoid. In the transformation of trypsin into a "zymoid" he sup- poses it loses its ergophore group, still retaining, however, its haptophore group, which serves to fix it to a protein molecule. There has been no lack of experiments with other ferments to apply to the enzyme problem the conceptions which have come in spite of all opposition to dominate our theories as to immunity. We may think as we will of these modes of thought ; the heuristic value of these and of similar schematic ideas cannot be controverted. Their justification can be maintained, however, only until something better can be substituted. 25 Action of Trypsin Upon Polypeptids. — Undoubted prog- ress has been accomplished by the systematic researches of Emil Fischer, Abderhalden and Bergell in determining the 23 A. Frouin and A. Compton, Compt. rend., 153, 1032, 1911. M C. Oppenheimer, Fermente, 3d ed., pp. 185-202, 1909 ; cf. also S. G. Hedin, Biochem. Journ., 1, 474, 484, 1906; 2, 81, 1907; L. Michaelis and H. Davidsohn, Biochem. Journ., 86, 280, 1910. 20 W. M. Bayliss, Arch. Sciences biol., St. Petersburg, 11, supplement 261, cited by O. Cohnheim, Nagel's Handb. d. Physiol., 2, 582, 1907. ADAPTATION OF PANCREATIC SECRETION 45 field of tryptic action upon definite proteins, particularly in relation to chemically pure Polypeptids ; the situation is com- parable to one in which of two unknowns in an equation, if one may somehow be eliminated, the theoretical possibility of a solution attained is probable. What are the structural peculiarities upon which the possibility of a given polypeptid being acted upon by trypsin depends? "The (molecular) structure of the individual compounds comes into considera- tion at once, ' ' says Abderhalden. 26 ' ' An instructive example is found in the relation of alanyl-glycin (CH 3 .CH(NH 2 ).CO. NH.CH 2 .C00H) and its isomer glycyl-alanin (NH 2 .CH 2 . CO.NH.CH ( CH 3 ) .COOH) . The former is split, the latter is not. The particular kind of amino acid is also of importance. In the dipeptids, for example, hydrolysis is favored if alanin acts as acyl. The resistance of dipeptids containing a-aminobutyric acid, a-aminovalerianic acid and leucin as acyl is very notable. The number of aminoacids concerned in the structure of a polypeptid is also of importance. The glycin group illustrates this nicely. Gylcyl-glycin, diglycyl- glycin and triglycyl-glycin are not split; while hydrolysis takes place in case of tetraglycyl-glycin. " It is of special interest, however, that pancreatic fluid splits only those Poly- peptids in whose molecular structure the natural optically- active aminoacids take part; and that the economy, if raceme bodies are at its disposition, may often be able to utilize only one of the two optically opposite components. This is true both for the mammalian body and for the low- est forms of plants (cf. Vol. I of this series, Chemistry of the Tissues, p. 15). Adaptation of Pancreatic Secretion to the Food. — The Pawlow school has assumed a general adaptability of the secretion of the pancreas to the particular type of food which may obtain at any given time; in other words, that M E. Abderhalden, Lehrb. d. phyaiol. Chem., 2d ed., 626-628, 1909; cf. Literature, thereto appended. 46 PROTEOLYTIC PANCREATIC FERMENT the pancreas adapts itself to its work in, as it were, an intelligent manner, by increasing its proteolytic ferment for meat, its amylolytic ferment for a meal of bread, or its lactose ferment for milk. This view, however, cannot with- stand criticism ; it bespeaks an effort to realize a harmonious principle pervading the arrangements of nature which goes somewhat too far afield. 27 Quantitative Determination and Ferment Law of Tryp- sin. — The realization that there are a number of physiological questions related with trypsin which rest upon the possibility of its quantitative determination, has, as in case of pepsin and by employment of similar principles, led to numerous attempts to accomplish this. Thus Metts' tubes of albumin or gelatin have been used. 28 Trypsin has been allowed to act upon dissolved casein, after Vollhard's method, 29 and from the increase of acidity of the resulting albumoses the degree of digestion has been determined by titration. Gross 30 dis- solves casein in soda solutions (as in E. Fuld's method), arranges a series of tubes with increasing amounts of trypsin, and observes after a time whether acidulation with acetic acid continues to give rise to turbidity. Jacoby 31 ob- serves the degree of clearing of deposits of ricin or edestin ; V. Henri 32 and Bayliss 33 follow the progress of digestion physico-chemically by determining the conductivity, and Brailsford Robertson 34 by determining the refraction index of a soda-casein-solution after precipitation of undigested casein by acetic acid. A simple and apparently very precise 27 Literature upon Adaptation of Pancreatic Secretion to Food : S. Rosen- berg, Handb. d. Biochem., 3', 138-140, 1910. 28 P. Hattori, Arch, internat. de Pharm., IS, 255, 1909. 20 W. Löhlein, Hofmeister's Beitr., 7, 120, 1905; Faubel, ibid., 10, 35, 1907. 30 O. Gross, Arch. f. exper. Pathol., 58, 157, 1908. 81 M. Jacoby, Biochem. Zeitschr., 10, 299, 1908. 82 V. Henri, and Larguier des Bancels, C. R. Soc. de Biol., 55, 563, 787, 866; Jahresber. f. Tierchem., 33, 512-514, 1903. 85 W. M. Bayliss, 1. c. 84 T. Brailsford Robertson, Journ. of Biol. Chem., 12, 23, 1912. PARENTERALLY INTRODUCED TRYPSIN 47 method has been devised by P. v. Grützner 35 on the principle of his method of estimating pepsin. Fibrin is stained with Spiritblau (diphenylrosanilin) and the color given to the digestive fluid when the fibrin is digested in a 0.1 per cent, soda solution is determined colorometrically. While the Schütz-Borissow rule may be found applicable within certain limits for the digestion of solid proteins, there seems to exist for dissolved proteins a simple ratio between the quantity of ferment and quantity of protein dissolved by it. "Both groups," thinks Palladin, 36 "are correct, those who believe that the Schütz-Borissow rule obtains for trypsin and those who think that Vollhard's rule of direct ratio applies. It is not of consequence which method is used. Probably the truth is that one ferment molecule does exactly as much work as any other, that n molecules will do n times as much as one in case no accidental inhibition occurs and the ferment molecules can have full access to their prey (if the term be permitted), that is to the protein, which is all that Grützner proved for pepsin originally. ' ' Toxicity of Parenterally Introduced Trypsin. — Refer- ence may here be made to a matter of general physiological interest, the toxicity which parenterally introduced trypsin or pancreatic tissue manifests upon the body. In spite of the resistance of living tissues to digestive fer- ments, mentioned in the preceding lecture, there unquestion- ably is a decided production of necrosis when trypsin is in- jected subcutaneously. "When the pancreas of a dog under- goes necrosis death quickly ensues, and that whether the pancreas was that belonging to the animal or a foreign gland transplanted under aseptic precaution. It is very interest- ing, as discovered by Achalme, that animals may be im- munized to a high degree against this toxic influence by 30 A. Palladin (Physiol. Institut., Tübingen), Pflüger's Arch., 134, 337, 1910; W. Waldschmidt, ibid., 143, 189, 1911. 30 P. Palladin, 1. c, p. 364. 48 PROTEOLYTIC PANCREATIC FERMENT previous gradual treatment with trypsin. G. v. Bergmann has been able to induce in dogs such a high grade of immunity that the animals were able to bear the implantation of an entire foreign pancreas, a procedure which in unpre- pared animals is followed by death within twenty hours at most. 37 It may be conjectured that the toxic effect of the parenter- ally introduced trypsin depends upon a sudden hydrolytic cleavage of the proteins in the living body produced in the same way as trypsin in vitro dissociates proteids. To de- termine this point experimentally the author with his col- league, Carl Schwarz, 38 has studied the effect of intra- peritoneal injections of emulsions of pancreas introduced under aseptic precautions. Although acute toxic symptoms were produced in the experiment animals, the latter could be protected by previous treatment with increasing dosage of trypsin, and rendered resistant to comparatively large amounts of the pancreatic material. By careful metabolic studies the investigators were able to prove that intra- peritoneal injections of large amounts of trypsin or pan- creatic tissue disturbs the nitrogen balance so that for one or two weeks the nitrogen excretion is irregular (a series of irregular increases and decreases). The total nitrogen output was, however, not altered, as might have been ex- pected if an exaggerated protein destruction and important catabolism of the tissue proteids had taken place. It is by no means safe to conclude that the marked toxicity of par- enterally introduced trypsin is to be referred to a direct influence upon protein-metabolism. According to Fischler, animals which have been previously treated with trypsin 37 P. Achalme, Ann. de l'lnstit. Pasteur., 15, 737, 1901 ; G. v. Bergmann, Zeitschr. f. exper. Pathol., 3, 400, 1906; G. Dorberauer, Beitr. z. klin. Chir., 48, 456, 1906; N. Gulecke, Arch. f. klin. Chir., 85, 644, 1908; G. v. Bergmann and N. Gulecke, Münchener med. Wochenschr., 57, 1673, 1910; D. Kirchheim (Cologne), Verh. d. Kongr. f. innere Med., 21 1 595, 1910. 88 O. v. Fürth and C. Schwarz, Biochem. Zeitschr., 20, 384, 1909. PARENTERALLY INTRODUCED TRYPSIN 49 react to very small amounts of phosphorus or hydrazin by suffering marked fatty degeneration of the liver. 39 The question of "antitrypsin" in the serum has been elsewhere discussed, and the author prefers not to return to this rather uninteresting subject. In what manner trypsin normally gains access to the blood circulation cannot easily be determined with certainty; especially when the body is flooded with the enzyme it has been recognized in the urine. 40 When it is remembered how little we know of the changes many chemically well-defined substances undergo in the animal body, it does not seem at all remarkable that the fate of so indefinite a substance as trypsin remains unknown. 39 Fischler and Wolff, 29. Kongr. f. innere Med., 19, IV, 1912. *°K. Bamberg (II Med. Clin., F. Kraus, Berlin), Zeitschr. f. exper. Pathol., 5, 7¥L, 1909; E. Graf von Schönborn, Zeitschr. f. Biol., 53, 386, 1910. CHAPTER III PROTEIN-DIGESTION IN THE INTESTINE Erepsin. — In the preceding chapters we have dealt in some detail with pepsin and trypsin. Our attention is now turned to a third ferment directly involved in protein-diges- tion, the erepsin of the intestinal secretion, the discovery of which is due to the acuteness of observation of Otto Cohnheim. Erepsin is an enzyme which does not act upon most of the native albumins, but reduces albumoses and peptones to crystallizable products. "In connection with the action of erepsin upon the individual intermediate substances between albumin and the amino acids, " Cohnheim observes, "it should be said that peptones, that is peptones corresponding to Kühne 's conception, lose the ability to respond to the biuret-reaction very quickly, within minutes or hours, when in contact with erepsin-solutions, very much more rapidly than I have ever observed when they are brought in contact with active pancreatic extract. Erepsin acts much more slowly upon the various albumoses, weeks elapsing before the disappearance of the biuret-reaction, which is in fact a rather deceptive phenomenon. Kutscher, Seemann and Weinland, who have worked with erepsin only upon albumin and who because of the slowness of the loss of biuret-reaction have denied any real importance to erepsin in digestion, are con- tradicted by these differences. ' ' The influence of erepsin is, however, not limited to the protein derivatives of higher molecular composition. Polypeptids are also split, as shown by the investigations of Abderhalden and his associ- ates and the studies of H. Euler. Thus the albumoses and peptones formed from ingested proteins by the action of 50 EREPSIN 51 pepsin and trypsin fall very quickly as prey to erepsin and are broken down into their final crystallizable dissociation- products. 1 At first 0. Cohnheim's brilliant discovery was questioned by many, but later was so fully confirmed 2 that its correct- ness cannot logically be doubted. Erepsin is neither iden- tical with trypsin nor does it come from the pancreas. Even where the pancreatic secretion cannot gain access to the intestine, as in case of a Thiry fistula 3 or after ligation of the pancreatic ducts, 4 a rich supply of erepsin is found in the intestine and its association with pepsin under these circumstances accounts for the continued albumin dis- sociation in the intestine. 5 If, however, the pancreas be completely extirpated or atrophied, general metabolic disturbances ensue of so marked a type that there can be no wonder that protein digestion should eventually suffer. It is a misconception to class erepsin among the autolytic tissue ferments. Some such erepsin-like influence may be met, according to Vernon, in many different tissues, which will be con- sidered in the following lecture; this is really a phenomenon due entirely to endocellular enzymes, whereas erepsin undoubtedly belongs in the intestinal secretion. It is, however, not a matter of much consequence about the name, if the facts of its capability are kept clearly in mind. 1 Literature upon Erepsin : 0. Cohnheim, Nagel's Handb. d. Physiol., 2, 583-585, 1907; Physiol, d. Verd. u. Ernährung, Berlin and Vienna, 1908, p. 217; F. Samuely, Handb. d. Biochem., 1, 555, 1909; Th. Brugsch, ibid., 3', 112- 115, 1910; C. Oppenheimer, Fermente, 3d ed., 181-184, 1909. ^Kutscher and Seemann, S. S. Salaskin, A. Falloise, J. H. Hamburger and E. Hekma, L. Tobler, M. Nagajama, Lambert, C. Foä, L. Weekers, E. Rau- bitschek, L. Langstein and Soldin, G. Amantea and others. 3 L. Weekers, Arch, intern, de Physiol., 2, 49 ; cited Centralbl. f. Physiol., 19, 90, 1905. ♦Th. Brugsch, Zeitschr. f. exper. Pathol., 6, 326, 1909; K. Glässner and A. Stauber (E. Freund's Lab.), Biochem. Zeitschr., 25, 204, 1910; E. Zunz and L. Mayer, Bull. Acad. de M£d. de Belgique (4), 19, 509; Jahresber. f. Tierchem., 35, 491, 1905. "Literature: O. Prym, Handb. d. Biochem., 3", 106-107 (1909). 52 PROTEIN DIGESTION IN THE INTESTINE There can be no objection to regarding erepsin in the light of a "heterolytic" ferment, however, even if it be not an "autolytic" one. From snch considerations it is evident that nature has fully provided means to insure the splitting of the proteid constituents of the food into their end-products. We are in position, therefore, to proceed a step further and take up the question whether there is any evidence that the provision can be traced as actually operative in normal digestion in further measure. 6 Extent of Protein Dissociation in the Intestine. — The classical investigation by Carl Ludwig and Salvioli should be recalled showing that peptone solution may disappear from the lumen of a loop of the small intestine (artificially perfused with blood) without peptone becoming demon- strable in the blood used in perfusion. Later, in 1881, Franz Hofmeister, whose pupil the author was, demonstrated the disappearance of peptone when brought in contact with gastric mucosa in extracorporeal conditions ; and N. Neu- meister made a similar demonstration for the intestinal mucosa. The interpretation of these fundamental and amply corroborated observations suggesting a resorption process, at a later date was modified by the discovery, mainly due to work by Kutscher and Seemann, Cohnheim, Cathcart and Leathes, 7 that, while albumoses and peptones disappear when brought in contact outside the body with intestinal mucosa, this is due not to their resorption but to their dissociation into more simple protein derivatives. Moreover, the investigations of these authors, and chiefly 6 Literature upon the Extent of Protein Dissociation in the Intestine: J. Munk, Ergebn. d. Physiol., 1, 310-317, 1902; O. Cohnheim, Nagel's Handb. d. Physiol., 2, 629, 1907; H. Lüthje, Ergebn. d. Physiol., 7, 800-804, 1908; O. Prym, Handb. d. Biochem., 3", 102, 1909; W. Biedermann, Winterstein's Handb. d. vergl. Physiol., 2", 1448-1449, 1911. 1 F. Kutscher and J. Seemann, Zeitschr. f. physiol. Chem., 3> t , 528, 1902 ; 35, 432, 1902; 49, 298, 1907; O. Cohnheim, ibid., 33, 451, 1901; 36, 13, 1902; 1,9, 64, 1906; 51, 415, 1907; E. P. Cathcart and J. B. Leathes, Journ. of Physiol., S3, 462, 1905. PROTEIN DISSOCIATION IN THE INTESTINE 53 numerous studies prosecuted by Abderhalden, London and their associates, 8 left no doubt as to the constant presence of considerable amounts of aminoacids in the contents of the intestine (even if dissociation is apparently not complete), and too of notable quantities of polypeptoids. 9 The intes- tinal contents differ decidedly in this from the gastric con- tent, which, as a rule, shows no aminoacids at all or at most only traces. (In the instances in which these substances do occur in the stomach, as in the omasum of ruminants, the possibility of the existence of ferments in the food itself must be considered, 10 as it has been indicated from the work of the Ellenberger Institute that under certain circumstances autolytic ferments may relieve the animal intestine in part of its work of digestion and may act as substitutes for the body enzymes.) X1 The later advances in the chemistry of proteins, espe- cially Emil Fischer's ester method, formol-titration of aminoacids, etc., harmonize with these studies. The ester method cannot be applied to the determination of amino- acids in the intestinal content unless special provisions (as refrigeration during the estering process, etc.) are em- ployed, as in the process of estering it is possible that aminoacids may be split off from proteid substances. 12 It is impossible to deny that proteid substances are separ- ated into their final derivatives ; but it is another question whether resorption normally occurs only after such ad- vanced dissociation. Three possibilities must be taken into consideration. In the first place absorption of an impor- 8 Baumann, Funk, Kautzsch, v. Körösy, Medigreceanu, Oppler, Reemlin. Literature: 0. Prym, 1. c. 9 E. Aberhalden, Zeitschr. f. physiol. Chem., 74, 436, 1911. 10 E. Abderhalden, W. Klingemann and Th. Pappenhusen, Zeitschr. f. physiol. Chem., 71, 411, 1911. 11 W. Grimmer ( Ellenberger's Lab.), Biochem. Zeitschr., ! { , SO, 1907; cf. Literature, there appended. 13 B. O. Pribram (Vienna), Zeitschr. f. physiol. Chem., 71, 472, 1911; 72, 504. 1911. 54 PROTEIN DIGESTION IN THE INTESTINE tant part of the protein dissociation-derivatives as under- stood in the older conceptions may take place in the stage of "albumoses" and "peptones." If, however, this be not the case, and if the dissociation proceeds to the extent of producing crystallizable products, two further possibilities present themselves: either these crystallizable derivatives are absorbed as such, or they undergo a synthetic change, before entering the blood, into high-molecular protein derivatives in the intestinal wall. Passage of True Proteins and High-molecular Protein- derivatives into the Blood. — In considering the first of these possibilities it must be granted that under certain circum- stances high-molecular protein dissociation-products and, too, true proteins, can pass from the bowel into the blood. 13 The precipitin reaction and anaphylactic methods assure us of the possibility of determining, with precision hitherto un- dreamed of, the most minute quantities of foreign proteins in the blood. 14 We know today that it is possible under given conditions to determine by such methods proteid substances absorbed from the bowel, as, for example, after flooding the intestine with uncooked eggs, raw milk or blood-serum. The normal protective influence of the intestinal epithelium is ap- parently undeveloped in the newly-born; and, too, may be decidedly impaired by pathological processes. 15 It may be shown experimentally in vitro, too, that foreign serum, toxins, hemolysins, ferments and antiferments of various kinds diffuse more rapidly through an inflamed intestinal wall than through the normal wall of the bowel; this perhaps J * Literature upon Absorption of True Proteins and High Molecular Protein Dissociation-products from the Bowel: O. Cohnheim, Nagel's Handb. d. Pbysiol., 2, 624, 1907; H. Lüthje, Ergebn. d. Physiol., 7, 830-835, 1908; C. Oppenheimer and L. Pincussohn, Handb. d. Biochem., i ', 705, 1911; P. Nolf, Journ. de Physiol., Nov., 1907. 14 F. Micheli (Turin), Giorn. Accad. Med. Torino, 73, 205, 1910. 15 Ganghofner and J. Langer (Prague), Münchener med. Wochenschr., 51, 1497, 1904. PASSAGE OF TRUE PROTEINS 55 being related to a variation in the degree of swelling. 16 Where there coexists with the increased permeability of the intestinal wall a diminished retention by the urinary filter, passage of the foreign protein into the urine can be de- tected, 17 although this does not require special renal permeability as it has long been known that after sub- cutaneous injection of albumoses, albumin and similar sub- stances these substances may at times pass directly into the urine. 18 It goes without saying that such facts are not only of physiological interest but are also of importance in medical practice. For instance, repeated rectal administra- tion of egg albumin may under certain circumstances induce in experiment animals a fatal marasmic condition with anaphylactic phenomena. 19 The well-known attempts of Behring to introduce anti-bodies of various kinds into chil- dren in milk is in direct relation with the question in hand. A full discussion of the subject can be found in a study made by UfFenheimer in the laboratory of Max Gruber. 20 Borchhardt has been able after feeding to recover in the blood two proteids, recognizable in minute amounts because of their characteristic chemical peculiarities, hemielastin and Bence-Jones albumin (v. Vol. I, of this work, The Chemistry of the Tissues, p. 511 ). 21 And yet Abderhalden has failed to find elastin in the blood, tissues or in the urine after administration of large quantities. 22 18 E. Mayerhofer and E. Przibram (R. Paltauf'3 Instit., Vienna), Zeitschr. f. exper. Pathol., 7, 247, 1909; Biochem. Zeitschr., 2k, 453, 1910; M. Loeper and Ch. Esmonet, C. R. Soc. de Biol., 6>t, 445, 1908. " R. Hecke (M. Gruber's Labor., Munich), Münchener med. Wochenschr., 56, 1875, 1909. U H. de Waele and A. J. J. Vandevelde (Ghent), Biochem. Zeitachr., SO, 227, 1910. » L. Petit and J. Minet, C. R. Soc. de Biol., 6k, 22, 1908. -"A. Uffenheimer, Arch. f. Hygiene, 55, 140, 1905. 21 L. Borchhardt, Zeitschr. f. physiol. Chem., 51, 506, 1907; 57, 3C5, 1908; L. Borchhardt and H. Lippmann (Med. Clinic, Königsberg) , Biochem. Zeitschr., 25, 6, 1910. 21 E. Abderhalden and Rüehl, Zeitschr. f. physiol. Chem., 69, 301, 1910. 56 PROTEIN DIGESTION IN THE INTESTINE The numerous positive findings 23 of albumoses in the blood of animals during digestion are contradicted by a series of other studies in which the higher molecular prod- ucts of protein dissociation were entirely missed in the blood at the height of digestion. 24 A recent controversy be- tween E. Abderhalden and E. Freund in reference to this discrepancy brings out distinctly the difficulties involved both in method and in the significance of the results of ex- perimentation. 25 It is extremely difficult to coagulate fully the entire mass of hsemic proteins; and small protein residua which have escaped coagulation may very easily come to be looked upon as "albumoses." Then, too, as dis- cussed in the previous volume of these lectures (Chemistry of the Tissues, p. 246), the possibility exists that non- coagulable proteid substances ("serornucoid," etc.) may be formed in the blood itself. And besides, autolytic ferments exist throughout the body, and where small amounts of albumose-like products are found in the blood they may as well have come from autolysis of the hsemic proteins as from the intestine as resorbed products of digestion. And finally it may be very properly maintained that, even if small quan- tities of digestion albumoses are positively detected, this does not contradict the proposition that the bulk of the products of digestion is resorbed only after complete dissociation. 23 G. Embden and F. Knopp, Hofmeister's Beiträge, 3, 120, 1903; L. Lang- stein, ibid., 3, 373, 1903; G. v. Bergmann and L. Längstem, ibid., 6, 27, 1905; F. Kraus (E. Freund's Labor.), Zeitschr. f. exper. Patbol., S, 52, 1906; E. Freund, ibid., 4, 3, 1907; O. Scbumm, ibid., 4, 453, 1904; Erben, Zeitschr. f. Heilk. (Inn. Med.), 24, 70, 1903. 24 R. Neumeister, Zeitschr. f. Biol., 24, 272, 1888; E. Abderhalden and C. Oppenheimer, Zeitschr. f. physiol. Chem., 42, 155, 1904; E. Abderhalden, Funk and London, ibid., 51, 269, 1907 ; O. Cohnheim, Nagel's Handb. d. Physiol., 2, 626, 1907; P. Morawitz and R. Ditschy, Arch. f. exper. Pathol., 54, 88, 1906; S. B. Shryver (Univers. College, London), Biochem. Journ., 1, 123, 1906. 25 E. Abderhalden, Biochem. Zeitschr., 8, 368, 1908; E. Freund, ibid., 7, 361, 1908; 9, 463, 1908; 11, 541, 1908. ALBUMOSES AND AMINOACIDS 57 Resorption of Iodized Proteins. — The author, in associ- ation with M. Friedmann, has attempted to solve this difficulty by a study of the resorption of iodized proteid substances. 26 Isolation of no particular metabolic product containing iodine was sought, but rather the iodine dis- tribution. One can in this way determine after admin- istration of an iodized protein what part of the iodine present at a given time in the intestinal content, the intes- tinal wall, in the blood and urine, exists in the form of in- coagulable organic substances which can be precipitated by phosphotungstic acid (albumoses and peptones), in the form of incoagulable organic substances not precipitated by phos- photungstic acid (amino acids) , and finally how much exists in inorganic combination. The results obtained indicate that at least the bulk of the determined iodo-proteid sub- stance (iodo-proteic acid) in absorption from the intestine of a cat is so completely dissociated that the iodine in the intestinal wall and blood appears, not in form of iodized albumoses or peptones, but as inorganic alkali iodides. Objections to the Resorption of Albumoses and Amino- acids. — Otto Loewi 27 has pointed out that as a matter of fact the toxicity of albumoses and peptones introduced directly into the blood circulation (referring to the reduction of blood coagulability and the lowering of vascular tone in the splanchnic area, shown by the investigations of Schmidt- Mühlheim, Fano and others) is contradictory to the idea that albumin is taken up in the form of peptone. To the objection that any important formation of aminoacids in the intestine would represent a waste of chemical tension energy lost to no purpose in the splitting process, Loewi has shown from Rubner's calorimetric determinations that in such a process certainly not above 10 per cent, of energy is lost, 26 O. v. Fürth and M. Friedmann (Vienna), Arch. f. exper. Pathol. (Schmiedeberg's Festschrift), p. 214, 1908. 27 O. Loewi (H. H. Meyer's Labor., Marburg), Arch. f. exper. Pathol., 48, 327, 1902. 58 PROTEIN DIGESTION IN THE INTESTINE and therefore that it is not in any sense a ' ' purposeless waste of energy." Residual Nitrogen. — It might very properly be supposed that, in case the aminoacids resulting from the advanced pro- tein dissociation are actually resorbed as such, it would be a matter of little difficulty in an animal in the midst of diges- tive activity to detect a notable increase of residual nitrogen in the blood in the form of incoagulable compounds. 28 As a matter of fact, however, this is very difficult. G. v. Bergman and Langstein 29 have calculated that, if, as supposed, tissue assimilation proceeds in full harmony with resorption, only a few hundredths of one per cent, of aminoacids in the portal blood will suffice to cover the total nitrogen transportation from intestine to the blood required for protein metabolism. 0. Cohnheim believes that it may be concluded from observa- tions upon the rapidity of meat digestion and the rapidity of blood circulation that even with the most active absorption and if no other tissues were involved not more than 0.03 g. of albumin or protein dissociation products could be taken up in a litre of blood. 30 According to an investigation con- ducted under the direction of Hofmeister 31 the residual nitrogen of the blood may be reduced to three fractions: urea, "albumoses" precipitable by tannin, and aminoacids (not precipitated by tannin) . The bulk, actually about three- fourths, of the residual nitrogen in the blood of either the fasting or of the digesting dog, consists of urea. Of the other two fractional parts that of the aminoacids always shows definite increase during digestion. The albumose fraction is an inconstant one ; even after feeding albumoses no increase was noted indicative of their entrance to the 28 Literature upon the Incoagulable Nitrogen-containing Bodies of the Blood Serum: P. Morawitz, Handb. d. Biochem., 2", 80-86, 1909. 29 1. c. 30 O. Cohnheim, Physiol, d. Verd. u. Ernähr., p. 227, 1908. 31 H. Hohlweg and H. Meyer (Physiol. Chem. Instit., Strassburg), Hof- meister's Beitr., 11, 381, 1908. SYNTHESIS OF THE PRODUCTS OF DIGESTION 59 blood in unsplit state. Folin found on resorption of glyco- coll or alanin from the stomach or large intestine an increase in the residual nitrogen, usually too of the urea, in the blood. 32 There is another series of observations which are at least not contradictory to the supposition that the amino- acids pass into the blood. Pringle and Cramer 3S found the blood of a digesting animal to contain a somewhat higher residual nitrogen than that of a fasting animal. Embden and his pupils have been able to detect the presence of small quantities of aminoacids in the blood and their passage into the urine by means of the naphthalin-sulphochloride method. 34 In pathological states the aminoacids in the blood can doubtless be increased. According to Neuberg and Richter 35 in acute yellow atrophy of the liver amino- acids may be found in such quantities as to suggest that perhaps in some way owing to the loss of hepatic function a further change of the catabolic products of proteids (split in the intestine into crystallizable products) fails to take place. In uraemia, too, at times the aminoacids are appar- ently increased in the blood. Moreover it should not be overlooked that 0. Cohnheim 36 in his studies upon invertebrates, octopods particularly, came to the conclusion that albumin is completely catabol- ized in the intestine and resorbed in the form of aminoacids. These may thereafter according to requirements undergo either combustion or synthesis in the tissues. Synthesis of the Products of Digestion. — Assuming then, as we seem fully justified in doing, that the dietary protein undergoes an advanced grade of cleavage in the ;: 0. Folin, W. Denis and H. Lymann (Harvard Med. School), Journ. of Biol. Chem., 12, 141, 253, 259, 1912. 53 H. Pringle and W. Cramer, Journ. of Physiol., 37, 158, 1908. '-* G. Embden and H. Reese, Hofmeister's Beitr., 7, 411, 1905: A. Bingel, Zeitschr. f. physiol. Chem., 57, 382, 1908. 35 C. Neuberg and Richter, Deutsche med. Wochenschr., 190 Jf, 499. 56 0. Cohnheim, Xagel's Handb. d. Physiol., 2, 629, 1907. 60 PROTEIN DIGESTION IN THE INTESTINE intestine, the further question arises whether the re- sultant cleavage-products do not undergo either locally in the intestinal wall or in their passage thence into the blood a synthetic reconstruction in such manner that the nitrogen from the intestine comes to be found in the circulatory fluids, not in the form of aminoacids, but in high-molecular com- binations. Attention may be called to the fact that Van Slyke 37 has recently applied his delicate nitric acid method of determination of the aliphatic amino-group (cf. Vol. I of this series, Chemistry of the Tissues, p. 18) to the detec- tion of aminoacid nitrogen in the blood. By this method it has been established that even large amounts of aminoacids injected intravenously disappear very quickly from the blood without anything like an important part appearing in the urine. For example, of twelve grams of alanin injected intravenously there remained after half an hour only a few decigrams in the blood, the rest being apparently transferred into the tissues. Van Slyke concludes from his investiga- tions that the supposition of synthetic change of the amino- acids in the bowel wall is superfluous and that the smallness of the increase of aminoacids in the blood of digesting ani- mals is entirely explicable by the avidity with which these substances are taken up by the tissues and further elaborated by them. The results obtained by efforts to arrive at a conclusion in regard to synthetic processes from analytic comparison of the composition of the intestinal mucous membrane of fasting and digesting animals are in no sense consistent. 38 It should always be remembered, however, that a number of direct observations indicate the possibility of condensation processes in the intestine. 37 D. D. Van Slyke, and G. M. Meyer (Rockefeller Instit., New York), Journ. of Biol. Chem., 12, 399, 1912. 88 F. Botazzi, Arch, di Fisiol., 5, 317, 1908; cited in Biochem. Centralbl., 8, 374, 1908; E. S. London, Zeitschr. f. physiol. Chem., 61, 69, 1909; P. Glagolew (St. Petersburg), Biochem. Zeitschr., 32, 222, 1910. SYNTHESIS OF THE PRODUCTS OF DIGESTION 61 Kutscher and Seemann 39 have determined that rela- tively simple abinret substances are included in the ex- tractives of the intestine, which split when treated with boil- ing mineral acids with the production of leucin in consider- able amounts. This observation may perhaps have some bear- ing upon the possibility of the leucin from the intestinal mucous membrane (and of many other simple products of protein cleavage) having previously undergone a process of coupling directly in the intestinal wall. Bearing upon con- densation processes, the extensive observations of Danilew- ski and his students (vide supra, p. 30) upon plastein formation should be noted. In albumose solutions autolysed with intestinal mucous membrane an increase of coagulable nitrogen at the expense of incoagulable nitrogen has been found, 40 interpreted tentatively as transformation of albumoses into serum albumin. A mixture of the digestion- products of casein has been observed to "jell" under the influence of intestinal secretion supposedly due to the action of a ferment. 41 Ernst Freund observed that upon bringing together fresh horse-serum and a solution of Witte 's pep- tone a portion of the albumoses become coagulable, and, further, that of the serum-proteins only euglobulin shows this characteristic, and that after addition of the peptone to serum the euglobulin fraction diminishes, while the pseudo- globulin and albumin fractions increase. 42 Italian authors have stated that precipitates are given by various tissue ex- tracts, some with blood-serum, some with peptone ; and have suggested the former type of precipitation as an expression of a binding of the circulating dietary protein with the tissue proteins. 43 "* F. Kutscher and J. Seemann (Physiol. Instit., Marburg), Zeitsch. f. physiol. Chem., 85, 432, 1902. • Grosmann (Kurajeff's Lab., Charkow), Hofmeister's Beitr., 6, 192, 1905. 41 E. S. London, Zeitschr. f. physiol. Chem., 74, 301, 1911. 42 E. Freund, Wiener med. Wochenschr., 1905, No. 47 ; 1909, 108. •"Pacchioni and Carlini (Pediatric Clinic, Florence), Arch, di Fisiol., 2, 297, 1905; abstract, Biochem. Centralbl., 4, 1490, 1905-06. 62 PROTEIN DIGESTION IN THE INTESTINE Interesting as such observations in themselves may be, the greatest caution should be observed in interpreting their physiological significance. That the physico-chemical rela- tions in as complicated colloid system as blood serum on the addition of a second, no less complex system, like Witte 's peptone, may be decidedly disturbed is in nowise surprising. It is very questionable whether any sort of conclusion is justifiable from such static change as to the actual physiology of absorption. Parenteral Introduction of Protein. — E. Freund 44 is of the opinion that the very fact of elaboration of parenterally introduced albumoses by the tissues, etc., bespeaks collabor- ation of the intestine. "Five minutes after intravenous injection of Witte 's peptone, renal elimination being pre- vented, only about half the injected substance remains in the blood. The fate of the peptone remaining in the blood appar- ently depends upon whether or not the intestinal blood ves- sels be traversed or not. If these are closed off the great bulk of the injected incoagulable substance remains in the blood . . ." Other experiments, 45 as those conducted by von Körösy, upon the fate of parenterally introduced pro- teins in conditions of intestinal exclusion are, however, not confirmatory of Freund 's assertion; according to these all protein to undergo cleavage in the organism must first pass the intestine and there undergo a particular preparation. Eeturning to the major question, as to the ability of the intestine to dissociate protein to its elementary building- stones: there can no longer be the least doubt of this if Abderhalden^ investigations are to be accepted. 46 The older view that proteid nitrogen is absorbed exclusively in the form of ' ' albumoses ' ' and ' ' peptones ' ' is no longer ten- able in the light of this knowledge. Another question, how- 44 E. Freund and H. Popper, Biochem. Zeitschr., 15, 272, 1909 ; G. Töpfer, Zeitschr. f. experimental Pathol., 3, 45, 1906; E. Freund and G. Töpfer, ibid., S, 633, 1906; E. Freund, ibid., 4, 1, 1907. 45 K. v. Körösy (Budapesth), Zeitschr. f. physiol. Chem., 62, 68, 1909. 40 Cf. E. Abderhalden, Zeitschr. f. physiol. Chem., 7 4, 436, 1911. PROTEIN SYNTHESIS FROM CLEAVAGE PRODUCTS 63 ever, upon which there is no unanimity, is to what stage maximal cleavage actually proceeds in physiological condi- tions. Many authors retain the older view "that," as E. Freund 47 says, "the body does not follow a single restricted principle, but manifests a many-sided ability in correspon- dence with the varying demands upon it." "Just as in ordinary household activities there is need not only of small firewood but also of larger timber, so the organism makes use of the protein material in form of all kinds of cleavage- products without first reducing all to the smallest grade." Protein Synthesis from Products of Advanced Cleavage of Protein. — The view that all protein undergoes advanced cleavage before resorption assumes the possibility, for main- tenance of the body in nitrogen equilibrium, of its being pro- vided, not with protein, but with the sum total of the lowest products of protein cleavage. The credit of having first experimentally proved this possibility belongs to Otto Loewi. In 1902 Loewi, working in the laboratory of Hans Horst- Meyer, showed experimentally by feeding with pancreatic tissue autolysed to the point of loss of biuret reaction that the aggregate of the biuret-free end-products can be substituted for dietary protein (that is, enters into the composition of every part of the body protein undergoing metabolic disintegration). 48 At first this fundamental proposition was received with widespread doubt ; but later, especially following the experiments of Henriques and Han- sen 49 and, above all, the studies of Abderhalden and his school it has been adopted as beyond doubt. 50 47 E. Freund., Wiener klin. Wochenschr., 1905, No. 47. ** O. Loewi ( Pharmacolog. Instit., Marburg), Arch. f. exper. Pathol., 58, 303, 1902. «»V. Henriques and C. Hansen, Zeitschr. f. physiol. Chem., -J5, 417, 1904; 49, 113; 5k, 406, 1907. 60 Literature upon the Synthesis of Protein from the Low Protein Cleavage Products: H. Lüthje, Ergebn. d. Physiol., 7, 805-830, 1904; P. Rona, Handb. d. Biochem., 4', 540-560, 1910; E. Abderhalden, Synthesis of Cellular Building stones in Plant and Animal, Berlin, J. Springer, 1912, 128 pp. 64 PROTEIN DIGESTION IN THE INTESTINE Abderhalden^ Experiments. — Abderhalden and his asso- ciates have by a long series of carefully conducted experi- ments so fully solved many of the problems relating to this field of our study that it is possible to regard it as fairly comprehensible. It is known today that by appropriate and long continued combined action of pepsin, trypsin and erepsin proteid matter may be completely (that is, comparably to total acid hydrolysis) dissociated, and that a mixture of the products of such process is capable of maintaining in nitrogen equilibrium for many weeks ani- mals and human beings, whether in course of growth, in conditions of pregnancy or of lactation. The integrity of the hepatic function is moreover not a matter of necessity in connection therewith ; for Abderhalden and London were able to maintain in nitrogen balance a dog with Eck 's fistula upon a diet made up of advanced protein cleavage prod- ucts. Contrary to the belief that only the aggregate of split products from digestive ferment action and not those from acid hydrolysis are capable of replacing protein, Abder- halden has been able to keep in full nutrition for fourteen days a dog fed upon meat completely hydrolysed by boiling with strong sulphuric acid. Bemoval of tryptophane from the mixed digestive products rendered the mixture unfit for maintenance of nitrogen balance. The same inefficiency was found in employing a mixture of aminoacids obtained by cleavage of silk, this proteid being characterized by its high proportion of glycocoll, alanin and tyrosin, and its poverty in a number of the important "building stones" of the typical protein molecule. Similar results were obtained with gelatine, a substance rich in glycocoll, containing very little alanin, and neither tyrosin nor tryptophane. If the glycocoll of the split gelatine is proportionately diluted by addition of the other building-stones which occur in small quantities the combination may be made to be entirely equivalent to protein. It may be questioned whether the dissociated protein is capable of replacing normal protein ABDERHALDENS EXPERIMENTS 65 not only in a qualitative but also in a quantitative sense. Experiments of E. Voit and Zisterer, 51 in which the matter of "economy" comes forward, apparently bear upon this point, uncleft casein being superior to digested casein and the latter superior to casein hydrolyzed by acid. With ad- vance of cleavage the physiological nutritive value of a sub- stance apparently decreases, larger and larger amounts at any rate being required for maintenance of nitrogen balance. On the other hand a recent series of experiments by Abder- halden, Frank and Schittenhelm would indicate that protein equivalence of the combined cleavage products is a complete one. Acquiescence may be granted to the first of these in- vestigators only in the sense that positive results in such experiments are always of more significance than are nega- tive ones. Experiments carried on by Buglia 52 in Botazzi's laboratory clearly confirm the view that the products of meat digestion can be substituted for the meat itself without noteworthy difference of result in nitrogen fixation and in- crease of body weight in growing animals. Finally Abderhalden in a very interesting experiment has fed animals upon a general diet, not merely proteid sub- stances, in state of complete cleavage. Dogs were given, be- sides the fully split protein, the cleavage products of fat (glycerol and the higher fatty acids), monosaccharids, cholesterol, the structural units of nucleinic acid and the necessary ash constituents. In the course of experiments extending over more than two months the experiment ani- mals not only maintained their nitrogen equilibrium but actually increased in weight. This is an example of indefatigable and purposeful labor ending in the solution of a great problem. If one reflects, 51 E. Voit and J. Zisterer (Veterinary School, Munich), Zeitschr. f. Biol., 58, 457, 1910. 62 G. Buglia (F. Bottazi's Lab., Naples), Zeitschr. f. Biol., 57, 365, 1911. 5 66 PROTEIN DIGESTION IN THE INTESTINE we may often find that the very greatest advances in physiology may be formulated in a few simple words ; in this case they run: "the problem of the role of foodstuffs of complicated structure is solved by their most simplified structural units." 53 Application of these Results in Sickroom Dietary. — These results are not only of interest to physiologists, but are of extreme importance in practical medicine. Abder- halden, Frank and Schittenhelm 54 have actually prevented nitrogenous loss for weeks in a patient by rectal feeding with beef split by the combined action of trypsin and erepsin until the biuret reaction no longer showed. This proves the possibility of introduction into human beings of their daily nitrogenous requirement without bringing the parts con- cerned with protein cleavage into activity. The nitrogen may thus be introduced in fluid form in small volume for quick and complete resorption. It may be predicted that in the future the employment of preparations of this sort will find a field in the treatment of gastric ulcer, stenoses, can- cerous and ulcerous processes of any sort wherever they may exist in the digestive tube. Where there is occasion to spare the digestive tract, theoretically it is unquestionably more rational to introduce the physiologically definite mixture of the end products of protein cleavage than to administer the usually indefinite prepared foods with which people in re- cent years have been so richly favored (largely through in- sistent laudatory advertising and in a much less measure 53 E. Abderhalden, Synthese der Zellbausteine in Pflanz und Tier, p. 74, Ber- lin, J. Springer, 1912. Cf. therein (pp. 116-118) a summary of the studies conducted by Abderhalden in association with P. Bona, B. Oppler, E. S. London, J. Olinger, E. Meszner, H. Windrath, F. Frank, A. Schittenhelm, F. Glamser, D. Manoliu, and A. Suwa. Cf. also Zeitschr. f. physiol. Chem., 77, 22, 1912. M E. Abderhalden, F. Frank and A. Schittenhelm, Zeitschr. f. physiol. Chem., 63, 214, 1909; F. Frank and A. Schittenhelm (Med. Clinic, Erlangen), Münchener med. Wochenschr., 1911, 1288; Therap. Monatsch., 26, 112, 1912. AMIDES IN METABOLISM OF VEGETARIANS 67 upon the ground of actual scientific investigation). Of course every soluble protein preparation has in the end a cer- tain ' ' nutritional value ' ' ; whether the ' ' nutritional value" in any individual case bears any direct proportion to the price charged for the preparation is a matter which may here be gladly omitted. Unfortunately, however, as far as odor and flavor are concerned, the applicability of the cleavage prod- ucts of protein is far from what one would wish; and moreover, perhaps because of amines present, a variety of collateral effects are likely to appear and for the present de- tract from the therapeutic appreciation of these substances. After what has been said above, the possibility of various nitrogenous substances serving as partial substitutes for protein in nutrition may be readily understood. A number of investigations bearing upon this point, concerning the nutritive value of leucin, various albumoses, peptones, pro- tamines, etc., have been published. 05 It may at least be seen that such preparations may serve to conserve protein and that their value as more or less complete substitutes for pro- tein must primarily depend upon just what and how many of the metabolically essential structural units of the protein molecule are missing from their atomic grouping. Relation of Amides in Metabolism of Vegetarians; Pro- tein Synthesis from Ammonium Salts. — Although the above questions, at least in a fundamental way, seem fairly solved, this certainly cannot be said of another matter, the relation of the amides and aminoacids in metabolism. Because of the wide distribution of aminoacids and amides in plants and owing to the fact that the determination of the value of the various foodstuffs (as turnips, molasses, etc.) is of very great 55 A. Ellinger (Physiol. Instit., Munich), Zeitschr. f. Biol., 33, 201, 1896; L. Blum (Hofmeister's Lab.), Inaug. Diss., Strassburg, 1901; V. Henriquez and C. Hansen, Zeitschr. f. physiol. Chem., 48, 383, 1906; 49, 113, 1906; P. Bona and W. Müller, ibid., 50, 2G3, 1906; J. B. Murlin, Amer. Journ. of Physiol., 20, 234, 1907-08; cf. therein literature appended. 68 PROTEIN DIGESTION IN THE INTESTINE practical interest in agriculture, an unusually large number of studies have been directed to the question of how far amides, especially as par agin (as W. Völtz and others have maintained), are capable of replacing the protein of food. 56 It is readily appreciable that amides and aminoacids may serve as conservers of protein. There is, however, a con- stantly recurring idea that at least in herbivora protein may be replaced by such amides. N. Zuntz, 0. Hagemann and other writers would find the key to the whole problem in the role of the intestinal bacteria. Introduction of asparagin and similar substances they believe not only serves to con- serve protein and preserve the dietary protein from the influ- ence of the bacteria; but the amides are a medium from which the bacteria because of their plant characteristics are able to construct protein. If in the end the bacteria die, this protein of theirs is of advantage to the vertebrate animal. In this manner not only asparagin, but ammonium acetate and many other ammonium salts may indirectly, of course, become a source of protein for the animal body. In the course of the last few months the question of the availability of ammonium salts and amides to enter into protein synthesis has been brought into prominence from the studies of E. Gräfe and of Abderhalden. E. Gräfe and Schläpfer from extensive metabolic studies determined that in dogs receiving a full diet of non- nitrogenous material not only nitrogen balance but actual nitrogen retention and increase of weight can be obtained by feeding ammonium citrate, without subsequent outpouring 60 K. Anderlik, K. Velich and VI. Stanek, K. Friedländer, S. Gabriel, O. Hagemann, V. Henriques and C. Hansen, J. Just, 0. Kellner, C. Lehman, A. Morgen, C. Beger and F. Westhauser, Mauthner, M. Müller, J. Munck, E. Peschek, G. Politis, B. v. Strusiewicz, F. Rosenfeld, VV. Tkär, W. Völtz, H. Weiske, N. Zunz. Literature: H. Lüthje, Ergebn. d. Physiol., 7, 828-830, 1908; E. Abderhalden, Vorlesungen, 2d ed., pp. 301-303, 1909; P. Rona, Handb. d. Biochem., //, 554-559, 1911; E. Peschek (Zootech Instit. of Agricul. School, Berlin), Pflüger's Arch., llß, 143, 1911. AMMONIUM SALTS IN PROTEIN SYNTHESIS 69 of the retained nitrogen taking place. 57 It would, however, be decidedly misleading to regard this as evidence indicating protein synthesis from ammonia and nitrogen-free material. The above findings have been confirmed by independent observations of Abderhalden to the extent that after addi- tion of ammonium acetate to a nitrogen-free diet consisting of rich fatty and carbohydrate constituents there may ensue a nitrogen retention. 58 In connection with the observations (to be discussed later in detail) of Knoop, Embden and their associates upon the transformation of a -ketonic acids into aminoacids by the fol- lowing schema: R— CH 2 R— CH, I I CO+NH, T^: CH NHg+O I I COOH COOH, it is not difficult to think of a shifting of the equation between aminoacids and ketonic acids by the introduction of am- monia in a way to bring about synthesis of aminoacids, the ketonic acids, however, to be derived from nitrogen-free food constituents. In further studies 59 Abderhalden, however, pointed out that it is a matter of great difficulty to draw any consistent conclusion from the results. In reality it turned out that even without nitrogen administration he was able by full diet of carbohydrates and fats to maintain for a long time constant body weight and, in fact, for short periods to induce increase of weight of the experiment animals; so that apparently it is inadmissable to come to any conclusions as to protein synthesis after administration of ammonium salts whether there be weight loss or weight increment. 57 E. Gräfe, Kongr. f. innere Med., Wiesbaden, 1912; E. Gräfe and V. Schlüpfer, Zeitsch. f. physiol. Chem., 77, 1, 1912; E. Gräfe, ibid., 78, 485, 1912; cf. claim for priority by W. Völtz, ibid., 79, 415, 1912. 58 E. Abderhalden, ibid., 7S, 1, 1912. K E. Abderhalden and P. Hirsch, Zeitschr. f. physiol. Chem., 80, 136, 1912. 70 PROTEIN DIGESTION IN THE INTESTINE In another series of experiments 60 Abderhalden fed gela- tin which is not a full equivalent for protein because the ani- mal cells are apparently unable to fonn de novo certain build- ing stones which are absent from gelatin. He then sought to increase the chance of protein synthesis in the organism during a full diet of carbohydrates and fats by adding am- monium acetate to the gelatin. But the nitrogen record constantly remained negative. "From these experimental results we may conclude," he says, "that the basis for protein synthesis is not to be found in ammonium salts with carbohydrate or fatty material. ' ' London's Studies. — It is impossible to conclude the sub- ject of protein digestion in the intestine without special con- sideration of the investigations which for years E. S. London with a number of collaborators has been conducting in the In- stitute of Experimental Medicine in St. Petersburg. London has developed the technic of intestinal fistulas to a high de- gree of perfection. He is not satisfied to provide a single fistula in the experimental animal ; his " polyfistulous dogs ' ' enable him to make comparative studies of various intestinal levels at one given time ; his " panchymotic dog, ' ' wearing in addition to a double-chambered duodenal canula a gastric and a jejunal canula, puts him in position to obtain coinci- dently the gastric secretion, the bile, the pancreatic juice and the intestinal secretion in proper isolation from each other. For a number of years London has been publishing the results of his studies in a very long series of contributions embracing a wealth of individual observations. 61 They take up the rate of protein catabolism, the resorption inten- sity and movement of the intestinal contents in particular 60 E. Abderhalden and A. E. Lampe, Zeitsckr. f. physiol. Chem., 80, 160, 1912. 61 E. S. London and associates, numerous publications in the last thirty volumes of Zeitschr. f. physiol. Chem. LONDON'S STUDIES 71 segments of the bowel, nitrogen partition in the intestinal contents, the completeness of food utilization, the results of exclusion of special segments of the intestine, of the bile and of the pancreatic secretion, protein digestion in mixed diet especially in presence of carbohydrates and fats, the specific adaptation of the digestive juices and the relative amounts of enzymes in the intestinal chyme in different diets, the dif- ferences between raw and cooked proteids, the behavior of various proteins (as serum albumin, egg albumin, muscle albumin, casein, gliadin, gelatin and elastin) and of digestive mixtures (removed through a fistula from one dog and introduced by a fistula into another dog), the resorption of individual aminoacids, the mechanism of secretory flow, the influence of blood pressure and other physiological factors upon secretion of the individual digestive juices, and other similar subjects. He has endeavored, too, to deduce a mathematically fixed relation between the different secretory factors (as the relations between the amount of food, the concentration of the gastric juice, the quantity of the bile and pancreatic juice, the alkali-content of the pancreatic and intestinal secretions, the nitrogen-content of the different secretions and the digestion time of solid albumins) in which as in chemistry of the ferments "quadrate root rules" (in Arrhenius' sense) play an important part. 62 The author confesses that he does not regard himself as fitted to criticise with full appreciation this remarkable wealth of undoubtedly very valuable observations. It would be a real service were London to take upon himself the duty of critically reviewing them in their proper relations and of bringing out clearly the lines of thought he has followed, which are now so divided among so many separate publica- tions that the outsider is bound to lose their connection. c2 Consult also E. London and C. Schwarz, Zeitschr. f . physiol. Chem., 68, 34G, 1910; L. Popielski, ibid., 71, 186, 1911. 72 PROTEIN DIGESTION IN THE INTESTINE This done, we may well hope that from these studies and others dealing with related matters (of which special men- tion may be made in passing of those of Cohnheim, 63 E. Zunz, 04 Brugsch, 05 A. Scheunert, 66 and K. Glässner 67 ) a well rounded picture of protein dissociation in the intestine in each of its phases will gradually take shape. 68 Summary. — If in conclusion another glance be cast back over the long way behind us, it will be realized that as the actual accomplishment of an almost interminable expendi- ture of effort and experimental work we may say that we know that by the combined action of pepsin, trypsin and erep- sin the protein in the alimentary canal can be effectively split into its most simple structural units and that a mixture of these constituent fragments is not only qualitatively but quan- titatively in every sense equivalent to the protein as supplied intact. To be able to say that means something, but by no means all. Abderhalden says very properly "that up to this time it is entirely impossible from examination of the intestinal contents to draw exact conclusions as to the degree of catabolism of the food, because besides the products of complete cleavage we must take into consideration also the intermediate substances." He states explicitly, too, "from our finding that a combined mixture of aminoacids is capable of fully maintaining protein metabolism for weeks and months we cannot conclude at once that necessarily in 63 R. Baumstark and O. Cohnheim (Physiol. Instit., Heidelberg), Zeitschr. f. physiol. Chem., 65, 477, 483, 1910. 64 E. Zunz, Bulletin de l'Acad. Roy. de Med. de Belgique, 30, April, 1910; cf. here Literature and review of author's numerous earlier contributions in same connection. 65 Th. Brugsch, Zeitschr. f. exper. Pathol., 6, 326, 1909. 6n A. Scheunert, Pfhiger's Arch., 121, 169, 1908 ; 139, 131, 1911; 1^1, 411, 1911. 67 K. Glässner (F. Kraus' Clinic, Berlin), Zeitschr. f. klin. Med., 52, 361, 1904. 68 Cf. also V. Lieblein (Prague), Zeitschr. f. Heilk., Abt. f. Chirurgie, 1906; F. Keller, Inaug. Diss., Breslau, 1909, abstract in Centralbl. f. Physiol., 23, 616, 1909. SUMMARY 73 normal conditions the cleavage of protein by digestion in the gastro-intestinal canal ultimately extends to the stage of aminoacids. ' ' 69 We lose track, practically under our very eyes, of the split products of the protein molecule as they pass out from the intestine into the blood. Accumulation of albumoses or of aminoacids in the blood of an individual in the course of digestion under entirely normal and physio- logical conditions has not been established beyond doubt, and on the other hand has not been definitely disproved. There is some justification therefore in the suspicion that there occurs a protein reconstruction in the wall of the intestine. The author believes, too, that it is by no means improbable that both the smaller and the larger cleavage products of the protein molecule may enter the circulation, be carried to the various organs and in the latter be further elaborated ac- cording to the local requirements, either being condensed into new protein molecules, or in further dissociation finally passing into various end-products (ammonia, carbon dioxide and water). The biological serum reactions (precipitin-reactions.etc.) have led to an appreciation of the almost endless multiplicity and specific peculiarities of the proteid substances with which nature deals. Axiomatically we come to the assump- tion, from the fact that every individual contains in the various fluids and tissues an enormous number of chemically variant proteid substances, that doubtless no two different kinds of animals contain exactly identical proteins. This tremendous multiplicity may be appreciated if we think of the equally tremendous number of variations in which the great numbers of "mosaic stones" of the protein molecule may enter into combination in its construction. That a given individual, no matter the extreme variety of Proteids entering into its food, is capable, always and under 68 E. Abderhalden, Synthese der Zellbausteine in Pflanze und Tier, pp. 53 and 71, Berlin, J. Springer, 1912; cf. Zeitschr. f. physiol. Chem., 78, 382, 1912. 74 PROTEIN DIGESTION IN THE INTESTINE all conditions and for the whole period of its life, of main- taining the specificity of its own peculiar proteins can be understood and conceived of, as far as the author is per- sonally concerned, only by adopting the idea that every proteid in the food is probably, before it can be assimilated, broken down into aminoaeids. This conception must be acknowledged, however, to be purely a personal one, and is offered only as such. In the last count every man can think only with his own brain. CHAPTER IV PROTEOLYTIC AND PEPTOLYTIC TISSUE FERMENTS AUTOLYSIS After the consideration in the previous chapters of the processes of protein digestion in the stomach and intestine, and with the future consideration of the fate of the final nitrogenous cleavage products in the intermediate metab- olism before us in succeeding ones, the present discus- sion may very properly be devoted to the protein- digesting tissue ferments. At this point study of that mys- terious change which is inclusively spoken of as "autolysis" may serve in part to bridge the great gap, or perhaps in more exact verbiage to conceal it, between the two phases of the metabolic process. It has been pointed out that the prod- ucts of cleavage of the protein molecule are lost sight of at the very moment when they are resorbed through the wall of the bowel; and their first traces reappear when the end- products of metabolism begin to escape from the body. That which lies between is the unexplored and completely un- known field of the intermediate metabolism. The hope of throwing some light from a single point of view upon a little part of this field, as with the light-cone of a projection lamp, lends special interest to the study of the phenomena of autolysis. Even if these expectations, at best provisional ones, fail of fulfilment, the enigmatic process is in itself suf- ficiently interesting to temporarily hold our attention. As early as 1890 Ernst Salkowski recorded the occur- rence of post mortem self -digestion ("autodigestion") in animal tissues. He noted, for example, that in a pulp- like suspension of tissue in chloroform water autodigestive processes take place, with disappearance of coagulable albumins to give place to their cleavage products. Then ten years later in Hofmeister 's laboratory Martin Jacoby 1 1 M. Jacoby (F. Hofmeister's Lab., Strassburg), Zeitschr. f. physiol. Chem., SO, 149, 174, 1900. 78 76 TISSUE FERMENTS investigated more exactly the bearing of these phenomena in relation to various physiological and pathological processes ; since when they have been the subject of general attention. Thereafter "autolysis" (a term introduced by Jacoby which has since been adopted generally) has remained an important subject in the list of problems of physiological pathology. At this point we may as well attempt to explain the process in as practical a manner as possible, whatever the outcome. Autolysis a Complex Process. — It is essential that we keep in mind that the autodigestive processes are really of complex nature, for the reason that they are not the result of the isolated action of any one given enzyme but of a number of ferments. Side by side in their operation are the protein- splitting "tryptases," "erepsines" (concerned in complet- ing the dissociation of the high molecular cleavage products of the protein molecule), "arginases," ammonia-splitting " deamidases," "nucleases" splitting the nucleins, the mys- terious "oxidases," and besides these undoubtedly many more for which we do not even have a name. It is not at all remarkable therefore that in the study of autolytic mixtures we have to do with a decidedly "mixed group"; along with the typical cleavage products of proteins and nucleinic acids, and besides the derivatives of these (as guanidin, tetra- methylendiamin and aminobutyric acid) are to be met a variety more of products (as lactic acid, succinic acid, volatile fatty acids), the origin of which is by no means certain. 2 Antiseptic Difficulty in Autolysis Experiments. — There is, however, one very great difficulty in conducting ex- periments in autolysis, namely, that of insuring antisepsis. 2 A. Magnus-Levy, Hofmeisters Beitr., 2, 261, 1902; S. Isaak (F. Hof- meister's Lab.), Inaug. Diss., Strassburg, 1904; M. Schenk (Physiol. Instit., Marburg), Wochenschr. f. Brauerei, 1905, Xo. 16; abst. Centralbl. f. physiol. 19, 519, 1905; P. A. Levene, Zeitschr. f. physiol. Chem., J t l, 393, 1904; Amer. Journ. of Physiol., 11, 437, 1904; 12, 276, 1904. AUTOLYSIS EXPERIMENTS 77 Only within the last few years has this difficulty come to be definitely realized and technically avoided, thanks to the studies of Salkowski and his pupils. 3 For instance, it might be supposed that when a finely divided organ is undergoing autodigestion in a medium well saturated with chloroform and toluol, there would be no danger of the unwelcome pres- ence of bacteria. In actual practice, however, such exact care has by no means been insisted upon ; and it has usually been supposed that if a few drops of toluol or a few bits of thymol were added to a thick emulsion of some organ it would be quite safe to let it stand in the incubator for some months and that the danger of bacterial invasion would be surely and for all time eliminated by such a purely symbolic perform- ance (for that is actually about all it represents) . It is quite obvious why "discoveries" prospered and multiplied in our literature on the same scale as did the bacteria in the pots and jars of the experimenters. But in addition, even with precautions to avoid such gross technical faults, other dif- ficulties often arose in the way of microbic contaminations. H. C. Jackson 4 pointed out that fresh canine livers even if removed with the utmost precaution are rarely entirely sterile, but are apt to contain an anasrobic organism similar to the hay bacillus which does not grow on the ordinary culture media but which grows readily in "sterile" tissues. Jackson believes that the appearance of lactic acid, succinic acid, butyric acid, hydrogen and sulphuretted hydrogen (as noted, for example, by Magnus-Levy in "aseptic autolysis," and their origin referred by him to the corporeal carbohy- drates and fats) is due to the presence of microorganisms in the autolysing mixtures. Here again we are confronted with that intolerable dilemma besetting every point and every side of the chemistry of fermentation : either we are destroying the normal physiological course of the processes by introduc- 3 E. Salkowski, Yoshimoto, Kikkoji and others, Zeitschr. f. pkysiol. Chem., 63, 109, 1036, 1909. 4 H. C. Jackson, Journ. of Med. Research, 21, 281, 1909. 78 TISSUE FERMENTS ing disinfecting agents which in the very nature of things cannot be without effect upon the enzymes ; — or else we keep on, making allowance as we may for the danger of bacterial invasion. Is Autolysis the Continuation of an Intra-vital Process? — It may be assumed with certainty that no one would have devoted as much effort and care to these rather bothersome subjects if it were not for the impression that just here there is a chance to remove one part of the intermediate metabol- ism from the dark interior of the living economy into the transparency of the test tube. It was believed that in the phenomena of post-mortem autodigestion there may be rec- ognized a continuation of a normal intra-vital process, that of the physiological protein dissociation which takes place in the living cell. The first point therefore to be determined is whether such a belief is or is not justified. 5 Autolytic tissue ferments, it must be accepted, have been met throughout the animal and vegetable kingdoms gen- erally, wherever any pains have been taken to recognize them, in the lowest as well as in the most highly organized living entities. Ferments have been differentiated which, on the one hand, act best in acid reaction, and on the other those best acting in alkaline conditions a - and ß-proteases of Hedin and Rowland). Vernon emphasizes "erepsines" above other proteases, and proposes to estimate the relative amount of enzyme in tissue extracts colorimetrically by application of the biuret test (supposing the time required to cause the biuret reaction to disappear from a given quan- tity of a standard solution of Witte 's peptone to be in reverse proportion to the amount of ferment present) . By compari- 6 Literature upon the Physiological Significance of Autolytic Processes: M. Jacoby, Ergebn. d. Physiol., 1, 225-229, 1902, and Handb. d. Biochem., 2', 175-182, 1910; E. Salkowski, Deutsche Klinik, 11, 147-182, 1907; H. M. Vernon (Oxford), Ergebn. d. Physiol., 9, 147-158, 1910; J. Wohlgemuth, Handb. d. Biochem., 3', 180-181, 1910; C. Oppenheimer, Die Fermente, 3d ed., pp. 242- 252, 1910. IS AUTOLYSIS AN INTRA- VITAL PROCESS? 79 son of various organs Vernon found the duodenal mucous membrane richest in erepsin, that of the lower portions of the intestine poor in erepsin ; of the other organs the kidney showed the highest content of ferment. It can be readily appreciated that there is difficulty in attempting to deal with all of the observations upon autolysis as it were under one heading, because of the extreme com- plexity of the chemical processes concerned. It may be re- garded as a step in advance that R. L. Benson and H. G. Wells 6 have perfected a physico-chemical method of follow- ing continuously the course of autolysis by determining the freezing-point and electrical conductivity of the autolysate, in place of depending exclusively upon the increment of incoagulable nitrogen or the disappearance of the biuret reaction. The whole idea of the physiological significance of the autolytic ferments is in close relation with the proposition that increase in the "physiological activity" of an organ is somehow related with increase of its intrinsic autolytic capacity. Accepting provisionally the correctness of such a statement, if we endeavor to satisfy ourselves as to the actual basis thereof, it is astonishing to find upon how little foundation of fact it rests. A few observations from Hof- meister 's laboratory bear upon the subject. That autolysis in the tissues of children of low vitality proceeds more slowly (measured by increase of incoagulable nitrogen) than in those of normal individuals, 7 and that the autolytic activity of a functionating mammary gland is higher than of a rest- ing gland 8 (cf. Vol. I of this series, p. 361, Chemistry of the Tissues) should be mentioned; and in addition a few of Vernon's observations 9 are applicable. According to this author the average quantity of "erepsin" in a tissue is prac- 6 R. L. Benson and H. G. Wells, Journ. of Biol. Chem., S, 61, 1910. T E. Schlesinger (F. Hofmeisters Lab.), Hofmeister's Beitr., J h 87, 1903. 8 P. Hildebrandt (F. Hofmeister's Lab.), Hofmeister's Beitr., 5, 463, 1904. •1. c. 80 TISSUE FERMENTS tically constant and virtually independent of diet ; neverthe- less it varies with the state of functional activity of the tissue. In fetal tissues it is at minimum, but the amount of erepsin rapidly increases as development proceeds, reach- ing the maximum soon after birth. It is asserted, too, that the increased protein destruction which is brought about by excessive thyroid substance entering the system is also mani- fested by increase of post-mortem autolysis 10 ; although these statements have been opposed. Addition of thyroid material has not been found to directly increase autolysis (consult Vol. I of this series, p. 450, Chemistry of the Tissues ) . It has been found that autolysis is decidedly more active in case of animals which have fasted before death than in those that were well nourished. 11 It is possible to interpret this as a continuation of the tissue destruction which is going on in marked degree in fasting ; but one might on the other hand be justified in assuming just the opposite, namely, that the " functional activity" of the tissues is lowered in states of fasting while their digestive ability is increased, in line with the fall in quantity of erepsin in the tissues of hibernating animals and persons reduced by disease which Vernon very satisfactorily demonstrated. 12 A number of objections have been presented against regarding autolysis as a physiological or vital process. It is said that autolysis cannot possibly be a direct continuation of a vital process because it does not appear immediately when death occurs, but after a period of latency of several hours. 13 Then, too, the predilection of the autolytic ferments for an acid reaction has been made an objection 14 ; (although this is answered by the fact that even with the natural reaction 10 Cf. G. Bayer (M. Löwit's Lab., Innsbruck), Sitzungsbericht d. Wiener Akad., US'", 181, 1909. 11 J. E. Lane-Claypon and S. B. Schryver, Journ. of Physiol., 81, 169, 1904. 12 H. M. Vernon, Journ. of Physiol., 33, 81, 1905. 13 Lane-Claypon and Schryver, 1. c. 14 H. Wiener (J. Pohl's Lab., Prague), Centralbl. f. Physiol., 19, 349, 1905. DEAMIDIZING TISSUE FERMENTS 81 of the blood and tissue-fluids autolytic processes can pro- ceed 15 ). In a study carried out in the laboratory of Julius Pohl 16 it has been shown that allantoin, which appears in large amounts in autolysis of dog's liver 17 and for which there is no reduction capacity on the part of the canine economy, appears nevertheless in but very small amounts among the normal excretory products. (Of course, if autolysis were actually a complete duplicate of intravital changes, we would necessarily expect that allantoin would normally be excreted as a product; yet autolysis might well be only one part of a vital process and it might well be that in conditions where the other components, particularly the vital oxidations, are absent it would not necessarily give rise to exactly the same end-products as found in life.) To the persistent question of exactly why, if autolysis actually occurs in the living body the living cells are not digested, here again we have presented, one after the other, anti- ferments, the immune bodies of the serum, the alkaline con- ditions, etc., in short the whole arsenal of problematic hypotheses with which, as the author has previously in- sisted, we are in the vain habit of trying to conceal our ignorance of the exact reason why the stomach, intestine and pancreas are prevented from digesting themselves. Deamidizing Tissue Ferments. — The atmosphere sur- rounding this subject is rather unattractive and obscure, making it a field to which the author is glad to devote no more time than is actually necessary. Before proceeding to better understood matters, however, he wishes at least briefly to refer to the deamidizing ferments. As originally observed by Martin Jacoby 18 in Hofmeister 's laboratory, in the course 15 J. Baer and A. Loeb, Arch. f. exper. Pathol., 53, 1, 1905; A. v. Drjewecki, Biochem. Zeitschr., 1, 229, 1906; Preti, Zeitschr. f. physiol. Cliem., 52, 485, 1907. 16 R. Poduschka (Pohl's Lab., Prague), Arch. f. exper. Path., 4> f , 59, 1900. 17 Borrissow (Baumann's Lab.), Zeitschr. f. physiol. Chem., 19, 499 (1904). W M. Jacoby (F. Hofraeister's Lab., Strassburg), Zeitschr. f. physiol. Chem., 30, 149, 1900. 6 82 TISSUE FERMENTS of autolytic cleavage of tissue proteids there occurs a much more marked production of ammonia (presumably from the non-fixed nitrogen which is split off as ammonia by hy- drolysis) than in acid hydrolysis. Thereafter Sigmund Lang 19 in the same laboratory determined the autolytic separation of ammonia both from acid amides (as asparagin, COOH— CH 2 — CH.NH 2 — C0NH 2 , and glutamin) and from aminoacids. It is now recognized that deamidization of the first of these series takes place very readily in alkali- hydrolysis according to the schema : E.CONH 2 -f H 2 = R.COOH + NH 3 . The author has not failed to obtain this result in any of his studies, conducted in association with M. Friedmann, in quantitative comparisons of autolytic asparagin cleavage in various animal tissues. 20 The fre- quent occurrence of amide-cleaving ferments in vegetable tissues, which are rich in acid amides, has been fully estab- lished by a number of investigators. 21 Of more interest than this group of enzymes are the deamidases , which are capable of splitting off from an aminoacid its firmly attached nitro- gen, which successfully resists the cleaving power of boiling fuming hydrochloric acid. Just as yeasts or moulds can split ammonia from aminoacids and transform them into oxy-fatty acids 22 (according to the schema: B.NH 2 E.OHK | + H 2 = NH 3 -f| ) so precisely the same COOH COOH/ r J changes can apparently take place in autolysis of animal tissues. 23 Cohnheim has observed a demidization of this sort in the passage of aminoacids through the living intes- 19 S. Lang (F. Hofmeister's Lab., Strassburg), Hofmeister's Beiträge, 5, 320, 1904. 20 0. v. Fürth and M. Friedmann, Biochem. Zeitschr., 26, 435, 1910. 21 K. Shibata, N. Castoro, H. Pringsheim, W. Butkewitsch, A. Kiesel. Literature: O. v. Fürth and M. Friedmann, 1. c. 22 H. Pringsheim, Biochem. Zeitschr., 12, 15, 1908. 23 S. Lang, 1. c, O. Schumm, Hofmeister's Beitr., 7, 175, 1906; F. Simon (Salkowski's Lab.), Zeitschr. f. physiol. Chem., 70, 65, 1910; W. Lindemann (Physiol. Instit., Halle), Zeitschr. f. Biol., 55, 36, 1910. IMPORTANCE OF AUTOLYSIS 83 tinal wall of fishes in the course of resorption; 24 which calls to mind the oft-repeated statement as to the excessive pres- ence of ammonia in the portal blood as it leaves the in- testinal wall. The author sometimes noted in the course of the above mentioned autolytic experiments an amount of ammonia obtained from asparagin by autolytic cleavage with intestinal mucous membrane, so large that it surely represented more than the loose amido-nitrogen present and probably was in part derived from cleavage of the closely- combined nitrogen of the aminoacid. The author acknowl- edges, however, on further consideration that perhaps, in spite of every ordinary precaution, microorganisms may have played some part — a thought which materially disturbs his satisfaction in this as well as in other similar results. Importance of Autolysis in Various Pathological Processes. — It must be accepted, therefore, that the impor- tance of autolysis in physiological processes is by no means satisfactorily established. With feelings of grateful satisfac- tion akin to those of the traveler who after the discomfort of a boggy moor finds firmer foothold again beneath his feet, we approach the question of the significance of autolysis in pathology. An important example of an autolytic process taking place in the living body has come to be recognized through Martin Jacoby's studies of the condition of the liver in phosphorus poisoning, 25 and very probably, too, in acute yellow atrophy of the liver which is very similar in its manifestations to phosphorus poisoning. 26 In both processes an impression is given that some toxic agent has impaired the vitality of the hepatic cells, and that the auto- **0. Cohnkeim and F. Makita (Heidelberg), Zeitscbr. f. physiol. Chem., 61, 189, 1909; 0. Cohnheim, Sitzungsber. d. Heidelberger Akad. d. Wiss., matb.- naturw. Klasse, 1911, 30 Abh. 26 M. Jacoby, Zeitschr. f. physiol. Chem., SO, 174, 1900; O. Porges and Przibram, Arch. f. exper. Patbol., 59, 20, 1908. 28 C. Neuberg and P. F. Richter, Deutsche med. Wochenschr., 1904, No. 14; A. E. Taylor, Journ. Med. Research, 8, 424, 1902; Zeitschr. f. physiol. Chem., 84, 580, 1902; H. G. Wells, Journ. of Exper. Med. 9, 627, 1907. 84 TISSUE FERMENTS lytic ferments thereafter are free to "complete the burial" (cf. Vol. I of this series, p. 558, Chemistry of the Tissues). Apparently the proteins in the course of the process become impoverished in their basic fractions 27 (especially in argi- nin), presumably because the basic moiety being a relatively instable component is rather readily lost in the catabolism of the protein molecule. 28 As the dissociation advances the final cleavage products, the aminoacids, are found in such quantity in the circulation that their presence in the blood and elimination in the urine become striking. In the course of these changes the exaggerated autolysis is by no means limited to the liver, although in the latter it is most manifest. Hsemic alterations in phosphorus poisoning, such as the loss of fibrinogen and of coagulability, have also been referred to autolytic changes. P. Saxl, in the Vienna Physiological Institute, has shown that even if dead tissues are treated with yellow phosphorus their autolysis is in- creased and the appearance of cellular " fatty degeneration" is induced, the fat present in the constitution of the cells becoming histologically visible. 29 Besides phosphorus there are undoubtedly many other toxic agents capable of increasing autolysis. Thus H. Gr. Wells 30 recognized extensive autolytic changes in the liver of a man dead from chloroform poisoning; a fact explicable from a study made in the laboratory of H. H. Meyer 31 indi- cating that a great number of narcotic substances, either in liquid or in gaseous form, are capable of hastening and increasing tissue autolysis. Apparently this is dependent 27 A. Kossel, Berliner klin. Wochenschr., 190.'t, 1065 ; A. J. Wakeman (Labor, of Kossel, Heidelberg, and of Herter, New York) , Journ. of Exper. Med., 7, 292 (1905) ; H. G. Wells, 1. c. 28 J. Wohlgemuth, Biochem. Zeitschr., 1, 161, 1906. 29 P. S'axl, Hofmeister's Beitr., 10, 447, 1907 (under direction of O. v. Fürth in Physiol. Instit., Univ. of Vienna). 30 H. G. Wells (Chicago), Journ. of Biol. Chem., 5, 129, 1909. 81 R. Chiari (H. H. Meyer's Lab., Vienna), Arch. f. exper. Pathol., 60, 255, 1909. AUTOLYSIS AND THE REGRESSIVE CHANGES 85 upon the lipoid-solvent ability of such substances which causes extensive changes in the intrinsic permeability of the cellular protoplasm. Another observation which should be noted in the same connection is that of L. Hess and P. Saxl 32 that tissue autolysis is apparently increased by the addition of diphtheria toxin, tetanus toxin and also of tuberculin (after a period of inhibition) ; from which these authors are disposed to suggest that toxines exert an influence toward protein cleavage. Further prosecution of similar studies is very desirable in order to determine to what extent the re- sults are due directly to the toxines and not to other coincident circumstances. Autolysis and the Regressive Changes in the Living Body. — The above is, however, by no means the only bearing of autolysis upon pathological processes. Its importance in the liquefaction and resorption of pneumonic exudates was recognized by Friedrich v. Müller and by 0. Simon 33 ; and there can be but little error in assuming that autolysis plays an important part generally wherever tissue structures, cellularized exudates, tumors, fibrinous clots, tissue grafts, etc., are resorbed in the living body. The author has had occasion (Vol. I of this series, p. 370, Chemistry of the Tissues) to point out that it is reasonable to believe that autodigestion likewise takes part in such physiological regressive processes as the involution of the uterus after delivery. The whole subject has such extensive practical bearing that it is really astonishing how little is actually known with reference to it. If autolytic processes occur thus widely in the living body it may naturally be expected that the system may be flooded with the products of autolytic digestion. From this 32 L. Hess and P. v S'axl (v. Noorden's Clinic, Vienna), Wiener klin. Wochenschr., 21, 248, 1908; cf. also A. Barloceo, Pathologica, 2, 195; abstracted in Jahresber. f. Tierchem., J,0, 887, 1910. 33 F. Müller, Verh. des 20. Kong. f. innere Med., 1902, 192; O. Simon, Deutsch. Arch. f. klin. Med., 70, 604, 1901. 86 TISSUE FERMENTS standpoint it is not without interest to note the appearance in the urine of albumoses after liquefaction of pneumonic exudates, in connection with large abscesses, and in instances of softening of tumors. Eamond 34 observed after ligation of the circulation in the hind leg of a rabbit for some hours that general disturbances follow when it is reestablished (dyspnoea, rapid pulse, etc.), perhaps due to autolytic prod- ucts which have gained access to the circulation. It seems quite possible, too, that "autointoxication" following the resorption of products of regressive autolytic changes may be an important factor in human pathology (to mention only the common term, "resorption fever") ; although it is true we have but little positive knowledge in this connection. It would not be hard to fancy, for instance, that the system is not indifferent to the influence of a wave of cholin and certain of its conversion products (v. Vol. I of this series, p. 190, Chemistry of the Tissues) which might find origin in auto- lytic cleavage of tissue lecithids (even if in a general way we know that the digestion products of the latter are harm- less after passing through the intestinal wall). Relation to Bacterial Processes. — In a further sense auto- lytic processes may have important pathological application in the fact that certain bacterial endotoxines, as those of the typhoid and cholera organisms, become active only after being set free by destruction of the bacteria themselves. It seems very likely to the author that there is an important meaning in the antibacterial and antitoxic influence of tissue autolysates (for example, the autolysate of lymph glands has a detoxifying influence upon tetanus toxine), as observed first in Hofmeister 's laboratory. 35 Proteolytic Ferments of Leucocytic Origin. — Within the past few years especial attention has been very properly devoted to a particular kind of autolytic fer- "F. Ramond, Jour, de Physiol., 10, 1052, 1908. «Conradi, Hofmeister's Beitr., 1, 193, 1901; L. Blum, ibid., 5, 142, 1904. PROTEOLYTIC FERMENTS 87 ments, tlie proteolytic leucocyiio enzymes, and their relation to suppuration. The way the tissues break down in these latter processes and the " eating" influence of the pus upon the surrounding tissues, striking even for non-medical peo- ple, is sure to suggest the idea of an association of fermenta- tive changes. As a matter of fact, in a suppurative process the digestive ferments of the leucocytes, of the bacteria, of the blood plasma, and, too, those of the disintegrating tissues, may all take part. 36 The importance of each par- ticular factor is naturally difficult of definition. Some would have it that the greater tendency of a staphylococcus infec- tion (in comparison with a streptococcus infection) to produce suppuration is due to a lower proteolytic power on the part of the latter microorganisms. 37 It is probably quite correct to ascribe to the white blood corpuscles an especially marked digestive power. It is thought that in this respect the polynuclear leucocytes are more important than the mononuclears; and that the lymphocytes develop scarcely any proteolytic power. 38 According to Jochmann and others the proportion of proteolytic enzymes in tissues and essential body fluids depends very largely upon the number of leucocytes present in them. By placing the tissues of either the lymph glands, spleen, bone-marow or the blood of myeloid leukaemic cases, or material from tuberculous lymph nodes with mixed infection, or staphylococcus pus in the incubator for a time on a Loeffler's plate, their diges- tive power is shown by delle-erosion in the medium. In contrast the tissue of normal lymphatic glands and, too, of those in lymphatic leukaemia, and unmixed tuberculous 38 H. G. Wells, Chemical Pathology, p. 93, 1907. 87 Knapp, Zeitschr. f. Heilk. (Chirurgie), 23, 236, 1902. ^Literature upon Leucoproteases and Antileucoproteases: C. Oppen- heimer, Die Fermente, 3d ed., pp. 216-221, 1910; cf. especially the works of Opie and Barker, Jochmann and Müller and their associates (Ziegler, Locke- mann, Kantarowitsch, Kolaczek), and also M. Fiessinger and P. L. Marie, Journ. de Physiol., 11, 613, 1909. 88 TISSUE FERMENTS caseous material are said to be inactive. In contrast to the typical leucoprotease which is active in weakly alkaline solutions, a ferment derived from the mononuclear leucocytes, active in acid solutions, has been described. Attempts have been made to " isolate" the ferment by precipitating with alcohol the autolysates of pus, spleen, marrow, etc., with subsequent solution of the precipitate in dilute glycerine, and to differentiate it from trypsin. Ex- periments have been made to ' * quantitatively estimate" the proteolytic leucocytic ferment by separation of the white blood corpuscles in citrated blood by centrifugation, dis- solving them in water, and allowing the ferment to act upon a solution of casein, the loss in casein thus occasioned being thereafter determined by the aid of a specific precipitin serum. 39 There is little need to call attention to the lack of exactness naturally inherent to all such experiments. To briefly refer to the matter of antileucoprot eases : were it correct to hold that suppurative destruction of tissue is due to the digestive action of the leucoproteases, it would be entirely logical to hope to overcome suppuration by the anti- ferment of leucoprotease or by the "normal antiferment" of the blood serum. As it was believed, following Jochmann, that antileucoprotease is identical or at least very similar to antitrypsin, attempts have been made to deal with suppura- tions by antitrypsin treatment. 40 The author has already (Vol. I of this series, p. 560, Chemistry of the Tissues) pointed out the more than problematic nature of ' ' antitryp- sin" and has not been impressed with the idea that the practical results of antitrypsin treatment have been brilliant enough to set aside all theoretical doubt. Effect of Extrinsic Factors Upon Autolysis. — Although the effort to influence autolysis within the living body has 88 M. Franke (Lemberg), Wiener klin. Wochenschr., 1910, No. 33. 40 Cf. Jochmann and W. Bätzner, München, med. Wochenschr., 1908, 2473 ; R. Chiarolanza, Med. Naturw. Arch., 2, No. 1, 1909. EFFECT OF EXTRINSIC FACTORS 89 had, even in the treatment of abscesses, no dazzling results, studies upon the influence exerted by extrinsic factors upon autolysis are full of practical interest in view of the unques- tionable importance the process has in every question of tissue resorption (v. supra). Only the most interesting references will be briefly made here from a rich literature bearing upon the subject. 41 It is known from experiment that autolysis, which can set in at the natural reaction of the animal juices, is very notably increased by addition of small amounts of acid, even of so weak an acid as carbonic acid. 42 From this it may be concluded that autolysis is relatively dependent upon the in- tra-vital and postmortem proportion of acid in the tissues ; that, for instance, the insignificance of autolysis in em- bryonal tissues is perhaps due to the low power of these tis- sues to form acid. 43 The idea has been suggested that the increased tissue-protein destruction seen in the asphyxias may have some connection with an increased autolysis made possible by the increased carbonic acid present under such circumstances. 44 As blood serum 45 exerts an inhibitory in- fluence upon autolysis (which is, however, in no wise specific, and inheres moreover to other colloids, as egg-albumin and gelatin 46 ) it may be supposed that this factor may be involved in a regulative way over catabolism of the tissue proteins. It should not be forgotten, however, that the rela- tion supposed to exist between the latter and autolysis is at least for the present entirely hypothetical. 41 Literature upon Effect of Extrinsic Factors upon Autolysis: C. Oppen- heimer, Die Fermente, 3d ed., pp. 245-246, 1910. ** S. Arinkin (Salkowski's Lab., Berlin), Zeitsclir. f. physiol. Chem., 53, 192, 1907; S. Yoshimoto, ibid., 58, 341, 1909. 43 L. B. Mendel and C. S. Leavenworth, Amer. Journ. of Physiol., 21, 69, 1908. 44 L. Bellazzi (Ascoli's Lab., Pavia), Zeitschr. f. physiol. Chem., 57, 389, 1908. 48 J. Baer, Arch. f. exper. Pathol., 56, 68, 1906. 46 W. T. Longcope, Journ. of Medical Research, 18, 45, 1908. 90 TISSUE FERMENTS In addition to the above it has been discovered that the alkaline earths as well as phosphorus, as above stated, hasten autolysis 47 ; arsenic, 48 quinine 49 and quite a number of other drugs manifest, at least when present in considerable quantities, an inhibitory influence. Many drugs seem to accelerate in very small dosage, but to inhibit when in higher proportions. The influence of colloidal metals 50 in stimulating auto- lysis, made known by observations of Ascoli in Pavia and his associates, seems to the author worthy of much con- sideration. Eecently a contribution by C. Neuberg and Caspari has attracted considerable attention, dealing with the results obtained in treating mouse cancers with colloids of the heavy metals. A very rapid softening and liquefac- tion of the tumors was produced, and in a number of cases actual tumor destruction was attained, apparently without danger to the life of the experiment animals (v. Vol. I of this series, p. 555 ff, Chemistry of the Tissues). One cannot but hesitate to spoil the pleasure this fine result inspires by stating the fact that mouse cancers are incomparably more benign than the analogous tumors of man (a point which Wassermann explicitly brings out in warning against em- ploying the method in human therapy, in an article announc- ing a similar result attained in animals by the use of selenium preparations). There is food for further thought in the fact that «L. Launoy, C. R. Soc. de Biol., 62, 487, 1907; L. Brüll (V. Noorden's Clinic, Vienna), Biochem. Zeitschr., 29, 408, 1910. 48 L. Hess and P. Saxl, Zeitschr. f. exper. Pathol., 5, 89, 1908. *° E. Laqueur, Arch. f. exper. Pathol., 55, 240, 1906; cf. also Biochem. Centralbl., 8, 564, 1909; Centralbl. f. Physiol., 22, 717, 1909; G. Izar (Ascoli's Lab., Pavia), Biochem. Zeitschr., 21, 46, 1909. 60 M. Ascoli and C. Izar, Biochem. Zeitschr., 6, 192, 1907; 7, 142, 1907; 10, 356, 1908; H, 491, 1908; 17, 361, 1909; L. Preti, Zeitschr. f. physiol. Chem., 58, 539, 1909; 60, 317, 1909; G. Izar, Biochem. Zeitschr., 22, 371, 1909; M. Truffi, ibid., 23, 270, 1910, and earlier publications from the same laboratory. PROTEOLYTIC TISSUE FERMENTS 91 besides radium, 51 consideration of which in the treatment of tumors has been given in an earlier lecture (v. Vol. I of this series, pp. 557-558, The Chemistry of the Tissues), two other great blessings to man, mercury and iodine, are to be numbered among the agents capable of increasing autolysis. While the addition of iodide of potassium to an autolysing mass is without effect, a very decided increase of hepatic autolysis has been attained in animals by long preadminis- tration of the salts of iodine. 52 May it not be that the favor- able influence, confirmed in hundreds of thousands of cases, which this agent exerts upon luetic processes, is in some way connected with stimulation of autolysis? The feW observations here referred to give us, of course, no justifica- tion for such a conclusion. But as a pointer for continued investigation they deserve constant reflection. Abderhalden 's Investigations Upon Proteolytic Tissue Ferments. — We are indebted for very real progress in the study of proteolytic tissue ferments to the extensive investi- gations of Emil Abderhalden and his army of collaborators. 53 By his method of work, testing the tissue enzymes, not as was always done before upon indefinable proteins, but upon numerous well-defined Polypeptids, Abderhalden, it may be said, dragged the whole problem from the depth of a dismal cloud-mass into an enlightened level; and it is not a diffi- cult thing to prophesy that when this series of studies has 61 J. Wohlgemuth, Berliner klin. Wochenschr., 1904, No. 26, 704; S. Löwenthal and E. Edelstein, Biochem. Zeitschr., 14, 484, 1908. 62 L. Kepinow (Instit. for Cancer Investigation, Heidelberg), Biochem. Zeitschr., 37, 238, 1911; L. B. Stookey, Proc. Soc. Exp. Biol., 5, 89; abst. in Jahresb. f. Tierchem., 88, 842, 1908. 83 E. Abderhalden, in association with C. Brahm, H. Deetjen, A. Gigon, M. Guggenheim, A. Hunter, K. B. Immisch, A. Israel, E. Kämpf, A. H. Koelker, W. Manwaring, J. S. McLester, F. Lussana, L. Pinkussohn, A. Pringsheim, B. Opp- ler, A. Rilliet, P. Rona, B. Schilling, A. Schittenhelm, J. G. Sleiswyk, E. Stein- beck, J. Teruuchi, L. Wacker, W. Weichhardt. Literature: E. Abderhalden, Lehrb. d. physiol. Chem., 2d ed., pp. 266-268, 1909; and Synthese der Zellbau- steine in Pflanze und Tier, p. 119, Berlin, J. Springer, 1912 ; C. Oppenheimer, Die Fermente, 3d ed., pp. 172-176, 1910. 92 TISSUE FERMENTS reached a definite conclusion many fundamental problems of physiology and of pathology will be regarded very differently than is now possible. Detection of Proteolytic Tissue Ferments, by Employing Glycylty rosin, Silk-peptone and Glycyltryptophane. — Men- tion may first be made of some part at least of the progress in technique contributed by Abderhalden in this field. A useful method of determining peptolytic tissue fer- ments is based upon their action on a solution of some Poly- peptid, which, like glycyltyrosin or a given silk-peptone, contains a relatively insoluble aminoacid. The ferment solution to be tested or a section of an organ is put in a 25 per cent, solution of silk-peptone and placed in the incubator after toluol has been added ; after a few hours presence of the ferment manifests itself by the separation of tyrosin. The tyrosin is to be found limited to the cortical surface of sections of renal tissue, for example, the medulla remaining free; in fatty kidneys the tyrosin separation is distinctly decreased. It is equally simple to determine the presence of peptolytic ferments in sections of vegetable material. An- other process which is also available for microchemical work is based upon the fact that a Polypeptid containing trypto- phane, as glycyltryptophane, when treated with bromine water will yield a violet color, not at once, but after the separation of the tryptophane. Optical Method. — The " optical method" introduced by Emil Fischer and Abderhalden has yielded excellent results in this field; the author has previously referred to this (Vol. I of this series, p. 555, Chemistry of the Tissues, and in the present volume, Chapter II) . This method demands the use of a suitably sensitive polarization apparatus with which to follow and accurately determine each step of the course of cleavage of optically-active Polypeptids in which the indi- vidual " building stones" are loosened from their structural positions in the complexly constructed Polypeptid. An illus- OPTICAL METHOD 93 tration may serve to make the method clear. 54 The tripeptid d-Alairyl-glycyl-glycin shows in aqueous solution a specific rotation of + 30°. This substance can give rise in cleavage to d-Alanyl and to glycylglycin. The former rotates light but -f- 2.4°, and the latter is optically inactive. When cleav- age occurs therefore there is a sudden decrease in the rota- tive power. Quite a different result is seen, however, if a cleavage occurs which produces giycocoll and d-alanylglycin. The latter of these two substances is characterized by a very high rotative power, of + 50°, and this type of cleavage therefore shows itself by access of optical rotation. Emil Fischer and Abderhalden made the important state- ment that racemic Polypeptids are split asymmetrically by * proteolytic ferments. Examining a dipeptid, NH 2-CH.C0 NH.CH.COOH, ft ma y ^ e geen there are ^ vo asymmetrically placed carbon atoms. From this it follows, in accordance with the well-known principle of van't Hoff, there exist four isomers, which group themselves into two racemoid bodies, thus : d-Alanyl-1-leucin < — > 1-Alanyl-d-leucin (racemic body A). d-Alanyl-d-leucin < > 1-Alanyl-l-leucin (racemic body B). It has developed that the two racemic compounds behave entirely differently toward pancreatic juice. Only that one which presents the combination of the natural aminoacids d-alanin and 1-leucin — that is, racemic body A — is split, and this is attacked asymmetrically as only the combination d-Alanyl-1-leucin is split ; while the combination of 1-Alanyl- d-leucin remains unchanged. In this respect the proteolytic ferments of animal tissues are shown to be analogous in their influence to that of the pancreatic juice. &; E. Abderhalden, Lehrb. d. physiol. Chem., 2d ed., pp. 266, 626, 1910. 94 TISSUE FERMENTS Inhibition of Proteolytic Processes by the Products of Protein Cleavage. — It has long been known that the action of proteolytic ferments is inhibited by the presence of pro- tein cleavage products. It has been repeatedly shown that the inhibitive influence is due to the optically active amino- acids, in which this power resides, which exist in the proteins. It is suggested in explanation that these aminoacids (and only these) bind the ferment and thus prevent its fixing its object of prey, the protein molecule. This is merely sug- gested because the test tube is incapable of affording a true picture of the proteolytic processes which are going on in the living body. It should be realized that in the living body, whether proteolysis is taking place in the digestive canal or in the tissues, the cleavage products of protein are removed, as they are formed, from further contact with the proteolytic enzyme, and thus the inhibition inevitable in the test tube is avoided. Moreover a natural prolongation of proteolytic cleavage may be conceived from the fact that ceteris paribus the longer molecular chains are more readily preyed upon than short ones; with equal amounts of ferment and equal molecular concentration of Polypeptids, a tetrapeptid is split more quickly than a tripeptid, and the latter in turn more quickly than a dipeptid. Classification of Proteolytic Ferments. — By substitu- tion of definite Polypeptids for high-molecular proteins a basis will be found for construction of a rational classi- fication and differentiation of proteolytic ferments. We know today, for example, that pepsin is incapable of acting upon any of the hitherto demonstrated Polypeptids ; that trypsin will separate some of them but by no means all; while erepsin can dissociate even those Polypeptids which, like glycylglycin, successfully resist trypsin. Keep in mind that every cell and every cellular combination in animals and plants, from infusorium and bacterium up to PEPTOLYTIC POWER OF BLOOD SERUM 95 the highly differentiated organs of man, is in reality a chem- ical laboratory, in which proteolysis is going on in myriads of different forms ! Think of the crudeness of our attempts to classify all these heretofore ! Many a time in the last ten years when busily engaged in collecting material for a com- parative chemical physiology of the lower animals, has the author lost his temper on seeing how learned men, in endless controversy as to whether this or that secretion possess a ' ' peptic " or ' ' tryptic ' ' character, quarrel among themselves, without the least thought on the part of any one of them of some better trophy than that of their trivial "clearly reddened" or " distinctly blued" litmus papers. It is to be hoped that as the methods elaborated by Abderhalden come into general appreciation it will all be very different. It is quite likely that these methods will not be as easy of prose- cution as litmus-paper processes ; and it is to be expected, too, that facility in them will require progressively a more and more comprehensive chemical training. Peptolytic Poiver of Blood Serum. — In conclusion refer- ence may again be made to the promise held out by the recent methods of study, of information regarding the active proteolytic agencies in the blood serum. Abderhalden and his collaborators have discovered that subcutaneous or in- travenous injection of specifically foreign but not of specifi- cally homologous protein increases the peptolytic capacity of blood serum. The normal serum of a dog, for example, is incapable of catabolizing silk-peptone. Its proteolytic ability is apparently increased by parenteral introduction of horse serum, gliadin, casein, diphtheria toxin, tuberculin, but not by dog serum. Serological studies in this line, ap- plying the optical method, are full of suggestive promise for manjr of our important problems (as anaphylaxis). It is a matter of regret that further discussion of the theme cannot be pursued ; the whole subject is thus far too undeveloped to permit a definitive conclusion. 96 TISSUE FERMENTS Mere mention can be given a very interesting practical application of the optical method, that of its employment in the diagnosis of pregnancy. Abderhalden started out with the idea that if it be correct, as many gynecologists testify, that cellular elements of the chorionic villi pass into the cir- culating blood of pregnant females, these being in a sense foreign to the blood should increase the proteolytic capacity of the blood against them. Investigation actually corrobor- ated the assumption, it being possible to demonstrate by optical means that serum from gravid females has the power of inducing cleavage of a placental peptone (obtained by partial hydrolysis of human placental tissue by sulphuric acid), while the same power does not exist in the serum of normal, non-gravid individuals. The chemical diagnosis of pregnancy can be made, however, in much simpler manner, by dialyzing the serum of the pregnant woman in which bits of boiled placental tissue have been suspended against water, and then testing the dialysate for material responding to the biuret reaction. At the present time Abderhalden is still engaged in so far perfecting the method that it may be adapted to the requirements of medical practice. 55 55 E. Abderhalden and M. Kiutsi, Zeitschr. f. physiol Chem., 77, 249, 1912; E. Abderhalden, ibid., 81, 90, 1912. (Employment of triketohydrindenhydrate as peptone reagent.) CHAPTER V UREA. HIPPURIC ACID. EXCRETION OF AMINOACIDS UREA It has been the earnest wish of the author to present in logical sequence as fully as possible the world of biochemical fact and error; but, as has been said before, and that not without feelings of decided rebellion, at a single stride the darkly yawning chasm of intermediate metabolism must be passed, with the vast number of unsolved enigmas it harbors, and attention next directed to the end-products of protein metabolism. The present chapter has first to deal with urea, the most important of the nitrogenous end-products of mammalian metabolism. What do we know of the method and manner of urea- formation? Theories of the Formation of Urea in the Living Body. — Of the many theories which have been proposed to ex- plain the origin of urea there are today practically, in the author's opinion, only two worthy of consideration, that of Schmiedeberg and that of Hofmeister. The former, the "anhydride theory," regards urea as originating from ammonium carbonate by withdrawal of water : AMMONIUM CARBONATE AMMONIUM CARBAMATE UREA /0(NH 4 ) /NH 2 /NH 2 CO — >- CO — >■ CO \C(NH 4 ) — h 2 o \0(NH 4 ) — h 2 o \NH 2 . In this schema the carbamate of ammonium may be regarded as an intermediate product. Hofmeister, on the other hand, in his theory holds that urea is formed by an oxy dative synthesis, in which one of the components is supposed to be an NH 2 "rest" arising from oxidation of ammonia, and the other an NH 2 .CO rest. The hydrocyanic acid theory of Hoppe-Seyler, which for decades has trailed through the 7 97 98 UREA. HIPPURIC ACID. AMINOACIDS literature of the subject, may to-day be consigned finally to well deserved rest. There was a time when it was entirely proper to think it possible that urea may be formed in the body precisely as in Wohler's synthesis by transformation from ammonium cyanate; but after vainly searching for hydrocyanic acid for a number of decades in the intermediate metabolism the theory may be finally dropped, in the author's opinion, from current consideration. If we are to drag along the whole dead weight of wornout mistakes of former generations on our backs, how can we be expected to acquire the strength and vigor required for the grinding effort to reach the higher levels of the future? Ur amino acids. — The formation of certain conjugate products, the uraminoacids, suggesting the possible avail- ability of free CONH 2 complexes, may be regarded as favor- ing Hofmeister 's theory. Thus, as Salkowski discovered, aminobenzoic acid and sulphanilic acid introduced into the system, as, too, taurin, can attach CONH 2 groups to them- selves ; and even ingested tyrosin may be observed linked with such a complex (cf. Vol. I of this series, p. 47, Chemistry of the Tissues) : m = AMINOBENZOIC ACID SULPHANILIC ACID /NH 2 /NH.CO.NH 2 /NH 2 /NH.Co.NH 2 ; C«ILj — >CeH4 ; C6H4 — >-C6-H4 \COOH \COOH \HS0 3 \HSO s TAURIN CH 2 .NH 2 CH 2 .NH.CO.NH 2 CH 2 HS0 3 " CH 2 HSO, TYROSIN OH.CÄ— CH 2 OH— CsH*— ch 2 I I CH.NH 2 — > CH.NH.CO.NH 2 I I COOH COOH. But it has been proved that in alkaline reaction aminoacids with urea are very readily transformed into uraminoacids. 1 1 F. Lippich, Ber. d. deutsch, ehem. Ges., 39, 2953, 1906; 41, 2053, 2074, 1908. MECHANISM OF VITAL OXIDATION 99 R.NH, NH,\ R.NH.CO.NH, I + CO = NH 3 + I COOH NH 2 / COOH. It is sufficient, for example, to heat an alkaline urine contain- ing tyrosin to steaming to effect such a transformation. 2 One must believe therefore that we are no longer justified in any conclusions bearing upon the mechanism of urea formation from the formation of these conjugate products. From experiments in Hofmeister 's laboratory 3 it was determined that by perfusing the isolated living liver with taurin the linking of CONH 2 , as above suggested, does not directly take place; this group becoming available only if glycocoll is introduced at the same time. The CONH 2 group at all events appears from oxidation of the latter ; whether, however, from finished urea (as in the formation of a uraminoacid from aminoacid and urea in the laboratory) or from some precursor of urea, CHj.NH, CO.NH, • • • CO.NH, COOH COOH C0 2 , cannot at present be determined. Mechanism of Vital Oxidation of Nitrogenous Sub- stances. — Schmiedeberg 's anhydride theory is based on the assumption that protein is burned in the living body to its end-products, all the carbon eventually becoming C0 2 , all the hydrogen water, the nitrogen appearing as ammonia, just as in preparation of a protein for Kjehldahl determi- nation. At present the great riddle of the vital combustion proc- esses, it is safe to say, is incomprehensible to us ; and it is beyond our understanding how the living body manages at a temperature less than 40° C. to effect an oxidation which in the laboratory we are able to accomplish only by great heat 2 H'. D. Dakin, Jour, of Biol. Chem., 8, 25, 1910. 3 P. Philosophow (F. Hofmeister'a Lab., Strassburg), Biochem. Zeitschr., 26, 131, 1910; cf. therein Literature upon the Uraminoacids. 100 UREA. HIPPURIC ACID. AMINOACIDS or by powerful reagents like fuming sulphuric acid. This is one of life's great mysteries. That the living cell, however, after successfully performing the trick of burning protein into C0 2 , H 2 and NH 3 , can finally change the carbonate of ammonium into urea by withdrawal of two molecules of water (carbonate of ammonium necessarily resulting from the combination of the carbonic acid with ammonia in aque- ous solution), is apparently much less inconceivable; and from this stage forward it is rather hard to comprehend why Schmiedeburg 's theory should present any particular difficulties. Schmiedeburg 's famous pupil, v. Schroeder, whose work was ended by his premature death, was able to show by his frequently-quoted perfusion experiments that the isolated liver is able to transform not only ammonium carbonate but also the ammonium salts of organic acids, as formate of ammonium, into urea. Thereafter Salaskin dem- onstrated that aminoacids are liable to the same change. How this is actually accomplished and what intermediate products are produced are entirely unknown. We have no knowledge, for example, in the oxidation of glycocoll whether the oxygen combines first with the carbon or with the nitrogen, and whether glyoxylic acid (occasionally appear- ing in metabolism) is an intermediate product : 4 HYDROXYLAMINOACETIC ACID GLYOXYLIC ACID /OH COH CH.NH 2 = +NHj. COOH CH 2 .NH 2 COOH I COOHI\ CH 2 .NH.OH COOH Ammonium Carbamate. — As previously stated, in anhy- dration-production of urea ammonium carbamate, Co/q ^ H » may appear as an intermediate material. For considerable time this compound, occasionally found in the blood, urine *H. Eppinger (Hofmeister's Lab., Strasssburg), Hofmeister's Beitr., 6, 481, 1905. PLACE OF UREA FORMATION 101 and tissues, has been regarded as important in pathology, being looked upon as especially toxic and as responsible for the symptoms of intoxication appearing after administra- tion of meat to animals with Eck fistulas. The import and credibility of these ideas have been completely lost, however, since it has been shown that anywhere, as in the urine, where an ammonium salt in aqueous solution comes in contact with sodium carbonate ammonium carbamate is produced, the NH 3 and C0 2 being distributed proportionately to form ammonium carbamate and ammonium carbonate in con- nection with the disturbance of equilibrium and water transportation. 5 Place of Urea Formation. Exclusion of the Liver.— In literature the question of the place of urea formation occupies a very disproportionate amount of space. That a process of this sort actually takes place in the liver is amply proved by v. Schroeder 's experiments ; but it is by no means settled that the liver is the sole location in which urea is formed. Efforts have been made to come to some conclu- sion upon this point by study of the sequels of hepatic exclusion. Experiments along this line of inquiry by Nencki and Pawlow (with Hahn and Massen) in which they made use of the Eck fistula (cf. Vol. I of this series, p. 296, Chem- istry of the Tissues), have become famous. Here, too, we may class the experiments in Hofmeister 's laboratory in which the liver is destroyed by injection of acid into the biliary duct, and by ligation of the hepatic vessels. In addi- tion a number of observations upon nitrogen elimination in acute yellow atrophy, phosphorus poisoning and hepatic cirrhosis are of significance. 6 The conclusion from this 5 J. J. Macleod and H. D. Haskins (Cleveland), Jour, of Biol. (Jhem., 1, 319, 1905. 8 Literature upon Formation of Urea in the Living Body and its Relation to Defect of Hepatic Function: M. Jacoby, Ergebn. d. Physiol., 1, 532, 1902; A. Magnus-Levy, Noorden's Handb. d. Pathol, d. Stoffw., 1, 99-117, 1906; E. Weinland, Nagel's Handb. d. Physiol., 2, 481, 1907; J. Wohlgemuth, Handb. d. Biochem., 3', 183, 1910; A. Ellinger, ibid., S', 563, 1910. 102 UREA. HIPPURIC ACID. AMINOACIDS whole group of studies may be briefly condensed into the statement, that the liver can be practically excluded without abolishing or in any marked degree reducing the formation of urea. It is true that in these subjects there sometimes occurs a lowering of the urea (from 90 to 75 per cent, of the total nitrogen), with corresponding in- crease of ammonia (the latter increasing from about 3 to 5 per cent, of the total nitrogen to 20 per cent, or more). But on the other hand it is known that loss of hepatic function coincides frequently with an " acidosis," that is, with ac- cumulation of acid metabolites ; and it is not at all unreason- able to think that this may very satisfactorily explain the diminution of urea formation and coincident ammonia in- crease (v. inf.). In dog fish, in the tissues of which there is an unusually large proportion of urea, it has not been pos- sible to reduce this by extirpation of the liver. 7 Recent clin- ical observations have invariably indicated that ammonia elimination is very commonly heightened in severe liver affections, although scarcely more than in fever or in con- ditions of acidosis ; and it is altogether impossible to con- clude that observation of urea-ammonia-elimination affords a safe basis or deduction as to the hepatic function. 8 Generally speaking, the inclination at the present time is increasingly toward the belief that the ability to form urea is by no means confined to the liver, but is one of the common characteristics of all living cells, just as is the power of protein combustion. In very low types of life, in fact, urea does not appear as such in the excretory products ; instead we find uric acid, which may be conceived as built up of two molecular urea rests and one tri-carbon group. 9 1 W. v. Schroeder, Zeitschr. f. physiol. Chem., 14, 576, 1890. 8 W. Frey (Gerhardt's Clinic, Basel), Zeitschr. f. klin. Med., 72, 383, 1911. "Literature upon Excretion in Lower Animals: 0. v. Fürth, Vergl. chem. Physiologie d. nieder. Tiere, Jena, 1903. ALKALOSIS AND ACIDOSIS 103 Alkalosis and Acidosis. — The views as to the special intoxication picture which tends to manifest itself in Eck fistula animals after meat diet have recently undergone an unexpected change. F. Fischler, in Krehl's Clinic in Heidelberg, from observations based on a large amount of material, differentiates the toxic symptom complex into two groups, only one of which he is willing to at- tribute directly to the meat diet. The other group of symptoms he believes to be determined by degenerative changes in the liver, referable to lesions of the pancreas pro- duced in the course of the operation and to consequent escape of free pancreatic ferment. Against these latter features of the toxic complex it should be possible to prepare the animal by appropriate immunization with trypsin. As far as the features of meat intoxication are concerned (thus in some sense isolated in purer form), Fischler states that he has never met among his Eck fistula dogs, in spite of ex- cessive meat feeding, excretion of an acid urine ; and because ostensibly administration of phosphoric acid may prevent or cure the toxic features, he believes it may be assumed that an "alkalosis" is of importance in their production, a patho- logical influence of alkaline material upon the tissues. 10 As before stated other authors have held to the idea of an acidosis in the study of this and of allied conditions, and have explained the increased ammonia elimination in con- sonance therewith. The same view is applied, for example, in the hepatic lesions observed by Gautrelet after injection of methylene blue or sodium fluoride. 11 It is not hard to un- derstand how a disinterested observer might be unable to resist a feeling of general distrust in such a position. And yet the difference may be only an apparent one ; and perhaps the following may approximate the truth. One should recall 10 F. Fischler (Med. Clinic, Heidelberg), Deutsch. Arch. f. klin. Med., 104, 300, 1911. 11 J. Gautrelet, with K. Mallie" and H. Gravelat, C. R. Soc. de Biol., 60, 551, 714, 1906. 104 UREA. HIPPURIC ACID. AMINOACIDS (v. supra., p. 82) that, after ingestion of a meal rich in protein, deamidization processes may begin at once in the in- testinal wall, and the portal blood thereby loaded with am- monium salts passes to the liver ,where these are transformed into urea. 12 But what if the liver be excluded or so seriously altered that formation of urea does not take place in the organ as normally from the ammonia which is swept into it with the portal blood? In that case the general circulation, of course, will next be flooded with ammonia, probably in the form of carbonate. As doubtless the liver does not monop- olize the matter of forming urea, a part is probably taken up by other organs and in them changed into urea, while an- other portion of the ammonia may perhaps be rendered inert by acids which the organism mobilizes. We know that the organism protects itself against an excess of acid by mobil- ization of alkaline material ; and it is perhaps possible, vice versa, that it may protect itself against an alkaline excess by mobilization of acid substances, or possibly by elaboration of unusual kinds and quantities of acids, as in the acidosis actually seen in a number of hepatic affections (icterus catarrhalis) . 13 If, however, both methods of protection are insufficient or act too slowly, the result may ultimately be an alkaline intoxication, an "alkalosis." The author wishes, however, not to be misunderstood as saying that this is actually true; these suggestions are presented only in the way of tentative explanation. The important part taken by ammonia in correction of excessive acidity in the living body was clearly recognized a number of years ago by Walter, in Schmiedeberg 's labora- tory. The usually more marked acid-resistance of carni- vores, in contrast with vegetarian animals, is apparently 12 K. Kowalevsky and M. Markiewicz, Biochem. Zeitschr., 4> 196, 1907; cf. therein the older literature, especially in reference to the differences of view of Salaskin and Zaleski and of Biedl and Winterberg. "N. Janney (F. v. Müller's Clinic, Munich), Zeitschr. f. physiol. Chem., 16, 99, 1911. POSTCCENAL UREA EXCRETION 105 fully explained by the difference of food. According to Eppinger it is not difficult to poison with acid dogs kept on protein-free diet; conversely the inherently low acid-re- sistance of rabbits and sheep is at once raised by food rich in protein. 14 That a similar defensive influence is manifest also from injected urea and aminoacids is denied by Pohl. 13 Arginase. — A small cleavage fraction of the nitrogen in the protein molecule arises, not by the roundabout way of total dissociation and combustion, but by direct separation from the arginin-group by the action of a special ferment, arginase of Kossel. In an earlier lecture (Vol. I of this series, p. 94, Chemistry of the Tissues) some attention was given to the mode of action of the latter. Experiments on protamines have indicated that arginase is able to seize upon not only the free arginin but to attack even that which is present in the interior of the protein molecular structure. Racemic arginin undergoes asymmetrical cleavage ; creatin and guanidin are not affected. 16 It is probable that some of the older statements in literature with reference to fermen- tative production of urea 17 in tissue extracts are explicable as due to the influence of arginase. Postccenal Urea Excretion. — The catabolism of ingested protein in the normal organism occurs so promptly that the process is complete within the course of a few hours. Ob- servations upon the excretion of urea from hour to hour after meals, conducted in the laboratory of Ernest Freund, have shown that in normal individuals it reaches its maxi- mal grade as early as four to five hours after the ingestion "H. Eppinger, Wiener klin. Wochenschr., 1906, No. 5; Zeitschr. f. exper. Pathol., 3, 530, 1906; H. Eppinger and F. Tedesko, Biochem. Zeitschr., 16, 207, 1909. "J. Pohl and E. Münzer, Centralbl. f. Physiol., 20, 232, 1906; J. Pohl, Biochem. Zeitschr., 18, 24, 1909. "A. Kossel and H. D. Dakin, Zeitschr. f. physiol. Chem., Ifl, 321, 1904; 42, 181, 1904; H. D. Dakin, Journ. of Biol. Chem., 3, 435, 1907; Rieszer (Kos- sel's Lab., Heidelberg) , Zeitschr. f . physiol. Chem., 49, 210, 1906. 17 Ch. Richet, Chassevant, Spitzer, D. Löwi; cf. O. Löwi ( Hofmeister's Lab., Strassburg), Zeitschr. f. physiol. Chem., 25, 512, 1898. 106 UREA. HIPPURIC ACID. AMINOACIDS of nitrogenous food. If the ingested material has been in the form of protein already advanced in catabolism the maxi- mum is reached more quickly and the greatest urea excretion is to be noted within one to two hours. 18 Quantitative Estimation of Urea. — The very great physiological importance of urea is apparently sufficient reason for the fact that there has been no dearth of effort to improve the methods for its quantitative de- termination. 19 The old method of Liebig by titration with mercuric nitrate continues to appear here and there in litera- ture; but has become practically unimportant, and efforts to reinstate the method have attained but little success. 20 The most modern methods as a rule are based upon the prin- ciple of transforming the urea into ammonia by hydrolytic agents and distilling the ammonia. Other nitrogenous con- stituents present, especially those of basic nature, may be separated by phosphotungstic acid (Pflüger-Bleibtreu- Schöndorf method) or by phosphomolybdic acid (Haskin's method). 21 Some prefer to at least partially separate the urea from other constituents by the Mörner-Sjöquist method (precipitation by alcohol and ether in presence of baryta) . Hydrolysis of the urea is accomplished by heating with magnesium chloride and hydrochloric acid (Folin's method), 22 with lithium chloride and hydrochloric acid (Saint Martin), 23 with hydrochloric acid in sealed tubes (Salaskin and Zaleski), with sulphuric or hydrochloric acid 18 A. Stauber (E. Freund's Lab., Vienna), Biochem. Zeitschr., 25, 187, 1910. 18 Literature upon the Quantitative Estimation of Urea : P. Rona, Handb. d. biochem. Arbeitsmethoden, 3', 774-782, 1910; 5', 295, 1911; Neubauer-Huppert, Harnanalyse, 11th ed., article by W. Wiechowski, I, 560-576, 1910; C. Neuberg, Der Ham, article by A. C. Anderson, 1, 631-641, 1911; Ch. Sallerin, Journ. de Physiol., 1903, No. 2. 20 B. Glaszmann (Odessa), Ber. d. Deutsch, ehem. Ges., 39, 705, 1906. 21 H. D. Haskins, Jour, of Biol. Chem., 2, 243, 1906. 22 O. Folin, Zeitschr. f. physiol. Chem., 36, 333, 1902; 37, 548, 1903; E. P. Cathcart, Jour, of Physiol., 35, Proc. viii, 1906. 23 L. G. Saint Martin, C. R. Soc. de Biol., 58, 89, 1905. HIPPURTC ACID 107 in an autoclave (Benedict and Gephart, 24 and Henriques and G-ammeltoft 25 ), or very conveniently by heating with glacial phosphoric acid in open vessels (Braunstein), or finally by heating with potassium acetate and addition of acetic acid and zinc (Folin). 26 Naturally there are any num- ber of variations and combinations of these methods pos- sible. In spite of this apparent richness it must be confessed that all of these methods are indirect ; and that in case other related substances occur in addition to urea, with their nitrogen similarly combined in the structural molecule, it would be difficult to detect them against the urea, to say noth- ing of estimating them. Attempts to determine urea quantitatively by splitting it into carbonic acid and nitrogen by means of nitric acid, with subsequent determination of nitrogen by Dumas' method have been repeatedly proposed. 27 HIPPURIC ACID After the preceding presentation of the knowledge we at present possess of the formation of urea in the living body, our attention naturally is directed to other nitrogenous end-products of protein metabolism, of which hippuric acid may first be dealt with. As is well known hippuric acid is a combination product arising from the union of glycocoll and benzoic acid : CH 2 .NH 2 CHj.NH— CO.C«H 6 | + C6H s .COOH=H 2 + | COOH COOH. Source of Benzoic Acid, — As far as the benzoic acid com- ponent is concerned our knowledge is fairly established. It 24 S. R. Benedikt and F. Gephart, Journ. of the Amer. Chem. Soc, SO, 1760, 1908; P. A. Levene and G. H. Meyer, ibid., SI, 717; G. L. Wolf and E. Osterberg, ibid., SI, 421; F. W. Gill, F, G. Allison and H. S. Grindley, ibid., SI, 1078; abstract in Centralbl. f. Physiol., 23, 1909. 25 V. Henriques and S. A. Gammeltoft ( Copenhagen ) , Skandin. Arch., 25, 153, 1911. 24 O. Folin (Harvard Medical School), Jour, of Biol. Chem., 11, 507, 1912. "Th. Ekekrantz, with K. A. Södermann and S. Erikson (Stockholm), Zeitschr. f. physiol. Chem., 16, 173, 1912; 79, 171, 1912. 108 UREA. HIPPURIC ACID. AMINOACIDS may originate from one or other of two sources : First, from aromatic products of vegetable food, as cinnamic acid, hy- drocinnamic acid, quinic acid, etc., which in metabolism are catabolized to benzoic acid. It is not remarkable, therefore, that the quantity of benzoic acid, excreted in the form of hippuric acid, may be decidedly increased by free ingestion of vegetables and fruit (normally present in human urine in amounts of 0.7 to 1. gram daily, on mixed diet) ; and that herbivora eliminate a much larger quantity than carnivores. A second important source is Phenylalanin, one of the molecular structural units of protein. Apparently this compound (cf. Vol. I of this series, p. 47, et seq., Chemistry of the Tissues) readily undergoes complete dissociation in normal metabolism; however the phenylpropionic acid which is produced in intestinal putrefactions from Phenyl- alanin (cf. Vol. I of this series, p. 32, Chemistry of the Tissues) is oxidized freely into benzoic acid when it is resorbed. PHENYLALANIN PHENYLPROPIONIC ACID BENZOIC ACID HIPPURIC ACID C«H 6 - -CH 2 CH.NH, 1 CeH 6 . CH 2 CH 2 C 6 H 5 . COOH — CeHs. CO— NH.CH 2 COOH COOH COOH The importance of protein putrefaction to the production of hippuric acid and the fact that in dogs whose intestinal canal has been largely disinfected by means of calomel the hippuric acid may be materially lowered in the urine were fully recognized by Baumann. The history of the second component part of hippuric acid, glycocoll, is, however, incomparably more complicated than that of the benzoic acid. Probably a clearer conception of the processes here concerned can be had if herbivorous and carnivorous animals are considered separately, as it is becoming more and more evident, as Ernst Friedmann 28 has 28 E. Friedmann and H. Tachau (First Med. Clinic, Berlin), Biochem. Zeitschr., 35, 88, 1911. HIPPURIC ACID FORMATION IN HERBIVORA 109 stated, "that the synthesis of hippuric acid in rabbits not only occurs in different organs but also in a different chem- ical manner than in dogs." Hippuric Acid Elimination in Carnivora and in Man. — In Carnivora, from the classical perfusion studies of Bunge and Schmiedeberg, the kidney is the sole seat of hip- puric acid synthesis. It should here be recalled, however, that according to Schmiedeberg the kidney contains a fer- ment, histozyme, which is capable of splitting hippuric acid into benzoic acid and glycocoll, 29 which may be identical with the ferment synthesizing the hippuric acid, as we have come to recognize a number of examples of the reversibility of enzyme action. Generally speaking the phenomena in the Carnivora present nothing surprising. If the living animal be flooded with benzoic acid a large part is passed uncom- bined ; a portion unites with some otherwise unknown reduc- ing substance ; and that part which appears in the urine as hippuric acid is not so great that it cannot be explained by the amount of glycocoll preexisting in the protein molecule and separable by hydrolysis from the dietary and tissue protein. 30 (This amount of glycocoll corresponds to only 4 to 5 per cent, of the total nitrogen mobilized in protein catabolism.) In human beings, too, in the opinion of Brugsch 31 the findings after administration of benzoic acid apparently do not indicate any other mode of formation of hippuric acid, than by hydrolytic protein cleavage, to be at all convincing. At least the general opinion is distinctly contrary to such a belief. 32 Hippuric Acid Formation in Herbivora. — In herbivora an essentially different situation exists. The mutually 28 N. Mutch, Journ. of Physiol., U, 176, 1912. 30 Th. Brugsch and J. Hirsch, Zeitschr. f. exper. Pathol., S, 663, 1906. u Th. Brugsch and J. Tsuchija, Zeitschr. f. exper. Pathol. 5, 731, 737, 1909. 32 J. Lewinski (Minkowski's Clinic, Greifswald), Arch. f. exper. Pathol., 58, 397, 1908; Th. Brugsch, Zeitsch. f. exper. Pathol., 5, 731, 1909. 110 UREA. HIPPURIC ACID. AMINO ACIDS confirmatory statements of Magnus -Levy, 33 Wiechowski, 34 Ringer, 35 and Abderhalden 36 leave no doubt that when ben- zoic acid is introduced in quantity into the body of a her- bivorous animal about a third or more of the total nitrogen may be excreted as hippuric acid. It is true the economy is not operating under entirely normal condi- tions ; there is probably a higher cleavage of the intrinsic proteins under the toxic influence of the benzoic acid than normally. 37 But it seems altogether improbable that the small quantity of glycocoll of the protein molecule would be enough to form the nitrogenous moiety of the synthesis. (Abderhalden has shown, too, that the tissue protein of veg- etarians is no richer in glycocoll than that of carnivores.) 38 Wiechowski, who met in some of his experiments on rabbits with more than fifty, and once as much as sixty- four, per cent, of the total nitrogen coming from protein cleavage appear- ing as glycocoll, believes that these animals actually produce glycocoll, as he found that the synthesis increased, other things being equal, the longer the benzoic acid continued in circulation, and remained the same in continuous full-day in- toxication. He suspects, too, that in rabbits glycocoll is the precursor of a great, if not the greatest, part of the urea eliminated. E. Friedmann 39 has been able to show by per- fusion that the liver of the rabbit is capable of transforming benzoic acid into hippuric acid ; from which, as the amount of hippuric acid synthesis is not influenced by introduction 33 A. Magnus-Levy, Münchener med. Wochenschr., 1905, No. 45; Biochem. Zeitschr., 6, 523, 1907. 84 W. Wiechowski (Pharmacol. Instit., Prague), Hofmeister's Beitr., 7, 258- 262, 1905. 38 A. J. Ringer (Cornell Univ., New York), Jour, of Biol. Chem., 10, 327, 1911. 36 E. Abderhalden and P. Hirsch, Zeitschr. f. physiol. Chem., 78, 292, 1912; cf. also A. A. Epstein and S. Bookman (New York), v. infra. S 'A. A. Epstein and S. Bookman (New York), Jour, of Biol. Chem., 10, 353, 1911. 88 E. Abderhalden, A. Gigon and E. Strauss, Zeitschr. f . physiol. Chem., 51, 311, 1907. 3 »E. Friedmann and H. Tachau, 1. c. SYNTHETIC SOURCE OF GLYCOCOLL 111 of actual glycocoll, it may be deduced that the glycocoll com- ponent or a predecessor thereof originates in the liver of the rabbit under the influence of benzoic acid. Behavior of Benzoylated Aminoacids. — How are all these considerations to be harmonized? The possibility has been suggested that the benzoic acid primarily binds the NH 2 group of the aminoacids and adheres to it, the remain- ing carbon skeleton of the molecule undergoing disin- tegration : LEUCIN BENZOYL-LEUCIN r HIPPURIC ACID CH, CH, \ / CH CH3 CH3 \ / CH CH 2 .NH— CO.CeH» CH 2 1 — > CH 2 1 -^ COOH. CH.NH 2 1 CHNH- -CO.C.H, COOH COOH A number of feeding experiments by Magnus-Levy with benzoylated aminoacids cannot be regarded as supporting this theory. 40 Synthetic Source of Glycocoll from Acetic Acid and Ammonia. — At present appearances again favor the idea that glycocoll may originate synthetically from ammonia and acetic acid. R. Cohn 4 1 years ago observed in the laboratory of Jaffe in Heidelberg that /u.-nitrobenzaldehyde or /u-toluidine, introduced into a living rabbit, was transformed into /A-aminobenzoic acid, but that this combined with acetic acid: NITROBENZALDEHYDE AMINO-BENZOIC ACETYLAMINOBENZOIC ACID ACID C 5 H^ NOj NH 2 NH.CO.CH3 >■ CßH.jCr >• CeH,)^ COOH COOH. These examples of a combination of acetic acid with am- monia rests suggested the idea of determining whether the 40 A. Magnus-Levy, Biochem. Zeitschr., 6, 541, 1907. a R. Cohn, Zeitschr. f. physiol. Chem., 11, 274, 1892 ; Arch. f. exper. Pathol., 53, 435, 1905. 112 UREA. HIPPURIC ACID. AMINOACIDS rabbit is able to form glycocoll synthetically from ammonia and acetic acid ; and as a matter of fact a very appreciable increase in hippuric acid elimination was observed in several experiments in which ammonium acetate and benzoic acid were simultaneously administered. Although, it is true, these studies are not definitely conclusive, E. Friedmann 42 has recently declared the possibility of a synthetic formation of glycocoll from acetic acid and ammonia, with the very /COH \ actively combining glyoxylic acid I as a possible intermediate product. A simple synthesis of acetic acid /CH, \ with ammonia can result only in acetamide ( I ) but VCO NH 8 / /H never in glycocoll f^^^Y Here may be a special ( c0 &J VCOOH / VI X COOH instance of the previously discussed (v. supra., p. 69) synthesis of aminoacids from a-ketonie acids and ammonia following the schema : R R CO + NH, ^±. CH.NH 2 -f-0 COOH COOH. Glycocoll and Ornithin as Detoxifying Agents. — We have come to regard this synthetic union of glycocoll and benzoic acid as peculiarly important in that it may be looked upon as eliminating the toxic benzoic acid. It is well known that quite a number of other aromatic acids analogous to benzoic acid may combine with glycocoll in the economy. One of these, phenylpropionic acid (C 6 H 5 . CH 2 . CH 2 . COOH.), Dakin has shown to be very toxic to cats, 43 being transformed in the animal partly into acetophenone (C 6 H 5 .CO.CH 3 ) ; although its synthetic compound with glycocoll, phenylpropionylglycocoll ( C 6 H 5 .CH 2 .CH 2 .CO-NH. 42 E. Friedmann and H. Tachau, 1. c, p. 90. 43 H. D. Dakin (C. A. Herter's Lab., New York), Jour, of Biol. Chem., 5, 413, 1908. QUANTITATIVE ESTIMATION OF HIPPURIC ACID 113 CH 2 .COOH) is nontoxic. This suggests the thought that perhaps in another synthetic process which takes place in the body, between glycocoll and cholic acid, with formation of the glycocholic acid of the bile (Vol. I of this series, p. 306, Chemistry of the Tissues), glycocoll may be playing the part of a detoxifying agent. Benzoic acid introduced into living birds is, however, not rendered harmless by glycocoll; in birds another cleavage product of the protein molecule takes its place, as pointed out by the celebrated Königsberg phar- macologist, M. Jaffe (recently deceased), viz.: Ornithin /CH 2 NH 2 ' I CH 2 CH 2 |, which, as is known, originates from cleavage of CN.NH 2 \COOH arginin (Vol. I of this series, p. 10, Chemistry of the Tis- sues). 44 This appears in the urine as a dibenzoyl compound, ornithuric acid : CH 2 .NH — CH 2 — CHj— CH.NH— COOH CO.CeHs CO.C 6 Hs. Quantitative Estimation of Hippuric Acid. — Before leaving this part of the subject reference should be made to the method of quantitative determination of hip- puric acid, a substance of decided importance physiologi- cally. The hippuric acid in an animal fluid may be estimated as such ; or its glycocoll may be determined ; or the benzoic acid. The first of these plans is followed in the method of Bunge and Schmiedeberg, in which the hippuric acid is extracted by acetic ether from urine, purified by passing through animal charcoal, and weighed. Henriques and Sörensen employ their method of f ormol titration to estimate the glycocoll separated out from the hippuric acid. ** In the text referred to in the formula for Ornithin an unfortunate typo- graphical error occurs, in that one CIL group too many is shown. R 114 UREA. HIPPURIC ACID. AMINOACIDS The fact that hippuric acid is exceedingly unstable, being readily dissociated by evaporating the urine in weak alkaline reaction by heat, or by urinary fermentation, has led to the introduction of an increasing number of methods in which the hippuric acid is estimated as benzoic acid. The latter compound because of its stability and ready solubility in ether and other solvents offers certain advantages. 45 The method of Wiechowski 46 is exact but time consuming, the benzoic acid being separated by aqueous distillation. Steenbock 47 oxidizes the alkalinized urine with peroxide of hydrogen, separates the phenols with bromine water after acidulation, and removes the benzoic acid by shaking with ether; after evaporation of the ether the benzoic acid is sublimed in a special glass apparatus and weighed. Folin 48 oxidizes the urine with nitric acid, extracts with chloroform, washes the chloroform solution with a saturated salt solu- tion containing hydrochloric acid and determines the benzoic acid by titration with alcoholic sodium hydrate solution. Recently one of the author's pupil's, Hryntschak, 49 has elaborated what is apparently a very satisfactory method. The urine is oxidized after treatment with boiling sodium hydrate solution by an excess of potassium permanganate ; the manganese which separates is dissolved by sodium bisul- phite and sulphuric acid; and the clear colorless fluid then extracted with ether. After evaporation of the ether the benzoic acid is taken up with chloroform, and finally in pure, crystallized form is weighed. Control determinations of hippuric acid in known solutions show that the method is, 44 R. Cohn, Th. Pfeiffer, C. Bloch, R. Riecke, W. Wiechowski. Literature upon the Estimation of Hippuric Acid: Th. Hryntschak, Biochem. Zeitschr., 43, 316, 1912. Conducted under direction of O. v. Fürth. 46 W. Wiechowski, Hofmeister's Beitr., 7, 262, 1906. "H. Steenbock (Univ. of Wisconsin), Jour, of Biol. Chem., 11, 201, 1912. "O. Folin and F. F. Flanders (Harvard Med. School, Boston), Jour, of Biol. Chem., 11, 257, 1912. 49 1. c. AMINOACIDS IN NORMAL URINE 115 with careful practice of precautions, capable of recovering 95 to 98 per cent. ELIMINATION OF THE AMINOACIDS The above consideration of the part of glycocoll in inter- mediate metabolism serves to introduce another interesting problem, that of the conditions under which thea-aminoacids, the essential products of the catabolism of the protein mole- cule, may escape the normal combustion and appear in appreciable quantity in the urine. Aminoacids in Normal Urine. — That a-aminoacids, at least their optically active forms, which are normally struc- tural parts of the proteins, may easily undergo complete disintegration is beyond question and has been proven experimentally many times; although in case of artificial introduction of formed aminoacids it has been proved that a portion sometimes escapes as such in the urine. 50 Nor- mally, the amount of aminoacids in the urine is, of course, quite small. Different explanations have been proposed for this fact, as it has been determined that hippuric acid in standing urine is very readily subject to cleavage 51 from action of bacteria, and its glycocoll may then be detected by the naphthalinsulphochloride method. Other aminoacids than glycocoll have apparently thus far not been certainly obtained from normal urine. 52 However, it might be easily conceived how, if small quantities of glycocoll, which had escaped synthesis into hippuric acid or had been produced 50 E. Abderhalden and P. Bergell, Zeitschr. f. physiol. Chem., 39, 10, 1903; K. Stolte (F. Hofmeister's Lab., Strassburg), Hofmeister's Beitr., 5, 15, 1904; M. Plaut and H. Reese (under direction of G. Embden), Hofmeister's Beitr., 7, 425, 1905; E. Reisz, ibid., 8, 332, 190G; S. Oppenheimer, ibid., 10, 273, 1907; R. Hirsch, Zeitschr. f. exper. Pathol., 1, 141, 1905; E. Abderhalden and J. Markwalder, Zeitschr. f. physiol. Chem., 12, 63, 1911; E. Abderhalden, A. Furmo, E. Goebel and P. Stübel, ibid., 74, 481, 1911. a Y. Seo (Minkowski's Clinic, Greifswald), Arch. f. exper. Pathol., 58, 440, 1908. 62 E. Abderhalden and A. Schittenhelm, Zeitschr. f . physiol. Chem., $7, 339, 1906. 116 UREA. HIPPURIC ACID. AMINOACIDS by a fermentative cleavage of formed hippuric acid, were present in the blood and tissues, they might enter the urine. 53 Elimination of Aminoacids in Disease. — It is of more interest than the occurrence of small amounts of aminoacids in normal urine to know that in certain pathological con- ditions the excretion of these compounds has been found to be notably increased, as in some of the severe infectious fevers (typhus fever, spotted fever, scarlet fever, pneu- monia and small pox, but not in measles and diphtheria). 54 A number of investigators have found increased aminoacid excretion in phosphorus poisoning and acute yellow atrophy of the liver 55 ; a symptom which, as already stated, has been referred to the increased autolysis in the liver peculiar to these conditions. It is noted also in other severe hepatic disturbances, 56 as in poisoning by arsenuretted hydrogen, prussic acid, and in various degenerative changes in the liver, especially cirrhosis, cancer, fatty degeneration and syphilis. It was at one time hoped that the recognition of an increase in aminoacid elimination, especially after purposeful administration of aminoacids (as it were an " alimentary aminuria"), might be utilized in diagnosis as to the functional integrity of the liver. 57 How- B G. Embden and A. Marx, H'ofmeister's Beitr., 11, 308, 190S; A. Bingel (G. Embden's Lab., Frankfurt a. M.), Zeitschr. f. physiol. Chem., 51, 382, 1908; G. Forssner (F. v. Miiller's Clinic), ibid., 41, 15, 1906; F. Samuely, ibid., 47, 376, 1906; G. Oehler, Biochem. Zeitschr., 21, 48, 1909; A. v. Reusz, Wiener klin. Wochenschr., 22, 158, 1909. M v. Jaksch, Zeitschr. f. klin. Med., 41, 1, 1902; 50, 167, 1903; R. Erben, Zeitschr. f. Heilk., 25, 33, 1904; Zeitschr. f. physiol. Chem., 43, 320, 1905; Internat. Beitr. z. Path. u. Ther. d. Ernährungsstörungen, 2, 252, 1911; A. Primavera (Naples), Giorn. Internaz. di. Scienze Med., 30; abstract in Biochem. Centralbl., 9, No. 880, 1909-10. "Literature upon Metabolism in Phosphorus Poisoning, Acute Yellow Atrophy of the Liver, etc.: C. Neuberg, Handb. d. Biochem., 4", 336-337, 1910. M W. Frey (D. Gerhardt's Clinic, Basel), Zeitschr. f. klin. Med., 12, 383, 1911; F. Falk and P. Saxl (v. Noorden's Clinic, Vienna), ibid., 13, 131, 1911. N. Masuda (Fr. Kraus' Clinic, Berlin), Zeitschr. f. exper. Pathol., 8, 629, 1911. "K. Gliiszner, Zeitschr. f. exper. Pathol., 4, 336, 1907; H. Jastrowitz (Med. Clin., Prague), Arch. f. exper. Pathol., 59, 463, 1908. ELIMINATION OF AMINOACIDS 117 ever, it is doubtful at present whether any practical result will be attained. 58 Increase in the aminoacid excretion has also been met in a variety of conditions in which it is not at all evident that we are dealing with disturbances of the hepatic function, as in pregnancy, 59 gout, 60 diabetes, 61 leukaemia, 62 and after large hemorrhages. 63 Physiologically the excretion of aminoacids is apparently greater in infants than in grown persons. 64 It is worthy of special attention that an enormous access of aminoacids occurs in the urine of hibernating marmots, with corresponding reduction in the urea. 65 It may well be a time will come when it will be possible to recognize and appreciate a common basis for these heterogeneous facts ; but at the present as far as the author is concerned it is entirely impossible to offer any sug- gestion save by labored and arbitrary theorization. How- ever much the author realizes the importance of hypotheses towards future discovery, he feels that in this case the metabolic chemist has every reason for keeping close to the fundamental facts lest he risk loss of all the footing on which he stands. 68 Cf. H. Ishihara, Biochem. Zeitschr., 41, 315, 1912 (under direction of O. v. Fürth). 59 E. C. van Leersum, Biochem. Zeitschr., 11, 121, 1908; C. Rolla, Pathologica, 2, 575; abstract in Jahresber. f. Tierchem., 40, 1910; F. Falk and O. Hesky (Clinic of v. Noorden and Schauta, Vienna), Zeitschr. f. klin. Med., 71, 261, 1910. "•A. Ignatowski (Fr. v. Miiller's Clinic, Munich), Zeitschr. f. physiol. Chem., 4%, 388, 1904; cf. to the contrary, A. Lipstein (v. Noorden's Clinic, Frankfurt a. M.), Hofmeister's Beitr., 7, 527, 1906. "Mies, München, med. Wochenschr., 1894, 671; Nicola, Giorn. d. R. Acad, di Torino, anno 67, Ser. IV, Vol. 10, p. 83, 1904; E. Abderhalden, Zeitschr. f. physiol. Chem., U, 51, 1905: M. Labbe and H. Bith, C. R. Soc. de Biol., 71, 348, 1911. 42 Ignatowski, 1. c. 62 D. Fuchs (Klausenberg), Zeitschr. f. physiol. Chem., 69, 482, 1910. 64 F. W. Schultz, Jahresber. f. Kinderheilk., 72, Ergänzungsheft, 1910; R. Kadlich and P. Grosser, ibid., 75, 4. M K. Nagai (Verworn's Lab., Göttingen), Zeitschr. f. allgem. Physiol., 9, 306-334, 1906. 118 UREA. HIPPURIC ACID. AMINOACIDS Cystinuria and Diaminuria. — Our interest in the dis- covery of the aminoacids in the urine has taken on special interest since we have learned to look upon two rare and important metabolic anomalies, cystinuria and diaminuria, as belonging in the same category. 66 It has been known for a long time that cystin is excreted in the urine and may be found in concretions and in urinary sediments in its strik- ing crystalline form (regular hexagonal plates). E. Bau- mann and L. v. Udranzky have recognized in a case of cystinuria the presence of diamines in the urine. In the light of A. Ellinger's investigations, previously considered (v. Vol. I of this series, p. 34, Chemistry of the Tissues), there can be little doubt that in such a subject cer- tain of the " building stones" of the protein molecule come, so to speak, to the surface of metabolism which should or- dinarily undergo complete disintegration in its depths ; and that here we have to deal on one hand with the sulphur- CH 2 .S-S-CH 2 containing fraction of protein, cystin I CH.NH 2 CH.NH 2 ^COOH COOH and on the other hand with the diamines, putrescin (tetra- methylendiamine — CH 2 .NH 2 - CH 2 - CH 2 - CH 2 .NH 2 ) and cadaverin (pentamethylendiamine — CH 2 .NH 2 - CH 2 - CH 2 - CH 2 - CH 2 .NH 2 ), the first arising from Ornithin and the latter from lysin by separation of C0 2 . Ornithin is, repeating a statement previously made in the present lecture, a cleav- age derivative of arginin. The true significance of cystinuria and diaminuria was first correctly explained by the observa- tions of A. Löwy and C. Neuberg. These writers noted in an individual under their observation that he was unable to oxidize introduced mono- and di-aminoacids, which in the C6 Literature upon Cystinuria and Diaminuria: E. Friedmann, Ergebn. d. Physiol., 1, 16, 1902; F. Umber, Lehrb. d. Ernähr, u. d. Stoffwechselkr., 1909, pp. 385-389; J. Wohlgemuth, Handb. d. Biochem., 3', 192-195, 1900; A. Ellinger, ibid., 3', 660-664, 1910; C. Neuberg, ibid., It", 338, 1910; C. E. Simon and D. G. Campbell, Johns Hopkins Hospital Bulletin, 15, 365, 1904. CYSTINURIA AND DIAMINURIA 119 normal economy undergo complete destruction. While in- gested mono-aminoacids appeared in part in the urine un- changed, cadaverin was excreted when lysin was introduced and putrescin when arginin was given. According to Neu- berg we should recognize different grades of cystinuria, which is a pronounced familial diathesis: (a) mild cases who excrete cystin but who oxidize other aminoacids, 67 and (b) moderately severe cases by whom no mono-aminoacids at all are spontaneously excreted but in whom an alimentary aminuria and diaminuria exists. Only in (c) the most severe cases are the aminoacids spontaneously excreted, as in an instance observed by Abderhalden and Schittenhelm. Special interest attaches to the case of a cystinuric indi- vidual, who was able to use up aminoacids but poorly, dipep- tids to better advantage, but with still more efficience the more complex protein derivatives. 68 It can be seen from such a case that this anomaly need not be associated with serious metabolic faults and may continue for years without manifesting itself by evident symptoms. As previously stated (Vol. I of this series, p. 307, Chemistry of the Tis- sues), cystin is closely related with taurin, one of the two synthetic products of cholic acid occurring in the bile: CYSTEIN TAURIN CH 2 .SH CH 2 .HSO, CH.NH, — >■ CH 2 .NHj. COOH If cystin be administered to normal individuals a large part of its sulphur appears in the urine in oxidized form, as sulphuric acid. If, however, along with cystin sodium cholate be given, in the first place taurocholic acid in the bile is apparently increased, 69 and a correspondingly increased * Cases of Chas. E. Simon, C. Alsberg and 0. Folin, A. E. Garrod and W. H. Hurtley, H. B. Williams and C. G. L. Wolf. es A. Löwy, and C. Neuberg, Biochem. Zeitschr., 2, 438, 1906. «•G. v. Bergmann (F. Hofmeister 's Lab., Strassburg), Hofmeister's Beitr., 4, 192, 1904. 120 UREA. HIPPURIC ACID. AMINOACIDS amount of the sulphur of the cystin fails to undergo oxida- tion into sulphuric acid. In a cystinuric individual, however, this latter result of administration of cholic acid does not oc- cur, presumably depending upon a failure to synthesize taurocholic acid. 70 An interesting addendum to these obser- vations is afforded by a case of hepatic cirrhosis presenting a combination of acholia and cystinuria, interpreted as an instance of failure of normal taurocholic acid synthesis be- cause of disease of the hepatic parenchyma, as a result of which the available (for transformation into taurin) cystin passed unchanged into the urine. 71 Quantitative Determination of the Aminoacids. — The great number of physiological and pathological problems related to the excretion of aminoacids in the urine is sufficient explanation for the amount of attention devoted in recent years to the elaboration of methods for the isolation and estimation of these substances. For isolating the amino- acids the method of binding them with naphthalinsulpho chloride or with naphthylisocyanate is of service (Vol. I of this series, p. 15, Chemistry of the Tissues). For estimation of the aminoacid nitrogen the van Slyke method (ibid., p. 18), depending upon displacement of the aliphatic amino group by nitric acid, 72 will be found valuable; or, too, the formol-titration method of Henriques and Sörensen. The latter is based on the fact that a-aminoacids in excess quanti- tatively bind formaldehyde : R. CH. NH 2 H R. CH. N = c/ | + I = H 2 + | C O O H COH COOH. 70 E. C. Simon and D. G. J. Campbell, Johns Hopkins Hosp. Bull., 15, 365, 1904. 71 Observation of Morawsky, cited by J. Wohlgemuth, Deutsche Klinik, 11, 329, 1907. "P. A. Levene and D. D. van Slyke (Rockefeller Instit., New York), Jour, of Biol. Chem., 12, 301, 1912. This method serves to determine not only the free aminoacids, but also those combined in form of Polypeptids, etc., in the urine. DETERMINATION OF AMINOACIDS 121 If the solution be carefully neutralized before its trans- formation with formaldehyde, the masking of the basic NH 2 groups becomes evident at once by the development of an acid reaction ; and as a matter of fact this increase of acidity after addition of formol, which may be determined by titra- tion, is a precise measure of the amount of amino groups present. It cannot be questioned that the method, the technique of which has received much criticism and has been improved in a number of ways, is really a valuable addition to our scientific equipment. 73 73 V. Henriques and S. P. L. Sörensen, Zeitschr. f. physiol. Chem., 63, 27, 1909; 6Jf, 120, 1910; V. Henriques, ibid., 60, 1, 1909; S. P. L. Sörensen, Biochem. Zeitschr., 25, 1, 1910; H. Malfatti, Zeitschr. f. physiol. Chem., 61, 499, 1909; 66, 152, 1910; L. de Jager, ibid., 65, 185, 1910; 67, 1, 105, 1910; W. Frey and A. Gigon, Biochem. Zeitschr., 22, 309, 1909; T. Yoshida, ibid., 2S, 239, 1909; cf. also Neubauer-Huppert's Analyse des Harnes, 2d ed., article by A. Ellinger, 1, 641 to 647, 1910; Neuberg, Der Ham., article by A. C. Andersen, 1, 569-630, 1911. CHAPTER VI CREATIN AND CREATININ. OTHER URINARY BASES. OXYPROTEIC ACIDS. UROCHROME. CREATIN AND CREATININ In the current lecture two important end-products of metabolism will first be carefully considered, creatin and Creatinin. The importance of these two closely related sub- stances is at once apparent when we recall that a fully grown human being excretes as an average about one gram of Creatinin in the urine in the course of twenty-four hours, and that this substance, from a quantitative standpoint, is a prominent member of the group of excretory products which represent the nitrogen fraction escaping transformation into urea. In attempting to present a precise statement of the knowledge we possess as to the nature and importance of these substances, the first impression obtained by tracing the history of this metabolic problem is a very confusing and, indeed, a depressing one. If, however, one works with de- termination through the tangle of actual and seeming con- tradiction which involves the subject, he will gradually realize a pleasant satisfaction in learning that conditions are not really as bad as they at first appear. The pioneer work of the last ten years has basically cleared the ground, and if to-day a survey be made free view in more than one direction is found possible. Quantitative Estimation. — In the development of the creatin problem may be seen, as often is the case in physio- logical chemistry, how actual progress first became appreci- able after analytical chemistry had provided an applicable method of estimation with which the physiological investiga- tor could work with satisfaction. In this instance we are much indebted to 0. Folin 1 for his carefully elaborated O. Folin, Zeitschr. f. physiol. Chem., 41, 223, 1904. 122 RELATION BETWEEN CREATIN AND CREATININ 123 method for the determination of Creatinin. Jaffe 's reaction, with its bright red color produced by the addition of sodium hydrate and picric acid to Creatinin, is made use of in the method for colorimetric determination. Creatin may like- wise be estimated after complete transformation into Crea- tinin by chemical agents (as by the process of Benedict and Meyers 2 by heating the creatin with hydrochloric acid in an autoclave). 3 It would unquestionably be very desirable to discover a direct method of satisfactorily estimating creatin. Whether the proposal to utilize the orange color which diacetyl 4 (CH 3 .CO.CO.CH 3 ) strikes with creatin (but not with Creatinin) will prove successful for colorimetric determination remains to be seen ; the results reported are apparently not entirely equivalent to those obtained by Folin's method. Relation Between Creatin and Creatinin. — Solution of the creatin problem 5 has been retarded by the fact that / N(CH 3 )-CH 2 \ physiological relation between creatin J c(NH)/ j ) V NH 2 COOH/ / N(CH 3 )-CHA and its anhydride, Creatinin I c(NH)/ I ), has V NH CO / been repeatedly denied. It is, of course, proper to assign no particularly important place in physiological chemistry to ' ' feelings ' ' ; but to say the least we cannot well do entirely without them. In the author's judgment a discerning bio- chemical sense must necessarily tell us that two substances, like creatin and Creatinin, one of which may be transformed a F. G. Benedict and V. C. Meyers ( Wesleyan Univ. ) , Amer. Jour, of Physiol., 18, 397, 1907. 8 In the presence of sugar, where the method is attended with certain difficulties, according to W. C. Rose (Univ. of Pennsylvania), Jour, of Biol. Chem., 12, 73, 1912, the creatin may he changed into Creatinin by heating with phosphoric acid in the autoclave. 4 G. S. Walpole (Wellcome Research Lab.), Jour, of Physiol., 42, 301, 1911. 5 Literature upon Creatin Metabolism: C. A. Pekelharing, Centralbl. f. Stoffweehselkr., 1909, No. 8; A. Schittenhelm, Handb. d. Biochem., //, 535-539, 1910. 124 CREATIN AND CREATININ into the other by the simplest sort of chemical interference (boiling with acid) are bound to bear a direct physiological relation to each other. To-day such a relation may be looked upon as definitely proved from the works of Pekel- haring and his school ; and we are justified in assuming that the urinary Creatinin owes its origin to anhydration of the creatin (primarily appearing in the tissue protoplasm of the body or introduced with meat food). 6 This anhydration is not necessarily always a complete one, as greater or smaller amounts of creatin usually coexist with Creatinin in the urine. For this reason in biological studies it is never re- garded as sufficient to make an estimation of Creatinin alone> but to deal with the total amount of the two substances. In the urine of birds the Creatinin is of decidedly less impor- tance than creatin. 7 Creatase and Creatinase. — Added difficulty in the study of the problem arises from the fact that along with the change of creatin into Creatinin (apparently from the influ- ence of anhydrating ferments) there also occurs a decompo- sition of both substances in the tissues. Gottlieb and Stangassinger, 8 who are responsible for this important observation, ascribe it to the influence of special ferments (creatase and creatinase). Arginase is not involved in the process. 9 The chemical course of this process of decomposi- tion, which is also to be met in excised living organs, is unknown. The cyclically formed Creatinin is apparently affected with more difficulty than the creatin. Creatin which has been introduced parenterally into the circula- tion of mammals is partly decomposed in the body ac- «Cf. E. P. Cathcart (Glasgow), Jour, of Physiol., 39, 320, 1909; D. Noel Paton, ibid., 39, 485, 1910. 7 D. Noel Paton, 1. c. 8 R. Gottlieb and R. Stangassinger, Zeitschr. f . physiol. Chem., 52, 1 1907 ; 55, 295, 322, 1908. 9 H. D. Dakin (C. A. Herter's Lab., New York), Jour, of Biol. Chem., 3, 435, 1907. CREATIN-CREATININ ELIMINATION 125 cording to the studies of Pekelharing and his associates, 10 partly excreted without change, partly excreted after anhy- dration into Creatinin; and according to these writers the liver is apparently of importance both in the decomposition and also in the process of dehydration. Besides this mode of destruction, in experiments in which creatin or Creatinin has been introduced by the mouth decomposition may take place of either substance from the agency of bacteria in the intestine ; so that it is not at all surprising in such experi- ments if nothing but a fraction of the substances introduced appears in the urine. 1 x Endogenous and Exogenous Distribution of Creatin- Creatinin Elimination. — In trying to recognize the precise sources of the urinary creatin and Creatinin reference to a recent illuminating presentation of the subject by Lafayette Mendel 12 with the following schema based thereupon may serve to most quickly orient ourselves upon the subject : Preformed creatin from meat food Protein Food Protein Reserve Protein Tissue Protein (Circulating Protein) O O Creatin Portion Decomposed in Metabolism Urinary Creatin Urinary Creatinin. 10 C. A. Pekelharing and C. J. C. Van Hoogenhuyze, Zeitschr. f. physiol. Chem., 69, 395, 1910; cf. also P. A. Levene and L. Kristeller, Amer. Jour, of Physiol., %k, 44, 1909. u W. Czernecki (E. S'alkowski's Lab.), Zeitschr. f. physiol. Chem., U, 294, 1905; P. Nawiasky (M. Rubner's Lab.), Arch. f. Hygiene, 66, 239, 1908; R. H. A. Plimmer, M. Dick and C. C. Lieb, Jour, of Physiol., S9, 112, 1909-10. 12 L. B. Mendel and W. C. Rose, Jour, of Biol. Chem., 10, 249, 1911. 126 CREATIN AND CREATININ It is apparent from this that (just as we are accustomed to recognize an endogenous and an exogenous fraction of the purin bodies in the urine) we are in position to make an analogous differentiation in case of the creatin-creatinin excretion. The possibility of increasing the proportion of creatin and Creatinin in the urine by free intake of creatin in meat food or Liebig's meat extract has been repeatedly observed. This exogenous part can readily be excluded by starvation or by exhibition of food which does not contain creatin. In this connection the interesting fact has been developed that under such circumstances individual vari- ations may be observed and these may remain constant for a given normal individual for as long a time as a year, 13 reminding one of the observations of Bnrian and Schur, who were able to establish a similar individual constancy in case of the endogenous purins. Taking up next the question as to how far we are justified in assuming a relation between the disintegration of tissue protein and creatin formation in metabolism, it may be assumed, as indicated in the above schema, that neither the food protein nor the readily mobilizable "circulating" protein forms a source of creatin and its anhydride, Creatinin. This is directly apparent from the fact that the excretion of creatin-creatinin does not proceed in precisely parallel lines with the total protein exchange, 14 and more- over seems to be fairly independent of the intake of proteid food. Relation of Creatin-Creatinin Excretion to Decomposi- tion of Tissue Protein. — However, an increase in the excre- tion of creatin and Creatinin is to be observed in those condi- tions where (and here probably is the kernel of the whole problem) there is extensive decomposition of the tissue 13 O. Folin (Waverley), Amer. Jour, of Physiol., 13, 84, 1905; C. J. C. Van Hoogenhuyze and H. Verploegh (Physiol. Lab., Utrecht), Zeitschr. f. physiol. Chem., 57, 161, 1908; 59, 101, 1909. 14 J. Forschbach and S. Weber (Minkowski's Clinic, Greifswald), Centralbl. f. Physiol, u. Pathol, d. Stoffw., 1906, 569. DECOMPOSITION OF TISSUE PROTEIN 127 protein, as in starvation, 15 in fever, 16 in diabetes, 17 in poison- ing by phloridzin 18 and phosphorus, 19 after being in an atmosphere poor in oxygen, 20 and after strenuous muscular exertion. 21 It is extremely suggestive that this is especially true in the last mentioned condition if the muscular effort is performed after insufficient nutrition, at the expense of the part involved in the exertion. It appears that work does not necessarily induce increase of creatin-creatinin elimination in well fed human beings and animals ; but in conditions of hunger this is quite apt to be the result. In phloridzin diabetes the elimination of Creatinin is particu- larly increased when there is an insufficiency of carbohydrate in the food. It is a fact, too, that in protein starvation the creatin-creatinin elimination can be prevented by exhibition of carbohydrate (or especially of fat). 22 This recalls the well-known antagonism of carbohydrates to the elimination of acetone bodies; in both instances there may be recog- nized the ability of carbohydrates to satisfy the immediate needs of the body, as it were by payment of available small change, and thus to avert the necessity of liquidation of its fixed assets. That no direct relation, but merely a parallel- ism, exists between the elimination of acetone bodies "E. P. Cathcart (Glasgow), Jour, of Physiol., 39, 311, 1909; L. B. Mendel and W. C. Rose (Yale Univ.), Jour, of Biol. Chem., 10, 255, 1911. 16 H. Rietschel, Jahrb. f. Kinderheilk.. 61, 621, 1905; O. af Klercker (Lund), Zeitschr. f. klin. Med., 68, 22, 1909; A. Skutetzky (v. Jaksch's Clinic, Prague), Deutsch. Arch. f. klin. Med., 103, 423, 1911. 17 R. A. Krause (Edinburgh), Quarterly Jour, of Physiol., 3, 289, 1010; R. A. Krause and W. Cramer, Jour, of Physiol., 40, 1, 1910. 18 E. P. Cathcart, and M. R. Taylor (Glasgow), Jour, of Physiol., 41, 276, 1910. 19 G. Lefmann (Heidelberg), Zeitschr. f. physiol. Chem., 57, 476, 1908. 20 C. J. C. van Hoogenhuyze and H. Verploegh ( Pekelharing's Lab., Utrecht), Zeitschr. f. physiol. Chem., 41, 101, 1909. 21 Older literature upon the Relation Between Creatin and Muscular Ac- tivity: Liebig, Sarakow, Ranke, Xawrocki, C. Voit, Monari, Grocco, Moitessier, Gregor; cf. O. v. Fürth, Ergebn. d. Physiol., 2, 603-605, 1903. 32 L. B. Mendel and W. C. Rose (Yale Univ.), Jour, of Biol. Chem., 10, 213, 1911. 128 CREATIN AND CREATININ (acidosis) and that of Creatinin 23 is readily appreciable, the former process involving a drain upon the fat deposit, the latter upon the tissue proteins. Muscle as the Source of Creatin. — In harmony with the hypothesis of consumption of tissue protein in the construc- tion of creatin may be offered the observation, made long ago in the laboratory of Hoppe-Seyler, and recently con- firmed, 24 that in conditions of inanition the muscles are rich in creatin. That muscle may be the source of creatin may be directly inferred from the large amount of this substance contained in it. Marked increase of the creatin-creatinin content ensues in isolated frog's muscle upon nervous stimulation 25 ; and the excised functionating mammalian heart in Ringer's solution may discharge considerable quantities of creatin and Creatinin into the surrounding fluid. 26 From recent studies of Pekelharing and his pupils the impression is given that tonic contracture of a muscle is more favorable than the short contraction of ordinary muscular action to induce a high degree of cleavage production of creatin. 27 With the work of Gottlieb and his associates, the results of which are in consonance with this view, and those of Seemann (cf. Vol. I of this series, p. 148, Chemistry of the Tissues) before us, we may well assume as a working hypothesis (in spite of the negative results of Mellanby) that creatin can be produced by cleavage from a colloid precursor in muscle tissue not only by vital but also under certain circumstances by post 23 E. P. Cathcart and M. R. Taylor, 1. c. 24 B. Demant, Zeitschr. f. physiol. Chem., 3, 388, 1879; L. B. Mendel (Yale Univ.), Jour, of Biol. Chem., 10, 255, 1911. 26 T. Graham-Brown and E. P. Cathcart, Jour, of Physiol., 37, XIV, 1908. 28 S. Weber (Minkowski's Clinic, Greifswald), Arch. f. exper. Pathol., 58, 93, 1907. 27 C. J. C. Van Hoogenhuyze and H. Verploegh, Zeitschr. f. physiol. Chem., 1,6, 415, 1905; C. J. C. Van Hoogenhuyze, Jahresber. f. Tierchem., 39, 445, 1909; C. A. Pekelharing and C. J. C. Van Hoogenhuyze, ibid., 64, 262, 1910; C. A. Pekelharing, ibid., 75, 207, 1911. TEST OF HEPATIC FUNCTION 129 mortem autolytic processes. 28 In further course of auto- lysis the creatin undergoes partial anhydration into Creatinin; and thereafter both substances may be decom- posed by fermentation. Cleavage of Creatin from Other Tissues. — Assuming, for a time at least, the formation of creatin in muscle autolysis as proven, the further question presents itself as to whether production by autolytic cleavage is limited to the musculature or whether the same possibility resides in other tissues as well. Gottlieb and Stangassinger 29 (after observing in experiments in which they employed expressed tissue juices, that, as in case of muscle, so, too, in kidneys in an early stage of autolysis there appears an increase of the creatin and Creatinin present) have expressed the opin- ion, based upon perfusion experiments, that probably " quite a number of different tissues are to be considered as possible sources of the blood creatin. The formation of creatin in the perfused livers of well nourished animals is apparently quite important. . . . It is therefore entirely possible that the liver in lifetime may be an important site of creatin formation." The above statements bearing upon the topography of creatin formation are unmistakably very meagre. The fact that many tissues contain creatin, and, too, that many bacteria 30 are able to produce Creatinin, should perhaps be interpreted as indicating that we really have here to do with a widespread function of living cells. Test of Hepatic Function. — Our knowledge of the topog- raphy of the other phases of creatin metabolism (anhydra- tion of creatin into Creatinin, and the decomposition of both substances) is likewise by no means satisfactory. It 28 F. Urano (Hofmeister's Lab.), Hofmeister's Beitr., 9, 104, 1906; R. Gottlieb and R. Stangassinger, Zeitschr. f. physiol. Chem., 52, 1, 1907; A. Rothmann (Gottlieb's Lab.), ibid., 57, 131, 1908; J. S'eeman, Zeitschr. f. Biol., 55, 322, 1908; 69, 333, 1907; E. Mellanby, Jour, of Physiol., 36, 447, 190S. 28 R. Gottlieb and R. Stangassinger, Zeitschr. f. physiol. Chem., 55, 322, 336, 1908. so N, Antonoff, Centralbl. f. Bakter., 43, 209, 1907. 9 130 CREATIN AND CREATININ was hoped that the study of creatin metabolism would lead to a method of determining the functional condition of the liver, from the rather undefined idea that the liver was the site of enzymic anhydration of the creatin. In phosphorus poisoning, 31 and in degenerative processes involving the hepatic parenchyma, especially in hepatic carcinoma, 32 it is said that urinary creatin is increased, with reduction of Creatinin. However, exclusion of the liver from the portal circulation does not materially affect creatin metabolism. 33 One of the author's pupils, H. Ishihara, 34 was likewise un- able to detect any influence of the kind in the course of sub- chronic phosphorus poisoning in dogs ; and the author feels there is little to be expected from this point of view as far as a clinical test of the hepatic function is concerned. Relation of Creatin Metabolism to Processes in the Female Sexual Organs. — The recognition of a relation be- tween creatin metabolism and the cyclical processes of the female genital organs is of decided interest. Creatin ap- pears in the urine of women in increased amount after menstruation, but may be entirely absent in the intermen- strual period. It may occur in the latter part of pregnancy and, too, in the period of involution of the uterus after de- livery. 35 It would be decidedly gratuitous to attempt to explain these phenomena on the basis of a lowering of the anhydrizing ability of the liver; there is at least as much reason to suppose that they are due to changes in the myometrium itself; for that matter, however, the one 81 G. Lefmann, Zeitschr. f. physiol. Chem., 57, 468, 1908. 32 C. J. C. Van Hoogenhuyze and H. Verploegh, Zeitschr. f . physiol. Chem., 58, 161, 1908. 33 E. S. London and N. Bolgarski, Zeitschr. f. physiol. Chem., 62, 465, 1909 ; C. Towles, and C. Voegtlin (Johns Hopkins Univ.), Jour, of Biol. Chem., 10, 479, 1912. 34 H. Ishihara (Instit. of Physiol., Univ. Vienna), Biochem. Zeitschr., Ji, 315, 1912. 35 R. A. Krause (Edinburgh), Quarterly Jour, of Physiol., 4, 293, 1911; R. A. Krause and W. Cramer, Jour, of Physiol., Ifi, Proc. of Phys. Soc, XXXIV, 1911; J. R. Murlin (New York), Amer. Jour, of Physiol., 28, 422, 1911. ORIGIN OF CREATIN FROM ARGININ 131 hypothesis is as far from proof as the other. Experiments on the excised living uterus have, however, constantly indi- cated that the amount of creatin, in proportion to the weight of the uterine muscle, increases with the muscular activity which has been in play. 36 Possible Origin of Creatin from Arginin. — It must be confessed at the last that we really do not know the ultimate derivation of creatin. Apparently it originates from protein. Even if we actually had proved to our satisfaction that it is formed in the course of tissue autolysis, this would not prove, however, that it necessarily originates from the proteins. Other tissue components, known and unknown, might, with equal propriety, enter into question. If, how- ever, the graphic formula of creatin is examined and compared with that of arginin, CREATIN ARGININ /NH 2 /NH 2 C(NH)< C(NH)< X N(CH 3 ) X NH I \ CH 2 .COOH CH 2 CH, CH 2 CH.NH, COOH, our biochemical sense suggests (it must be confessed that even with the best of intentions we cannot get on without something of the sort) that a relation between the substances, even if not proved, is, a priori, extremely probable. Prom arginin, which forms one of the important constituents of the protein molecule, by a typical oxidation there may be / ,nh /NH2 \ produced guanidin acetic acid J ^nh— CH 2 ), which re- ^ cooir quires only a simple methyl binding for transformation into 36 Rübsamen and Gusikoff (E. Kehrer's Clinic, Berne), Arch. f. Gynäkol., 95, 461, 1912. 132 CREATIN AND CREATININ creatin. A whole series of examples may be appealed to to show that the living body actually is engaged in bringing about such a process of methylation. Metabolically from telluric or selenic acid, as Franz Hofmeister noted, 37 tel- lurium methide or selenium methide can be formed; from CTT pyridin, I , according to His, 38 may be produced HC 1 ), CH methylpyridylammonium hydroxide, hc'IIch; from di- N / \ CH 3 OH C TT ethylsulphide, Ns, according to Neuberg and Grosser, 39 diethylmethylsulphinium hydroxide /^^Xg/^* 13 \ j^ j s /C2H6V yCH 3 \ V^Ha 7 ' \)H/' rather difficult to appreciate precisely why, therefore, we should deny the possibility of methylation of guanidinace- tic acid in metabolism. Even the negative or varying results of experiments intended to influence the elimination of crea- tin and Creatinin in the urine and the amount of creatin in the muscles by administration of guanidin acetic acid or of proteins rich in arginin 40 prove but little. It is a well known fact that attempts are not always successful to re- produce at will the processes which accompany protein decomposition in the living body by experimental introduc- tion of their decomposition products. 37 P. Hofmeister, Arch. f. exper. Pathol., SS, 198, 1894. 38 W. His, Arch. f. exper. Pathol., 22, 253, 1887. 39 C. Neuberg and Grosser, 2. Tagung d. deutsch, physiol. Gesel., Cen- tralbl. f. Physiol., 19, 316, 1905. 40 W. Czernecki ( S'alkowski's Lab.), Zeitschr. f. physiol. Chem., kk, 294, 1905; M. Jaffe, ibid., 48, 430, 1906; G. Dorner (Jaffe's Lab.), ibid., 52, 225, 1907; O. af Klercker, Hofmeister's Beitr., 8, 59, 1906; E. Mellanby, 1. c. OTHER URINARY BASES 133 OTHER URINARY BASES M ethyl guanidin; Dimethylguanidin; Vitiatin. — Besides creatin and Creatinin small quantities of other bases closely related to them may be met at times in the nrine. In the Mar- NTT burg Physiological Institute methyl guanidin , C(NH)/ has been isolated from normal human urine in its relatively insoluble picrolonic acid combination; and in the urine of dogs, after ingestion of meat extract, dimethylguanidin, /NH 2 C(NH) v CH 3 , X N / has been found. Vitiatin, with its ascribed CH 3 /NH— CH 2 — CHa— N v structural formula, C(NH)< | XXNH), (cf. Vol. X NH 2 CH 3 / NH,' I of this series, p. 149, Chemistry of the Tissues) has also been met in the urine. 41 Small quantities of methylpyridin may occur, probably produced from vegetable foods ; pyridin compounds are in- troduced into the body with the nicotine of tobacco smoke and with some of the constituents of coffee. 42 Novain. — Then, too, small quantities of bases may appear in the urine, having a number of methyl groups linked to nitrogen. Kutscher 43 encountered novain (as previously stated in these lectures, Vol. I of this series, p. 151, this substance may be regarded as identical with carnitin, ca '\ \ CH 3 — N — — O I : reductonovain also occurs, re- CH 3 / CHa-CH^CHCOID-CC/ 41 F. Kutscher and Lohmann, Zeitschr. f. physiol. Chem., 48, 422, 1906; 49, 81, 1906; W. Achelis, ibid., 50, 10, 1906; F. Kutscher, ibid., 51, 457, 1907; R. Engeland, ibid., 51, 49, 1908. 42 F. Kutscher, 1. c. " F. Kutscher, 1. c. 134 URINARY BASES lated to novain in the same way as is neurin to cholin. These bases characteristically yield by cleavage trimethylamine when distilled with alkali. Trimethylamine. — In connection with the question whether trimethylamine occurs as such in the urine, Takeda, one of Kutscher 's pupils, has brought forward the necessity of employing a partial vacuum distillation method. The residue of the urinary distillate taken up in dilute hydro- chloric acid is separated into a fraction soluble in alcohol and one insoluble in alcohol; and from the latter the gold chloride combination of trimethylamine is obtained. 44 Takeda found that preformed trimethylamine does not exist in the urine of dogs and of horses, but may sometimes be present in human urine; it, however, always appears when alkaline ammoniacal fermentation takes place in urine. Erdmann was unable to detect trimethylamine in normal fresh human urine. However, it has been learned that if the urine is treated with concentrated sulphuric acid as if for Kjeldahl estimation, and the fluid alkalinized after decolori- zation and distilled, alkylamine, including trimethylamine, passes over with the ammonia. 45 The author's student, Kinoshita, 46 proceeded as follows: He distilled off under lowered pressure the volatile amines from the urine in presence of magnesia, taking them up in a receiver containing dilute hydrochloric acid. In the chloride residue which remained after evaporation of the distillate, consisting mainly of ammonium chloride and a small admixture of the chlorides of other volatile bases, the amount of alkyl in nitrogen combination was determined by the method of J. Herzig and H. Meyer, and as a conclusion 44 Takeda (Physiol. Instit., Marburg), Pflüger's Arch., 129, 82, 1909. 45 O. Folin, C. C. Erdmann (Waverley), Jour, of Biol. Chem., 3, 83, 1907; 8, 41, 57, 1910; 9, 85, 1911. 40 T. Kinoshita (University Physiol. Instit., Vienna), Centralbl. f. Physiol., 2k, No. 17, 1910. ARNOLD'S REACTION 135 from the preliminary findings it was held that the alkyl was really present in the form of trimethylamine. In conformity with the above the amount of trimethylamine recoverable by distillation from normal fresh urine was found extremely small. And, too, expectation of obtaining larger amounts after acid or alkaline hydrolysis was not fulfilled. Somewhat larger amounts of the base were found in spoiled urines; and the largest proportions (about 3 to 6 centigrams in the liter of urine) were met in a few urines which, with or without addition of toluol, had been standing for a long time at room temperature, from which it must be inferred that the cleavage production of trimethylamine is the result of a fermentative process acting on some "mother sub- stance. ' ' Doubtless the physiological significance of the ap- pearance of trimethylamine in urine has been much over- estimated by the earlier investigators. 47 Arnold's Reaction. — One more peculiar substance should be mentioned, which appears in the urine particularly after ingestion of meat or meat bouillon and which underlies the ' ' Arnold reaction. ' ' Upon addition of sodium nitroprusside and alkali a violet color is assumed, which changes to blue when acetic acid is added. The nature of this reaction is not known; and the substance producing it is, to say the least, very fugitive, disappearing from the urine in the course of a few days, even if the specimen be preserved by addition of sublimate. It has been stated that the reaction is a "typical meat reaction," but this probably cannot be maintained as it has also been observed after use of milk, cheese and eggs. 48 17 C. Serena and A. Percival, Jahresber. f. Tierchem., 29, 338, 1890; F. de Filippi, Zeitschr. f. physiol. Chem., 49, 433, 1906. 48 V. Arnold (Lemberg), Zeitschr. f. physiol. Chem., 49, 397, 1906; Th. Holobut (Lemberg), ibid., 56, 117, 1908; X. Buss, Inaug. Dissert., Zürich, 1910, cited in Centralbl. f. d. ges. Biol., 11, No. 1702; J. Caretti, Bull. Scienze Med., 80, 253, 1909. 136 URINARY BASES Urinary Rest-nitrogen. — If the nitrogen of the various known urinary constituents for a given specimen be added together and the total compared with the determined total nitrogen, will any noticeable difference be found? It has been shown that this actually is the case. By far the major portion of the nitrogen, estimated as from 87 to 95 per cent, of the total nitrogen ordinarily and in states of full diet, 49 is referable in man and the mammals to urea. After calculat- ing the fractions referable to uric acid, the purin bases and Creatinin, hippuric acid and ammonia, there still remains a nitrogen rest which in human urine has been estimated at from 2.5 to 8.5 per cent, by Donze and Lambling, 50 at about 5 per cent, by Folin, and 11 per cent, of the total nitrogen by Maillard. These figures are changed as soon as the diet becomes abnormal, the urea varying especially, falling, particularly where opportunity for an acidosis occurs, with corresponding increase in the ammonia. 0. Folin observed a fall of the urea nitrogen to 60 per cent, of the total nitrogen in as full as possible restriction of proteid metabolism by means of a starch-cream diet. But even this is probably far from the lowest limit which may be met. In an insane patient, who took almost no food, Folin found only 15 per cent, of the total nitrogen represented by the urea, and 40 per cent, as ammonia ; 51 one would hesitate to give credence to these ^ figures if they were not published by one who is a master of the technique of urinary analysis. It should be remembered in this connection that similar perverse metab- olism proportions have been observed in the hibernating marmot (v. supra., p. 117), and have been described as an enormous increase in the aminoacids at the expense of the urea. 49 Cf. B. Schöndorff, Pflüger's Arch., 117, 275, 1907. 60 G. Donze and E. Lambling, Jour, de Physiol., 5, 225, 1903; O. Folin, Amer. Jour, of Physiol., 13, 45, 1905; L. C. Maillard, Jour, de Physiol., 10, 1017, 1908. 61 O. Folin, 1. c. FRACTIONATION OF THE OXYPROTEIC ACIDS 137 OXYPROTEIC ACIDS It would seem that the bulk of the "non dose" in the urine, the undetermined nitrogen rest, is to be referred to the group of the oxyproteic acids. These substances have been dealt with in a previous lecture in connection with the subject of the elimination of the residual material of pro- tein metabolism in cancerous affections (Vol. I of this series, pp. 547-551, The Chemistry of the Tissues). The oxyproteic acids, discovered in the urine in 1897 by S. Bondzynski and R. Gottlieb, 52 constitute a group of protein derivatives containing both nitrogen and sulphur, and ap- parently of high molecular structure ; they seem to be char- acterized by an acid nature, the solubility in water and insolubility in alcohol of their baryta salts, and by their precipitation by mercuric acetate in weakly alkaline reac- tion. In general they no longer retain the character of Polypeptids, failing to give the biuret reaction, and not being precipitable, as are other protein derivatives of high molec- ular structure, by phosphotungstic acid in the presence of excess of mineral acid. Fractionation of the Oxyproteic Acids. — The investiga- tions of Bondzynski and his collaborators 53 indicated that separation of these acids is possible by precipitation as salts of the heavy metals, and in fact led to the diff erentiation of alloxyproteic acid (precipitated by acetate of lead), ant oxy- proteic acid (precipitated by mercuric acetate in acid re- actions) and oxyproteic acid (precipitated by the same in neutral or weakly alkaline reaction). These Polish investi- gators are also to be credited with the important determina- tion that the autoxyproteic fraction includes the yellow M S. Bondzynski and R. Gottlieb, Centralbl. f. d. med. Wiss., 1897, No. 33. 63 S. Bondzynski and K. Panek, Ber. d. deutsch, ehem. Ges., 85, 2959, 1903; S. Bondzynski, S. Dombrowski, K. Panek, Zeitschr. f. physiol. Chem., Jf6, 83, 1905; S. Dombrowski, ibid., 5> h 188, 1907, Bull, de l'Acad. de Cracovie; CI. des Sciences Math, et Natur., October, 1907; J. Browinski and S. Dombrowski, Jour, de Physiol., 10, 819, 1908; W. Gawinski, Zeitschr. f. physiol. Chem., 58, 458, 1909; S. Bondzynski, Kosmos, 35, 680, 1910. 138 OXYPROTEIC ACIDS urinary coloring matter, urochrome. Moritz Weiss 54 has shown in the course of his investigations (partly carried out in the Vienna Physiological Institute) that urochrome is produced from a chromogen, urochromogen, which is like- wise an oxyproteic acid. This chromogen is characterized by two striking peculiarities, viz., by its ability to pass over into urochrome upon oxidation (a faintly tinted yellowish- green solution assuming an intense yellow color when per- manganate of potassium is added drop by drop), and by the interesting fact that it is the particular substance involved in Ehrlich 's curious diazo-reaction. Very shortly after the Polish authors happened upon the fact that autoxyproteic acid will give the well known color reaction with the neces- sary diazo-reagents the relation between urochromogen and the diazo-reaction was determined by Weiss. It is therefore apparent that we are dealing with a de- cidedly confusing group of phenomena. In the hope, how- ever, of simplifying the current status of proteic acid fractionation, the following schema is presented : 55 PROTEIC ACID FRACTION (The group of substances which form baryta salts which are soluble in water, but precipitated by alcohol.) Alloxyproteic Acid Fraction (precipitated by acetate of lead) Autoxyproteic Acid Fraction (precipi- tated by mercuric acetate in weakly acid reaction) Dombrowski's Urochrome Urochromogen .(transformed by oxidation into true urochrome) Oxyproteic Acid Fraction (precipi- tated by mercuric acetate in soda- alkaline reaction) Colorless Alloxyproteic Acid. M M. Weiss (Alland Hosp.) , Wiener klin. Wochenschr., 1907, No. 31 ; Beitr. z. Klinik der Tuberculose, 8, 117, 1907; Biochem. Zeitschr., 21, 175, 1910; 30, 333, 1911, under direction of O. v. Fürth, Physiol. Instit. Vienna; Med. Klinik, 1910, No. 92; Miinchener med. Wochenschr., 1911, No. 25. M M. Weiss, Biochem. Zeitschr., 30, 338, 1911. PROTEIC ACID FRACTION 139 The alloxyproteic acid fraction apparently contains at times four substances: Dombrowski 's urochrome (recogniz- able by its precipitation by neutral lead acetate and by copper acetate, and the insolubility of its lead salt in dilute acetic acid), true urochrome of Weiss and the colorless allo- xyproteic acid of the Polish writers True urochrome, the normal yellow coloring matter of urine, is not the same as Dombrowski 's urochrome, and may be differentiated from the latter by the solubility of its lead salt in dilute acetic acid. In addition urochromogen, occurring in pathological urines, is found for the most part in this fraction (careful precipita- tion avoiding excess of lead acetate) ; but sometimes, it is true, it is met in the autoxyproteic acid fraction. Weiss 56 has devised a method by which it is possible to estimate by comparative colorimetry the amount of urochrome and urochromogen in a urine with great exact- ness. The urine is first freed of other coloring materials by saturation with ammonium sulphate (urobilin, hsematopor- phyrin and uroerythrin) ; and the color of the filtrate is then compared by means of a Dubosq colorimeter with a true yellow solution of known content. If urochromogen is pres- ent along with urochrome a dilute solution of permanganate is cautiously added as long as increase in the yellow color can be clearly recognized or until the Ehrlich 's diazo-reac- tion, if at first present, begins to fail ; thereafter the process is as above. The difference between the quantities of uro- chrome obtained before and after the full oxidation repre- sents the quantity of urochromogen present in the specimen. In examining urines with positive diazo-reactions there is always to be noted a direct proportion between the intensity of the diazo-reaction and the amount of urochromogen in the urine. The technical simplicity and the sensitiveness of the urochromogen reaction with permanganate makes it possible to substitute the diazo-reaction by the former. 50 M. Weiss, Biochem. Zeitschr., 30, 345, 1911. 140 OXYPROTEIC ACIDS Chemical Position of Urochrome. — It is at present not advisable to say more in reference to the chemical classi- fication of urochrome, the urinary coloring matter which is mainly responsible for the yellow color of normal urine, than that we are dealing with one of the substances in- cluded in the group of protein residual matter which from its characteristics of solubility and precipitability should be classed among the oxyproteic acids. There is a contradiction in the matter of its sulphur content between the statements of the above-named Polish authors,who found the coloring material to contain sulphur, and the results ob- tained in Hofmeister 's laboratory. 57 The latter indicate that the yellow material, which can be separated from the urine by animal charcoal and can subsequently be freed from the latter by glacial acetic acid (known as "uroipyrryV' because of the large proportion of pyrrol left when it is subjected to dry distillation), does not contain any sulphur. It may be thought that this contradiction may be explained on the supposition that the urochrome, which has its sulphur bound so loosely in the molecular structure that sulphuretted hydrogen separates, even without heating, when it is acted upon by alkali, 58 may likewise undergo cleavage in the course of the above process of demonstration with loss of its sulphur ; but this has not been proven. There may possibly be a close relation between such a sulphur partition and the peculiar reduction power of urochrome which can be directly estimated by titration by separation of iodine from iodic acid. 59 The suggestion that urochrome owes its coloring to one of the cyclic groups of the protein molecule included in some form in its structure (or a transformation product of such a substance) is not improbable. There is not the least foundation, however, for assuming a relation between 57 H. Hohlweg, K. E. Salomonsen, S. Mancini (Lab. of Physiol. Chem., Strassburg), Biochem. Zeitschr., IS, 199, 205, 208, 1908. 68 S. Bondzynski, S. Dombrowski and K. Panek, Zeit9chr. f. physiol. Chem., 46, 83, 1905; S. Dombrowski, ibid., 62, 358, 1909. 50 J. Browinski and S. Dombrowski, Jour, de Physiol., 10, 819, 1908. UROCHROME 141 urochrome and hamiatin and urobilin. Direct evidence of the cyclic nature of urochrome may perhaps be seen in the striking diazo-reaction of urochromogen. (A color reaction, less brilliant however, is given by the diazo-compounds with formed urochrome; it corresponds closely with the diazo- reaction of the normal urine. 60 ) The fact that when uro- chrome is boiled with hydrochloric acid melanin-like prod- ucts ("uromelanin") may be formed 61 is, if the author is correct in his previous statements as to the nature of melanin production (v. Vol. I of this series, p. 526, et seq.. Chemistry of the Tissues), certainly not contradictory of a cyclic character of urochrome. It suggests, too, that certain ring-form groups in the protein-molecule escape complete dissociation in the course of metabolism and may finally appear as urinary coloring materials. In the author's laboratory there are in course at present certain experiments bearing upon a possible part taken by histidin in the con- struction of urochrome ; an idea suggested by the fact that this compound is distinguished among the molecular cleav- age products (as also tyrosine) by responding to the diazo- reaction. True Urochrome and Dombrowski's Urochrome. — The marked tendency to undergo decomposition shown by the yellow coloring substance of the urine, is, moreover, the cause of the differences of opinion obtaining between M. Weiss and the Polish authors with reference to it. While the latter authors look upon the urochrome discovered by themselves as the peculiar urinary coloring matter, Weiss, as stated, differentiates between true urochrome and the uro- chrome of Dombrowski. The impression is that this latter product, which is isolated from urine by precipitating it by 60 K. Feri (Chvostek's Clinic, Vienna) recommends substitution of the Ehrlich reagent by azophen red, that is, paranitrodiazobenzolsulphate (NO-2.0Ä — N = N — ) 2 S'0 4 ; Wiener klin. Wochenschr., 1912, No. 24. 61 J. Dombrowski, Zeitschr. f. physiol. Chem., 5Jf, 188, 1907; 62, 358, 1909. 142 OXYPROTEIC ACIDS means of copper acetate, with subsequent dissociation of the copper compound by sulphuretted hydrogen, is nothing but an oxydation transformation product of the true urochrome. Browinski and Dombrowski 62 in their most recent publica- tion with special stress suggest "that urochrome is an un- usually easily decomposable compound and readily under- goes oxidation. Its precipitation from the urine and from pure solutions by means of cupric acetate is due to the forma- tion of a cuprous compound which is soluble in water; and can occur only with reduction of the copper acetate. The pre- cipitation is therefore incomplete, because necessarily the combination is formed with oxidation of a portion of the urochrome. ' ' Thus far it is possible to agree with the Polish authors. But with their further statement the author's views become directly opposed : "The oxidation product (of the urinary coloring matter) which remains in solution im- parts the faint yellow tint to the filtrates of copper precipita- tion peculiar to them. ' ' According to this the substance in the filtrate would be an insignificant remnant of the true urinary coloring matter which has escaped precipitation and has undergone oxidation. Actually, however, as Weiss has shown in his colorimetric estimations, 63 the opposite is the case. If Dombrowski 's urochrome (a sallow brownish yellow coloring substance) be removed, by far the greater part of the coloring matter remains in the filtrate, which shows the characteristic pure tint (yellow with a greenish cast) of the former [true urochrome]. Any unprejudiced person must grant that when one knows that a given sub- stance is very ready to undergo oxidation there is little propriety in using an oxidizing agent like copper acetate in isolating it. If, however, such an agent is employed and, too, if it has been clearly proved that oxidation has actually 62 J. Browinski and S. Dombrowski (Instit. of Med. Chem., Lemberg), Zeitschr. f. physiol. Chem., 77, 105, 1912. 63 M. Weiss, Biochem. Zeitschr., SO, 340, 1911. QUANTITATIVE DETERMINATION 143 been effected in the course of demonstration, it may un- doubtedly be said that nothing but an oxidation product can be expected as the result. It may be, too, that a portion of the true urochrome may undergo spontaneous decomposition in the urine with forma- tion of its oxidation product. In any case we must regard, not Dombrowski's urochrome, but Weiss 's so-called "true urochrome ' ' as the native urinary yellow coloring material. The attempt of the authors indicated to introduce a polemic spice in their latest publication can have not the least influ- ence upon the actual fact. Quantitative Determination of Oxyproteic Acids. — Our appreciation of the physiological role and signifi- cance of the various substances of the group of oxyproteic acids is of slow development. The reason for this is mainly that as yet we are unable to overcome the technical difficul- ties attending a quantitative determination of these sub- stances. In discussing the elimination of the oxyproteic acids and of neutral sulphur in cancerous disease (v. Vol. I of this series, pp. 547-551, Chemistry of the Tissues) this fact was fully presented. Recently objection has been raised by F. Erben 64 to the method of Ginsburg C5 for estimation of the oxyproteic fraction in urine, that besides the oxyproteic acids practically all the aminoacids are thrown down. There is something, however, against the correctness of this charge in the fact that Ginsburg has been unable to find more than minute amounts of aminoacid nitrogen in his oxyproteic acid fraction both by formol titration and, too, by Van Slyke's method 66 (v. Vol. I of this series, p. 18, Chemistry of the Tissues). Although Erben summarily characterizes Gins- burg's method as "useless," it should be observed that the method, although (as already stated, v. Vol. I of this series, 64 F. Erben (Vienna), Internat. Beitr. z. Pathol, u. Ther. d. Ernährungs- störungen, 2, 252, 1911. 65 W. Ginsburg (Univers. Physiol. Instit., Vienna), Hofmeister's Beitr., 10, 411, 1907. 68 Personal communication from W. Ginsburg, Halle a. d. Saale. 144 OXYPROTEIC ACIDS p. 548, Chemistry of the Tissues) far from an ideally exact mode of determination, is still undoubtedly of greater scope than that of Erben, who sought no more than the autoxy- proteic acids (by precipitation of the baryta syrup by mercuric acetate after faint acidulation with acetic acid). This mode of separation must be regarded as largely open to the personal equation, — if for no other reason because, as is well known, a part of the mercurial salts of the oxyproteic acids is soluble in a greater surplus of acetic acid. On the other hand it should be acknowledged that it would be a good thing if a modification of the Ginsburg method were worked out by ascertaining the possible admixture of urea and of aminoacids in the fraction of oxyproteic acids, and making correction therefor. Elimination of Oxyproteic Acids in Normal and Patho- logical Conditions. — With this in view it is best to state only in a provisional way that the quantity of oxyproteic acid in normal urine represents from 3 to 7 per cent, of the total nitrogen in case of adults, 67 and 10 per cent, in infants. 68 Probably the oxyproteic acids in the "rest nitrogen" of the blood are in greater proportion than they are in the urinary rest nitrogen (it is said that 40 per cent, of the rest nitrogen of the blood serum is here included) ; and it is probable that a part of the hsemic oxyproteic acids may be oxidized before they reach the kidney for elimination. 69 In disease an increased elimination of substances be- longing to the group of oxyproteic acids is usually met in cases in which an increased decomposition of cellular protein is proceeding under the influence of toxic metabolic disturb- ances. This may be expected in phosphorus poisoning, in 67 W. Ginsburg, 1. c; W. Gawinski (Instit. of Med. Chem., Lemberg), Zeitschr. f. physiol. Chem., 58, 454, 1909. 68 S. Simon ( Children's Clinic, Univ. Munich ) , Zeitschr. f . Kinderheilk., 2, 1, 1911. 09 J. Browinski (Instit. Med. Chem., Lemberg), Zeitschr. f. physiol. Chem., 58, 134, 1908; W. Czernecki, Bull, de l'Acad. de Cracovie, 1910, abst. in Jahresber. f. Tierchem., 40, 189, 1910; cf. also ibid., 39, 820, 1909. ELIMINATION OF OXYPROTEIC ACIDS 145 febrile infectious diseases, hepatic affections, the cachexia of cancer and in the later stages of tuberculosis. In any such condition an increased excretion of oxyproteic acids may be told from the increase in the elimination of neutral sulphur, 70 which, as previously pointed out (v. Vol. I of this series, pp. 548-550, Chemistry of the Tissues), may serve as an index for the former. The degree of complexity of the subject may, however, be inferred from the fact that, according to circumstances, now one, now another fraction of the oxy- proteic acids may predominate in a given case. Thus in tuberculosis a much greater amount of urochromogen is excreted than in cancerous cachexias. It is a well-known fact that a practical significance is ascribed to the appear- ance and disappearance of the Ehrlich diazo-reaction (now known to be referable to urochromogen) in the matter of prognosis of the course of tuberculosis. 71 In considering the diazo-reaction 72 it should be observed that besides urochromogen other substances may occur in the urine which yield color reactions with the diazo-bodies, as, according to Clemens, tyrosin (OH.C 6 H 4 .CH 2 .CH(NH 2 ). COOH) and p-oxyphenylpropionic acid (OH.C 6 H 4 .CH 2 .CH 2 . COOH) and p-oxyphenylacetic acid (OH.C 6 H 4 .CH 2 .COOH). Kutscher and Engeland state that even in normal urine after removal of the aromatic oxyacids by ether certain bodies may remain which form red products with diazo-benzol- sulphurous acid when in an alkaline soda solution. This N— c reaction apparently is peculiar to the imidazol nuclei, c c , N 70 Cf. M. Halpern, Centralbl. f. d. ges. Biol., 11, No. 2547, 1911; E. Salkow- ski, Biochem. Zeitschr., 82, 356, 1911; cf. M. Weiss, Münchener med. Wochenschr., 1911, No. 25. 71 F. Kutscher, Sitzungsber. d. Ges. z. Bef. d. ges. Naturw., Marburg, 4, 83, 1908; abst. in Centralbl. f. Physiol., 22, 516, 1908; R. Engeland (Physiol. Instit., Marburg), Münchener med. Wochenschr., 1908, 1643; M. Weiss and A. Weiss, Wiener klin. Wochenschr., 1912, No. 31. 72 Literature upon the Diazo-reaction in Urine : P. Clemens ( Bäumler's Clinic, Freiburg), Deutsch. Arch. f. klin. Med., 63, 74, 1899. 10 146 OXYPROTEIC ACIDS which may be found in the urine in the form of imidazol- CH.NHv II >CH C— N " aminopropionic acid (histidin), CH 2 , and of imidazol- CH.NH 2 COOH CH-NH V B >CH C N^ aminoacetic acid, | . It is said that the puzzling CH.NH 2 "urocaninic acid" of dogs' urine is also an imidazol deriva- COOH. acid' CH-NH V II >CH C N^ £j ve CH , obtainable from histidin by ammoniacal CH I COOH cleavage. 73 Other High-molecular Residual Substances. — In addi- tion to the oxyproteic acids there are other high-molecular metabolic residual substances of various kinds in the urine, about which we know practically nothing. E. Abder- halden and F. Pregl, 74 after freeing an alcoholic extract of urine of urea and other easily diffusible materials by dialysis, obtained a mixture of substances which contained no free aminoacids but after acid hydrolysis yielded a num- ber of typical protein cleavage products. It is impossible at the present to decide the extent to which these substances are related with the oxyproteic acids (which unquestionably hydrolytically yield aminoacids 75 ) or with typical Poly- peptids. It seems that they may be met in small amount in normal urine and somewhat more in disease, as in cancer 78 A. Hunter (Cornell Univ.), Jour, of Biol. Chem., 11, 537, 1912. 74 E. Abderhalden and F. Pregl, Zeitschr. f. physiol. Chem., 46, 19, 1905. 76 W. Ginsburg, Hofmeister's Beitr., 10, 441, 1907; J. BrowinBki and S. Dombrowski (Lemberg), Zeitschr. f. physiol. Chem., 77, 92, 1912. HIGH-MOLECULAR RESIDUAL SUBSTANCES 147 and hepatic affections (v. Vol. I of this series, p. 550, Chem- istry of the Tissues). The chemical position of the urinary constituent known as "uroferric acid," isolated by Sieg- fried's iron-peptone method, is at present indefinable 76 ; and the same may be said of a substance obtained by P. Häri 77 by precipitation by phosphotungstic acid, and the urinary colloids of Salkowski (v. Vol. I of this series, p. 550 r Chem- istry of the Tissues) precipitable by salts of the heavy metals and insoluble in alcohol. But it would be a mistake to assume that all nitrogenous colloids of the urine are of the nature of high-molecular protein derivatives. Investiga- tions in the laboratory of F. Hofmeister indicate on the contrary that among the nondialysable urinary constituents (which can be quantitatively estimated by diffusion methods employing very fine fibre capsules), we may meet even such materials as chondroitin-sulphuric acid (Vol. I of this series, p. 281, Chemistry of the Tissues) or nucleinic acid as im- portant compounds ; their quantity may be found increased in renal diseases, in eclampsia and in various febrile states (pneumonia). 78 It will probably be a long time before the misty atmos- phere at present enveloping these subjects, and in fact cover- ing them as in thick clouds, will be dissipated by the sun. However, even here there is beginning to be a little more light. 76 O. Thiele, H. Liebermann (Siegfried's Lab., Leipzig), Zeitschr. f. physiol. Chem., 31, 251, 1903; 52, 129, 1907. "P. Häri (Budapesth), Zeitscbr. f. pbysiol. Chem., 46, 1, 1905. 78 K. Sasaki, Hofmeister's Beitr., 9, 386, 1907 ; M. Savarg, ibid., 9, 401 ; 11, 71, 1907; Ch. Pons, ibid., 9, 393, 1907; U. Ebbecke, Biochem. Zeitschr., 12, 485, 1908; all from the laboratory of F. Hofmeister, Strassburg. CHAPTER VII PHYSIOLOGY OF PURIN METABOLISM Having in the last several lectures dealt with the nitrog- enous end-products of protein metabolism, we may next take up the difficult subject of purin metabolism. First, however, it should be said that the general mass of observa- tions bearing upon this phase of our subject is so huge that no honest man, even if he has busied himself with nothing else for years, dare feel that he has reached the bottom facts- and become thoroughly conversant therewith. The author would be presumptuous, moreover, being himself not con- tinuously engaged in this field, but concerned rather with the single purpose of tracing the general subject, to attempt to surround himself here with dogmatic assertions. The purpose of these lectures does not go beyond the arrange- ment and presentation of a picture of this world of phe- nomena so far as the writer has from honest study come to understand and appreciate them ; and it should not be for- gotten at any time that this picture may for other eyes have a very different aspect. In the end every man has a right to use his own eyes in inspecting the things about him ; only he should realize that his impressions in such case are essen- tially subjective ones. So much in introduction. Exogenous and Endogenous Formation of Uric Acid. — The first question to occupy us is — what do we know of the origin of uric acid in the mammalian body I In precise answer it might be replied : it originates from the free nuclein bases and the nuclein bases fixed in the molec- ular structure of nucleinic acids. These are in part intro- duced into the living body with food (exogenous fraction) and in part are freed by nuclear disintegration or other processes within the living organism from its molecular com- binations of which they are structurally a part (endogenous fraction). 148 CONVERSION OF ADENIN INTO URIC ACID 149 The recognition of this relation had its beginning in the classical studies of Horbaczewski in 1889 upon the formation of uric acid in the splenic pulp ; and slowly crystallized into its present form from a great number of investigations, among which those of Kossel and his school, and of W. Spitzer, H. Wiener, A. Schittenhelm, N. Jones, R. Burian, L. B. Mendel and their collaborators stand out prominently. 1 Conversion of Adenin and Guanin into Uric Acid. — As previously stated (Vol. I of this series, p. 112, Chem- istry of the Tissues), the physiological relation between the two bases in the nucleinic acid molecule, adenin and guanin, with hypoxanthin, xanthin and uric acid may be expressed by the following schema : ADENIN (C 6 H 6 N 6 ) GUANIN (C 6 H 6 N 6 0) N-C=NH N-C=0 'I SI C-N v NH=C C-Ns , I >C \ I N-C-N' N-C-N' HYPOXANTHIN (C6H4N4O) XANTHIN (C5H4N4O2) URIC ACID (C6H4N4O1) N-C=0 N-C=0 N-C=0 / \ / I / I C C Nv — ^ 0=C C-N. — > 0=C C-Nv. \ I >c \ 1 >c \ 1 >c=o. n-c-n/ n-c-n/ n-c-n/ The transformation of adenin and guanin into hypoxan- thin and xanthin by replacement of their NH group by an atom of oxygen is ascribed to ' ' deamidases ' ' whichmust effect changes of the type indicated in the formula : R : NH + H 2 = R : + NH 3 . Jones recognizes two of these enzymes, " adenase" and " guanas e." In the conversion of hypoxanthin into xanthin and the latter into uric acid, oxidizing ferments known as " ' xanthoxydases " are concerned. 1 Literature upon the Formation of Uric Acid from the Nucleins : H. Wiener, Ergebn. d. Physiol., 1, 575-606, 1902; F. Samuely, Handb. d. Biochem., 1, 565-566, 1909; A. Ellinger, ibid.,3', 575-576, 1910; A. Schittenhelm, ibid., -V, 490-514, 519-531, 1910; C. Oppenbeimer, Die Fermente, 3d ed., 5, 166-170, 370-372, 1910; H. M. Vernon, Ergebn. d. Physiol., 9, 162-167, 1910. 150 PHYSIOLOGY OF PURIN METABOLISM A great deal of careful work has been devoted to the study of the distribution of these ferments in various animal species, in experiments with tissue pulp. 2 Schittenhelm 3 was doubtless correct in questioning whether all the differ- ential detail involved should be accepted literally; and whether, if one of these ferments were missed in a given or- gan it would be justifiable to infer therefrom that it is also absent from the living organ. In some aspects, however, investigations of this sort may be productive of results. Guanin Gout in the Hog. — For example, it was found that in tissues of the hog "guanase" occurs only in small amount and that addition of guanin to extracts of spleen and liver was followed by but insignificant increase in the quantity of xanthin produced in course of the digestion ; but that adenin is freely converted into hypoxanthin. An interesting cor- relation may be recognized in the fact that normally in the urine of hogs the purin bases are more prominent than uric acid, and that there is a disease, guanin-gout of the hog, in which uric acid apparently disappears completely from the urine, and at the same time (in analogy to the deposit of uric acid in the tissues in human gout) guanin is deposited in the muscles, cartilages, and in the liver. The lack of ' ' guanase ' ' apparently interferes here with the normal formation of uric acid, which in the hog is for the most part further cata- bolized into allantoin. 4 Nucleases and Deamidases. — The chemical conversion of the purin bases must, however, be preceded by their cleavage from the molecule of nucleinic acid, which takes place in the nucleins of the food in the intestine through the agency of 2 Cf. especially the numerous studies of A. Schittenhelm, as, too, of W. Jones (with C. L. Partridge, W. C. Winternitz, C. R. Austrian, Amberg and others). S A. Schittenhelm, Handb. d. Biochem., 4', 509, 1910; W. Jones, Zeitschr. f. physiol. Chem., 45, 84, 1905. 3 A. Schittenhelm, Handb. d. Biochem., 4', 509, 1910; W. Jones, Zeitschr. f. W. Jones and C. R. Austrian (Johns Hopkins Univ.) , ibid., 48, HO, 1906; L. B. Mendel and J. F. Lyman (Yale Univ.), Jour, of Biol. Chem., 8, 115, 1910; A. Schittenhelm, Zeitschr. f. physiol. Chem., 66, 53, 1910; L. B. Mendel, and P. H. Mitchell (Yale Univ.), Amer. Jour, of Physiol., 22, 97, 1907. NUCLEASES AND DEAMIDASES 151 "nucleases" (cf. Vol. I of this series, p. 128, Chemistry of the Tissues). From recent experiments of London, Schitten- helm and K. Wiener, in which a series of dogs (normal, with- out stomach, without pancreatic juice — ligation of the excre- tory ducts of the pancreas, and without pancreas) were fed upon nucleinic acid, it was determined that the cleavage of the nucleinic acid is to be entirely ascribed to the ferments of the intestinal juice. The cleavage always stops at the stage of nucleosides, which undergo no further dissociation in the intestine, the purins remaining in combination with the sugar of the nucleinic acid molecule (as do also the pyri- midin bases) in all experiments. In the lower segments of the bowel the splitting influence of bacteria may be associated with that of the intestinal ferments. 5 The resorption of the purin bodies takes place by way of the blood, not with the lymph. 6 Cleavage of the nucleinic acids in the animal tissues is undoubtedly a very complicated process. Two kinds of nucleases involved are differentiated: "purinnucleases," which split off the purin bases, and " phosphornucleases," which separate phosphoric acid from the molecule; the nucleosides (combinations of purin bases and carbohy- drate), however, remaining intact. 7 It may be recalled (cf. Vol. I of this series, pp. 123-125, Chemistry of the Tissues) that the carbohydrate group in the nucleinic acid molecule is intercalated between phosphoric acid and the base : Phosphoric acid < Carbohydrate >. Base. Further complication of the situation arises from the fact that deamidization of the purin bases may also take place at this stage, provided they are still in organic combination, in 6 E. S. London, A. Schittenhelm and K. Wiener (Erlangen and S't. Peters- burg), Zeitschr. f. physiol. Chem., 77, 86, 1912. 6 J. Biberfeld and J. S. Schmid (Breslau), Zeitschr. f. physiol. Chem., 60, 292, 1909. 7 Amberg and W. Jones (Johns Hopkins Univ.), Zeitschr. f. physiol. Chem., 73, 407, 1911. 152 PHYSIOLOGY OF PURIN METABOLISM such manner that, according to Schittenhelm, 8 deamidization may involve either free or fixed purin bodies ; and for that reason it is essential to distinguish between purin- deamidases and nucleosiddeamidases. It may be seen that in the catabolism of the purin bases included in the nucleinic acid molecule into uric acid a great many different possibilities enter, and that we must assume a coordination of nucleases and nucleoside-splitting fer- ments, of purin-deamidases and nucleosiddeamidases, as well as a number of oxidases. According to Levene and Medigreceanu 9 the nucleinic acids are made up of nucleo- tids, a type of which, according to these authors, may be seen in guanylic acid (Vol. I of this series, pp. 125-128, Chemistry of the Tissues), to which they ascribe the simple constitution of phosphoric acid-pentose-guanin. They then differentiate in the fermentation-cleavage of nucleinic acids between nucleinases, which split up the nucleinic acid molecule into nucleotids, and nucleotidases, which in turn catabolize the nucleotids. What an enormous amount of work is still to be accom- plished before we can be in position to clearly define the physiological and pathological influence of any one of these factors ! As a matter of fact this is only the beginning of the difficulty. Nuclear Destruction and Urinary Purins. — The next question is that of the origin of the endogenous and exog- enous urinary purins, about which it is safe to say that veritable rivers of ink have run dry in advancing to a point where (and this is always a good sign) the matter can be properly stated in a few words. At present we know that the exogenous portion of the urinary purins depends in mammals upon the quantity of free or combined purins in the food, bearing in mind moreover the further conversion of 8 A. Schittenhelm and K. Wiener (Erlangen), Zeitschr. f. physiol. Chem., 77, 77, 1912. 9 P. A. Levene and F. Medigreceanu (Rockefeller Instit., New York), Jouiv of Biol. Chem., 9, 65, 375, 389, 1911. RELATION TO MUSCULAR ACTIVITY 153 uric acid, especially the formation of allantoin which is so prominent in many animals (vide infra). As far as the endogenous fraction is concerned it is known that the purin bases set free in the disintegration of cellular nuclei in all tissues eventually appear in the form of urinary purins. There is reason for avoiding a restricted concep- tion which would make the leucocytes, the muscles, the digestive glands 10 or the kidneys alone responsible for the endogenous production of uric acid ; it is better to look upon it as the expression of a continuous and general cellular wear. Precisely because this process of wearing out and gradual removal of cells is a very constant and regular one, at least when serious pathological changes are not present, a relative constancy in the endogenous urinary purin frac- tion is to be expected for each individual, as first observed by R. Burian and H. Schur and afterwards confirmed by a number of other writers. 11 Briefly stated, the active metab- olism of the growing body, the experimental exaggeration of glandular activity by pilocarpin, the increase of cell de- struction by use of Röntgen rays, and very many pathologi- cal disturbances like phosphorus poisoning, hepatic atrophic cirrhosis, jaundice, Eck's fistula, fever, leukaemia, etc., are all capable of increasing the endogenous fraction of urinary purins. 12 Relation of Purin Metabolism to Muscular Activity. — There is only one point which requires any further dis- cussion in this connection, namely, that of the relation of the purins to muscular labor, 13 a matter heretofore dealt with in 10 O. Siven (Helsingfors) , Pfliiger's Arch., 1^6, 449, 1912. n Cf. E. W. Rockwood, Amer. Jour, of Physiol., 12, 38, 1904; F. Mares, vide infra. 12 B. Hirschstein (F. Umber's Clinic), Arch. f. exper. Path., 57, 229, 1907; Th. Brugsch and A. Schittenhelm, Zeitschr. f. exper. Path., 4, 761, 1907; O. Siven (Helsingfors), Skan. Arch. f. Physiol., 18, 177, 1906; S. Bondi and F. König, Wiener med. Wochenschr., 1910, Nos. 44-45 ; F. Mares, F. Smetanka (Physiol. Institut. Czech. Univ., Prague), Pflüger's Arch., 13' h 59, 1910; 138, 217, 1911. 13 R. Burian, Med. Klinik, 1905, No. 6, and 1906, Nos. 19-21. 154 PHYSIOLOGY OF PURIN METABOLISM considering the chemistry of muscular tissue (v. Vol. I of this series, p. 159, Chemistry of the Tissues). It cannot be doubted that muscular activity has a part in the formation of the endogenous urinary purins. The fact, however, that Schittenhelm 14 has found no striking reduction of the endogenous amount in human beings with well marked mus- cular ati ophy would, however, contradict the idea that the bulk of these substances originates in muscle tissue. Siven, too, failed to recognize any characteristic influence of free muscular movements upon the endogenous purin metabolism in man 15 ; while Goudberg invariably found the uric acid increased in starving rabbits after muscular spasms pro- duced by faradism. 16 Finally it should be noted that mus- cular involvement in processes concerned with heat regula- tion is apparently more likely to influence the purin excretion than voluntary contractions. The increased uric acid excretion of fever and its nocturnal diminution are perhaps related to the same type of processes. 17 Synthetic Formation of Uric Acid in Birds and Reptiles. — Being in some measure satisfied of the production of uric acid through processes of oxidation from nucleins, attention should be directed to the synthetic production of uric acid. 18 It is known that the major portion of the nitrogen, which in mammals, amphibians and fish is excreted as urea, is repre- sented in birds, reptiles and also in many invertebrates 19 by uric acid, which is produced in them synthetically. What is known of the mechanism of this process? 14 A. Schittenhelm, Handb. d. Biochem., 4' 521, 1910. 15 V. O. Siven, 1. c. 18 A. Goudberg (Hamburg), Zeitschr. f. Neurol, u. Psych., 8, 487, 1912. " E. P. Cathcart, J. B. Leathes, E. L. Kennaway, cited by A. Ellinger, Handb. d. Biochem., 4', 576, 1910. 18 Literature upon the Synthesis of Uric Acid : H. Wiener, Ergebn. d. Physiol., 1, 606-615, 1902; A. Magnus-Levy, v. Noorden's Handb., 1, 126-129, 1906; A. Schittenhelm, Handb. d. Biochem., \ , 522-525, 1910. 19 Cf. O. v. Fürth, Vergl. chem. Physiol, d. niederen Tiere, pp. 258-303, Jena, 1903. LACTIC ACID IN URIC ACID SYNTHESIS 155 The skeletal plan of the uric acid molecule may be repre- sented as consisting of two fractions of urea fixed to a chain of three atoms of carbon : / NH 2 I NH 2 NH— CO CO C \(X) — ► CO C— NH X \ N n | NH / \ II >CO NH * \ Q NH * NH— C— NH/ Our ideas in reference to the urea rests required in the synthesis of uric acid are fairly clear. According to the investigations of Knieriem, Jaff e and Hans H. Meyer, as well as those of Schröder, there can be no question as to the abil- ity of the liver in birds to construct uric acid from ammonia salts, aminoacids, as well as from urea. The hypothesis that in the avian body, just as in mammals, the protein nitrogen is first broken down into urea, and subsequently as a secondary process is synthesized into a complex of two urea rests and a group of three carbon atoms is an entirely satisfying one. What is the significance of this triple group of carbon atoms? Minkowski's well-known experiment, in which he noted the appearance of lactic acid and ammonia in the urine of geese from which the liver had been extirpated, has forced lactic acid for the past nearly twenty years prominently into consideration, and has apparently harmonized the general subject. It has been proved that the appearance of lactic acid is due to the loss of the hepatic function ; as ligation of all of the hepatic vessels produces the same effect, but if even a single branch of the hepatic artery be open there is no forma- tion of lactic acid. To prove that the failure of uric acid for- mation is actually due to the exclusion of the liver and is not in some way a secondary result of accumulation of lactic acid in the body, requiring considerable ammonia for neutraliza- tion and thus interfering with synthesis of uric acid, we may recall the fact that (as Sigmund Lang was able to show in Hofmeister 's laboratory) introduction of alkali into geese with excluded livers is not followed by any increase in the 156 PHYSIOLOGY OF PURIN METABOLISM disturbed uric acid synthesis. H. Wiener has noted in de- hepatized birds that if the economy is experimentally flooded with urea, lactic acid and especially, too, a number of di- basic acids in whose structure there occurs a chain of three carbon atoms, as malonic acid (COOH.CH 2 .COOH), tar- tronic acid (COOH - CH(OH) - COOH) and mesoxalic acid (COOH- CO -COOH) are capable of giving rise to an increase in the uric acid elimination. The synthesis of uric acid in such case may proceed somewhat as follows : LACTIC ACID TARTRONIC ACID DIALURIC ACID URIC ACID CH 3 COOH NH— CO NH— CO I I /I /I CH.OH — ^CH.OH — > CO CH(OH) — > CO C— NH + urea \ | + urea \ || \ ro COOH COOH NH— CO NH-C-NH/^ U ' The recent recognition of a synthesis of uric acid from dialuric acid and urea 20 may be regarded as the capstone of the construction. An important recent investigation by Ernst Friedmann and H. Mandel 21 would indicate that we are still far from the truth. Contrary to Kowalewski and Salaskin, who found an increase in the amount of uric acid in the blood used in perfusion of the excised goose-liver after lactate of ammonia was added, the above named authors were un- able to in any way influence uric acid formation by introduc- ing either lactate of sodium and urea, or malonate of sodium and urea. On the contrary a remarkably large formation of uric acid was a striking feature when perfusion was per- formed with normal blood unchanged by any additions, clearly not dependent upon washing out of previously formed uric acid but indicative rather of an actual new formation. This brings up the question whether perhaps the synthesis of uric acid is not accomplished in a very dif- ferent manner than indicated in the above schema. It is 20 G. Izar (Ascoli's Lab., Catania), Zeitschr. f. pbysiol. Chem., 13, 317, 1911. 21 E. Friedmann and H. Mandel (First Med. Clinic, Berlin), Arch. f. exper. Pathol. (Schmiedeberg Festschrift), p. 199, 1908. SYNTHETIC PURIN FORMATION IN MAMMALS 157 very evident that the problem cannot be regarded at present as solved. It has been actually shown in Minkowski's laboratory that, as previously suspected, besides the synthetic formation in the avian body there is also an oxidation formation of uric acid in small amounts in complete analogy to the process in mammals. Synthetic Purin Formation in Mammals. — There is no evidence at all at present to indicate that uric acid is synthetically produced in mammalian animals and man along with its oxidative production, in analogy to the proc- ess seen in birds, as was asserted by H. Wiener. 22 However, there is no doubt as to the ability even of the mam- malian body to construct purin complexes in prepara- tion for their incorporation in the molecules of nucleinic acid. The most important points in this connection have been previously considered (Vol. I of this series, p. 129, Chemistry of the Tissues). Proof is available not only in case of the eggs of the silk moth and of the hen, the salmon in starvation, but also in case of growing sucklings and of rats fed upon purin-f ree diet, that the animal body is able to construct de novo the bases necessary for purin formation out of practically any components of the protein molecule ; and that it is not restricted exclusively to purin bases of exogenous origin. 22a But no one knows just how this con- struction takes place. It has not been successfully shown by feeding experiments that transformation takes place of de- N— C rivatives of the pyrimidin nucleus, 23 C C, (Vol. I of this N— C 22 P. Burian, Zeitschr. f. physiol. Chem., J@, 497, 1905; W. Pfeiffer (F. Hof- meister's Lab., S'trassburg, and Quincke's Clinic, Kiel), Hofmeisters Beitr., 10, 324, 1907. 22a E. V. McCollum (Wisconsin), Amer. Jour, of Physiol., 25, 120, 1909; L. S. Fridericia (Copenhagen), Skandin. Arch. f. Physiol., 26, 1, 1912; cf. therein Literature. 23 L. B. Mendel and V. C. Myers (Yale Univ.), Amer. Jour, of Physiol., 26, 77, 1910. 158 PHYSIOLOGY OF PURIN METABOLISM series, p. 113, Chemistry of the Tissues) or of a derivative Q n of the imidazol nucleus, | >C , histidin , 24 into the purin C— n/ N— C nucleus, C C— N v in which both the former nuclei are, \ I V N— C— N' so to say, included. Allantom as an End-product of Mammalian Purin Me- tabolism. — We come now to the most abstruse and debated portion of the uric acid problem, that of uric acid destruction in the body, uricolysis. For the past decade, in spite of all the study devoted to it, this problem has remained fixed, un- doubtedly for the reason, at present easily appreciable, that the dominating position which is occupied by allantoin in the nuclein metabolism in mammals was entirely overlooked. The ignorance which previously prevailed upon the general subject first began to lessen when Wilhelm Wiechowski con- tributed to metabolic investigation an exact method of de- termining allantoin and took up in definitive manner the problem of uric acid destruction in a series of very carefully conducted studies. 25 After it became known from repeated investigations 28 that living excised tissues of mammals are capable of de- stroying uric acid, Wiechowski proved that it is completely oxidized in the process into allantoin without undergoing further change : URIC ACID ALLANTOIN NH— CO NH 2 CO C— NH V — > CO CO— NH X \ II >co \ I >co. NH-C-NH/ HN-CH-NH/ 34 E. Abderhalden, H. Einbeck and J. Schmid, Zeitschr. f. physiol. Chem., 68, 395, 1911. 25 W. Wiechowski (J. Pohl's Lab., Prague), Hofmeister's Beitr., 9, 247, 295, 1907; 11, 109, 1907; Arch. f. exper. Pathol., 60, 185, 1909; Biochem. Zeitschr., 25, 431, 1910; cf. in this last the Literature upon Uricolysis. 26 Stokvis, Chassevant and Riebet, Ascoli, Jacoby, Schittenhelm, Burian, Austin, Almagia, Wiener. Cf. H. M. Vernon, Ergebn. d. Physiol., 9, 168, 1910. ALLANTOIN AS AN END-PRODUCT 159 That this is really the expression of a physiological process is evidenced directly by the fact that in the mammals carefully studied from this point of view (dog, cat, rabbit, hog, cow) excretion of uric acid and of the purin bases is decidedly overshadowed by allantoin, as shown from obser- vations of Schittenhelm, Abderhalden, Underhill, L. B. Men- del and others. Parenterally introduced uric acid is com- pletely excreted by dogs and rabbits, in by far the greater proportion as allantoin, and only in minor amount as uric acid. Nucleinic acid from thymus derivation, too, when in- troduced with the food, according to Schittenhelm 27 under- goes cleavage in the body of the dog in a way that the great bulk (93-97 per cent.) of its purins appear as allantoin, and only the small residual percentage is partitioned as uric acid and purin bases. The author's pupil, W. Hirokawa, 28 ob- tained similar figures when nucleinic acid was fed to dogs ; although the purins did not appear in the urine entirely free from residue. Likewise, too, in the pig 29 after adminis- tration of nucleinic acid the purins for the most part appear in the allantoin fraction. On the basis of these observations, supplemented by numerous older findings bearing upon the conversion of uric acid and nucleinic substances into allan- toin, 30 Wiechowski has been thoroughly corroborated in his statement that allantoin is to be regarded and accepted as an end-product of uric acid metabolism, and that besides allantoin no other products (neither oxalic acid, glycocoll nor urea) of the intermediate uric acid metabolism are met in mammalian animals. Further explanation is not necessary for appreciation of the fact that allantoin, an end-product of the normal vital catabolism of nucleoproteids and nucleinic acids, appears in the catabolism of the same substances in case of intoxication 37 A. Schittenhelm, Zeitschr. f. physiol. Chem., 62, 80, 1909. 28 W. Hirokawa, Biochem. Zeitschr., 26, 441, 1910; under direction of O. v. Fürth. »A. Schittenhelm, Zeitschr. f. physiol. Chem., 66, 53, 1910. 80 Salkowski, Minkowski, Th. Cohn, L. B. Mendel and others. 160 PHYSIOLOGY OF PURIN METABOLISM with protoplasmic poisons (hydrazin, hydroxylamine and semicarbazide) as well as in autolysis, as repeatedly observed by Borissow and by J. Pohl and his collaborators. 31 In what organs the change from uric acid into allantoin takes place is not known ; that it is not limited to the liver is proved by observations on dogs with Eck's fistulas. 32 While parenterally introduced nucleinic acid is appar- ently changed practically completely into allantoin by the mammalian body, this is not by any means always the case with the nucleinic acid introduced by the mouth. In rabbits only half or less of the nitrogen of the bases appears as allantoin in the urine under such circumstances. According to Wiechowski this may be easily explained by the fact that in the alkaline intestinal contents extensive destruction of allantoin takes place, apparently without necessity for inter- vention of bacteria, but certainly with such aid. On the other side of the intestinal wall, however, the allantoin is a stable product. Fate of the Intermediary Uric Acid in Man. — As far as uricolysis in the lower animals is concerned there is a fair unanimity of opinion; the more divergent, however, the opinions as to the destruction of uric acid in human metabolism. For a decade the physiology and pathology of purin metabolism remained unchanged under the domination of the hypothesis that a marked destruction of uric acid takes place in the human body. In conditions in which (as in gout) special accumulation of uric acid was assumed to exist, the fault was always referred to some lowering of the power of the tissues to destroy uric acid. In the course of the last few years these ideas have undergone appreciable change. Absence of Uricase in Human Tissues. — As has been said, uric acid destruction, with formation of allantoin, "Literature: W. Wiechowski, Hofmeister's Beitr., 9, 306, 1907. 82 E. Abderhalden, E. S. London and A. Schittenhelm, Zeitschr. f. physiol. Chem., 61, 413, 1909. ABSENCE OF URICASE IN HUMAN TISSUE 161 takes place with great intensity in the pulp of mam- malian tissues. (It takes place apparently in other verte- brates as well ; it is said that the liver of the ray is from this standpoint more capable than any other vertebrate tissue.) 33 There have been a number of attempts to isolate an actual ferment, "uricase" (as far as it is possible to speak of isolation in case of enzymes). 34 Wiechowski here made the important discovery that living excised human tis- sues are unable to break down uric acid under conditions in which the tissues of lower mammals prove efficient, and that the former therefore must be regarded as not conforming to the usual rule. This has been confirmed from so many sources 35 that there can be no doubt as to its correctness. 36 The objection raised that invariably a preuricase is present but is prevented from manifesting itself because of the an- tagonism of inhibiting substances can scarcely be accepted, if for no other reason than that other ferments which are active against purins, as xanthinoxydase and guanase, are found without any difficulty in the human tissues. "If," says Wiechowski, 37 "äs is amply confirmed by all observers, human tissues build up uric acid with ease from guanin and xanthin but do not catabolize the substance thus formed, in contrast to animal tissues, which oxidize the uric acid further into allantoin, it seems to me the assembling of these 33 V. Scaffidi (Zool. Station, Naples), Biochem. Zeitschr., 18, 506, 1909; 25, 296, 411, 415, 1910. 81 W. Wiechowski and W. Wiener (J. Pohl's Lab., Prague), Hofmeister's Beitr., 9, 247, 1907; F. Battelli and L. Stern (Geneva), Biochem. Zeitschr., 19, 219, 1909; G. Galeotti (Naples), ibid., 30, 374, 1911. 35 Battelli and Stern, Miller and Jones, J. G. Wells and H. J. Corper, cited by Wiechowski, Biochem. Zeitschr., 25, 434, 1910. 30 It would probably be going too far to deny entirely all possibility of a uricolysis in human tissues, as there have been positive findings also in this direction, as those of W. Pfeiffer, Croftan and Schittenhelm. Consult in refer- ence to the physiological importance of the subject: W. Wiechowski, Arch. f. exper. Pathol., 60, 199-200, 1907. " I believe that I am in position to conclude with a fair certainty," says Wiechowski, " that in carefully prepared experi- ment-conditions in which living excised animal tissues oxidize uric acid vigor- ously, human tissues are practically inactive." 27 W. Wiechowski, Biochem. Zeitschr., 25, 436, 1900. 11 162 PHYSIOLOGY OF PURIN METABOLISM findings constitutes a valuable scientific contribution to our knowledge of the fate of uric acid in human beings. ' ' Fate of Experimentally Introduced Purins in Human Metabolism. — Another point of considerable importance is the fate of artificially introduced uric acid in the human metabolism. After feeding nucleinic acid to human be- ings Schittenhelm, Brugsch and Frank have recovered as a rule the greater part of the purin base nitrogen in the urea fraction, a smaller portion in the uric acid fraction and only a very small proportion as nitrogen of the purin bases. 38 Al- lantoin which predominates in metabolism in animals is of little importance here, and is found only in very small amounts in the human urine. However, according to Wie- chowski's results, uric acid introduced subcutaneously is recovered for much the most part as such (up to 90 per cent.) in the urine. There is a difference of opinion as to the importance of this last result, between Wiechowski on the one hand, and Brugsch and Schittenhelm on the other. Schittenhelm be- lieves that because of its toxicity subcutaneous injections of uric acid are not suited for conclusions with reference to normal metabolic processes, and thinks it C3rtain that uric acid is in some proportion further catabolized in human me- tabolism, with production of urea. 39 This view agrees with that of Burian, who assumes that of the uric acid which enters the circulation from without or which is produced in the body by the breaking down of the nucleins, in man al- ways about half is destroyed and only the other half is excreted unchanged; for which reason he multiplies the amount of excreted uric acid by the "integrative factor 2" in order to obtain the total uric acid which entered the cir- culation in the course of a day upon purin-free diet. 40 M R. H. Plimmer, M. Dick and C. C. Lieb, Jour, of Physiol., 89, 98, 1909. »A. Schittenhelm, Handb. d. Biochem., 4', 516, 1910; cf. also L. B. Mendel and J. F. Lyman (Yale Univ.), Jour, of Biol. Chem., 8, 115, 1910. 40 R. Burian, Med. Klinik, 1905, No. 6, and 1906, Nos. 19-21. Consult in these and in the contributions of R. Burian and H. Schur the Literature. PURINS IN HUMAN METABOLISM 163 To this Wiechowski makes adverse reply. He acknowl- edges that the subcutaneous injection of uric acid severely taxes the metabolism. It is not impossible that a toxic ac- tion, which must be taken into consideration, but permitted to proceed, may afford correct conclusions in animal ex- perimentation ; in no case, however, can the metabolic dis- turbance produced explain why the greater part of uric acid which has been parenterally introduced into a human being is excreted unaltered. Schittenhelm 's 41 finding, that experimentally introduced allantoin is not affected in human metabolism, is an impor- tant one. Were uric acid catabolized in man, as in animals, in important degree into allantoin, we might expect that the latter would appear in the urine; but actually very small amounts only of this substance occur normally therein. 42 In harmony with earlier opinions of Otto Löwi 43 and of Soetbeer and Ibrahim, 44 Wiechowski concludes that inter- mediate uric acid is not broken down in man in any really im- portant amount. "For this conclusion the individual con- stancy of endogenous uric acid excretion is a favorable point. For how else can this be explained if, as shown, the reality of a so-called integrative factor of the amount decomposed is not to be assumed. And the established difference between the urine of mammals and of man on purin-free diet (in one case a rich and individually constant daily allantoin elimina- tion with very low uric acid elimination in the other a rich and individually constant excretion of uric acid in the twenty-four hours with mere traces of allantoin excretion) may be looked upon as entirely satisfactory and logical evi- dence of the inferred position of the human being in relation to uric acid." 45 Finally Wiechowski 45a comes to the point 41 A. Schittenhelm and K. Wiener, Zeitschr. f. physiol. Chem., 63, 283, 1909. 42 K. Ascher (Prague), Biochem. Zeitschr., 26, 370, 1910. 48 O. Löwi, Arch. f. exper. Pathol., /,//, 22, 1900. ** F. Soetbeer and J. Ibrahim (Heidelberg), Zeitschr. f. physiol. Chem., 35, 1, 1902. 46 W. Wiechowski, Arch, f . exper. Pathol., 60, 206, 1904. 46 a W. Wiechowski, .Biochem. Zeitsch., 25, 445, 1910. 164 PHYSIOLOGY OF PURIN METABOLISM that all the objections raised by Schittenhelm and others are unsound. ' ' I am compelled to remain of the same opinion as before, believing it to be well founded and not contradicted ; that is, that the changes in intermediate uric acid proceed in man qualitatively precisely as in the other mammals, that is, that it is transformed by oxidation into allantoin ; but quan- titatively they lag behind so much that uric acid must be regarded as the principal product of purin metabolism in man." Wiechowski 's view has very recently received important support from the results of a personal experiment conducted by Lewinthal in Friedrich v. Müller 's Clinic in Munich, 46 in which the investigator injected into himself xanthin (dis- solved in piperidin) and recovered the bulk (89 per cent.) in the urine mainly as uric acid. Siven, in Helsingfors, 47 like- wise concludes from experiments upon himself that the human body is devoid of uricolytic ability and that purins when they have once gained access to the circulation are to be looked upon as end-products of metabolism. In connection with the exceptional position of man as to purin metabolism it is instructive to note that the uricolytic power of the tissues of the macacus rhesus species of monkey is more like that of the tissues of other animals than of human beings. 48 According to "Wiechowski, in the lower monkeys, as in other mammals, allantoin is the principal end- product of purin metabolism. The urine of the chimpanzee, however, corresponds in this particular with human urine. 49 Decomposition of Purins in the Intestine. — The fact that dietary purins, whether free or combined, fail to appear either as uric acid or allantoin in the urine can- 40 W. Lewinthal (F. v. Müllers Clinic, Munich), Zeitschr. f. physiol. Chem., 77, 273-274, 1912. 47 V. O. Siven (Helsingfors), Pflüger's Arch., 1^5, 283, 1912. 48 H. G. Wells (Chicago), Jour, of Biol. Chem., 7, 171, 1909-10. 48 W. Wiechowski, Prager med. Wochenschr., 1912, 275. DECOMPOSITION OF PURINS IN INTESTINE 165 not be harmonized with the assumption that these sub- stances are to be regarded as end-products of intermediary metabolism, except by acknowledging the possibility of an important degree of purin destruction in the bowel. 50 As a matter of fact, we have no ground for doubting such a possibility. It has been shown that uric acid undergoes a spontaneous dissociation even at the low alkalinity of sodium bicarbonate when in a warm temperature, 51 and is even more readily catabolized in alkali solution when acted upon by an oxidizing agent : 52 URIC ACID TETRACARBONIMIDE CARBONYLDIUREA UREA NH-CO NH-CO-NH NH 2 NH 2 NH, CO C-NH CO CO CO CO CO \ || >CO — > I I — »■ I I — > i NH-C-NH NH-CO-NH NH-CO-NH NH,. The oxidation takes place in a strongly ammoniacal solution with separation of imino-allantoin, 53 NH-CH-NH.CO.NH, NH- CO < [-C=NH. It is doubtful, it is true, whether such oxidation-cleavage processes play any part in the usually strongly active reduc- ing intestinal contents. However, bacterial cleavage proc- esses must not be overlooked, an example of which may be seen in the action of the uric acid bacillus isolated from chicken excrement, producing a fermentation of uric acid into urea and carbonic acid as end-products. 54 Reconstruction of Uric Acid. — It is difficult to realize the 50 P. A. Levene and F. Medigreceanu (Rockefeller Instit., New York), Amer. Jour, of Physiol., 27, 438, 1911. 51 W. Wiechowski, Arch. f. exper. Pathol., 60, 200, 1907. "A. S'chittenhelra and K. Wiener, Zeitschr. f. physiol. Chem., 62, 100, 1909; cf. also R. Behrend, Ann. d. Chem., 333, 141, 1904; 365, 21, 1909. M G. Denicke, Ann. d. Chem., 3J t 9, 269, 1906. " C. Ulpiani and M. Cingolini, Gazzetta Chimica Ital., 1908, 34, 377, 1904; cit. in Centralbl. f. Physiol., 19, 166, 1904. 166 PHYSIOLOGY OF PURIN METABOLISM statements of Ascoli and Izar 55 to the effect that uric acid may reform after it has undergone a previous decomposi- tion. These authors observed the disappearance of sodium urate which had been added to hepatic pulp in the incubator when a stream of air was conducted through it. When the material was left in the incubator with air excluded it is said reformation of the uric acid took place. Control ex- periments are said to have shown that the newly formed uric acid did not come from any cleavage of nucleins belonging in the liver pulp, but from a real regeneration of the decom- posed uric acid. From what has been said (vide supra) it would be necessary to assume that in oxidation-destruction of uric acid in liver extracts allantoin would be formed. That this is the case has meanwhile recently been shown by Fasiani, although he was unable to confirm the regeneration of the uric acid. 56 The author prefers to postpone any definite opinion upon the matter until the conclusion of experiments now in course in his laboratory. Methylated Pur in Derivativ es. — Besides the typical purin bases there is a series of methylated purin derivatives pres- ent in small quantities in urine, which originate from the caff ein, theobromin and theophyllin of coffee and tea. Their nature has been fully demonstrated, in the first place by their synthetic production by Emil Fischer and, too, espe- cially by the studies of Kost, Albanese, Bondzynski and Gottlieb, Salomon, Krüger, J. Schmid and Schittenhelm. 57 The schema below printed may serve to indicate their rela- tion (in which, as in previous instances the writer has omitted for brevity hydrogen atoms and double bindings) : »M. Ascoli and G. Izar, Zeitschr. f. physiol. Chem., 58, 529, 1909; G. Izar, ibid., 65, 88, 1910. K Fasiani, Arch. Ital. de Biol., 57, 222, 1912. "Literature: A. Ellinger, Handb. d. Biochem., S', 579-581, 1910; A. Schit- tenhelm, ibid., k', 525-528, 1910. METHYLATED PURIN DERIVATIVES 167 CAFFEIN CHj— NH— CO / I /CH, CO C— N< \ I >co CH,— NH— C— N x PARAXANTHIN CH 3 — N— CO TEEOBROMIN N— CO / I /CH, CO C— N< \ I >co — N— C— W THEOPHYLLIN CH,— N— CO / I CO C— Ns CH, CH 3 — N --i-N> CO C-N-CH, N-C-N/ C0 1-MONOMETHYL- XANTHIN CH,-N— CO CO C— N v \ I >co N— C— W 7 — MONOMETHYLXAN- THIN ( = HETEROXANTHIN) N— CO CO C-N-CH, \ I \co N-C-N/ L/U ^X 3 — MONOMETHYL- XANTHIN N— CO CO C— N v \ I >co. CH S — N— C— N/ It is possible apparently that the demethylation, how- ever, can proceed further and may in the end lead to the pro- duction of uric acid and allantoin, for which reason the prohibition of coffee, tea and chocolate is probably always advisable in prescribing diet for a case of gout 58 ; although the methylxanthins are likely to increase the purins sooner than the uric acid in the urine. The methyl derivatives of xanthin are entirely absent from the urine of an individual who has abstained completely from coffee and tea ; but this is not true of epiguanin I (NH)c' N— CO ,CH C— N<^ i a guanin de- N— c— N x rivative which cannot originate from any of the known purins of coffee and tea. 39 It is possible that we are here dealing with an example of the methylation processes in the body which have been previously considered (v. supra, p. 132). Demethylation of methylated xanthin deriva- M A. Schittenhehn. Therap. Monatsch., 1910, 113; P. Fauval, Compt. Med., 142, 1428, 1906; 1^8, 1541, 1909. 80 M. Krüger, Biochem. Zeitschr., 15, 361, 1909. 168 PHYSIOLOGY OF PURIN METABOLISM tives has been directly observed in experiments with tissue pulp. 00 Quantitative Estimation of Uric Acid. — In concluding the present lecture a few words may be devoted to enumera- tion of recent advances in the analysis of the urinary purins. Besides the time-honored method of E. Ludwig and Salkowski, which, as is well known, depends upon precipitat- ing the uric acid by magnesia mixture and ammoniacal silver solution, the method of Hopkins has met with the most popularity. In the latter the uric acid is thrown down by ammonia and ammonium chloride as ammonium urate. The uric acid, freed from the urate by hydrochloric acid, may be weighed or determined by the Kjeldahl method. Folin and Schaffer have modified this method, by titration of the acid in sulphuric acid solution with permanganate. According to Eonchese the uric acid separated by the Hopkins method may readily be titrated with a solution of iodine in an alkaline medium, employing starch paste as an indicator. 61 Kowarski 62 proposes to free the uric acid from the urate of ammonium precipitated as in the Hopkins method, by means of hydrochloric acid, then to wash with water and alcohol until the reaction becomes neutral, and finally to titrate with n/50 piperidin solution against Phenolphthalein. According to Krüger and Schmid 6:i precipitation of uric acid and the purin bases with copper sulphate and acid sulphite of sodium has proved useful ; after breaking up the precipitate with sodium sulphide the uric acid may be caused to crystallize out by rendering the solution acid with sulphuric acid ; by which means it may be separated from the purin bases. His 's method of uric acid estimation consists in acidifying 60 Brugsch, Pincussohn and Schittenhelm, cited in Handb. d. Biochem., Jf', 528; Y. Kotake, Zeitschr. f. physiol. Chem., 57, 378, 1909; J. Schmid (Breslau), ibid., 61, 155, 1910. " A. Ronchese, C. R. Soc. de Biol., 60, 504, 1906. C2 A. Kowarski, Deutsche med. Wochenschr., 1906, 997. 6a J. Krüger and J. Schmid, Zeitschr. f. physiol. Chem., Ifö, 1, 1905. METHOD OF ESTIMATING ALLANTOIN 169 the urine with hydrochloric acid, inoculating it with a bit of uric acid and subsequently shaking the urine for a long time when the salt-like combined uric acid is separated, presum- ably completely. However, this method furnishes distinctly lower results than the Ludwig-Salkowski and other methods, probably because it leaves complex uric acid combinations intact. It may, however, possibly serve, if more fully elab- orated, to separate the uric acid which is in the blood in loose salt-like form from that in firmer combination. 64 Wiechowski's Method of Estimating Allantoin. — Refer- ence has been made to the very great importance of Wiechowski's method of determining allantoin, and the statement made that the introduction of this method, which had been worked out very precisely, had determined the turn- ing point in the historical development of the study of purin metabolism. The method consists first of removal of all substances from the urine which can be precipitated by means of phosphotungstic acid, acetate of lead and acetate of silver, after which the allantoin is thrown down by mercuric acetate with addition of sodium acetate after care- ful neutralization. In animal urine the nitrogen content of the precipitate may then be directly determined ; the impuri- ties in the precipitate being in such small amount that they are scarcely appreciable analytically in proportion to the relatively large amount of allantoin. However, in case of the very scant quantities of allantoin in human urine, the conditions are quite different. In this case simple nitrogen determination of the precipitate cannot be used, and it is essential to have recourse to purifying processes to enable us to separate and weigh the allantoin in crystalline form. 65 M A. Nicolaier and M. Dohrn, Deutsch. Arch. f. klin. Med., 91, 151, 1907; B. Bloch (Med. Clinic of W. His, Basel), Deutsch. Arch. f. klin. Med., 83, 499, 1905 ; Meisenburg, ibid., 87, 425, 1906. 06 W. Wiechowski, Hofmeisters Beitr., 11, 121-131, 1907; Biochem. Zeitschr., 25, 446-453, 1910. CHAPTER VIII PATHOLOGY OF PURIN METABOLISM Having attempted in the previous lecture to orient our- selves in relation to the present status of the theory of nor- mal purin metabolism, we may at once proceed to picture to ourselves the nature of gout as it is viewed to-day. Quite naturally the author prefers to limit the present discussion to the basic physiological chemical problems, and would therefore refer to the recent monographs on the subject for the various details, particularly those involving the clinical symptoms, the pathological anatomy and questions as to the therapy concerned in this affection. 1 Increase of Uric Acid in the Blood of the Gouty. — The views held as to the nature of this puzzling anomaly of metabolism are so changing that it is by no means easy for the biological chemist to find a fixed point in the flood of phenomena. Perhaps such a point of inception may be rec- ognized in the increase of uric acid in the blood in gouty subjects. It is true that even at the present day there are earnest persons who doubt whether uric acid, the local depositions of which characterize the morphological picture of the disease, is to be looked upon as the actual materia peccans of gout, and whether it does not play merely a col- lateral and secondary part. But, for at least a half century, the fact pointed out by Garrod in 1848 that the blood of gouty individuals is at times richer in uric acid than that of normal persons has maintained its position. G-arrod's primitive experiment (he demonstrated by his "thread test" that threads laid in blood serum to which an acid was added would, from the crystallization of uric acid, after a time be 1 H. Wiener, Ergebn. d. Physiol., 2, 377-432, 1903; O. Minkowski, Die Gicht, Vienna, 1903 (in Nothnagel's Handb. d. spez. Pathol.) ; W. Ebstein, Die Natur u. Behandl. d. Gicht, 2d ed., 1906 ; C. v. Noorden, Handb. d. Pathol, d. Stoff- wechsel, 2d ed., 2, 138-188, 1907 ; F. Umber, Lehr. d. Ernähr, u. d. Stoffwech- selkr., pp. 262-340, 1909; A. Schittenhelm, Handb. d. Biochem., If' 529-535, 1910. 170 FORMATION OF URIC ACID 171 covered over with glistening crystals) has given place to more modern methods. Yet the more recent investigators, as Klemperer, Magnus-Levy and Brugsch, have confirmed his original statement that uric acid is usually increased in the blood of the gouty. This holds good in a remarkable manner, according to Brugsch and Schittenhelm, 2 for the gouty even on purin-f ree diet ; from which therefore it may be inferred that the accumulation of the acid in the blood results from an abnormality in the usual course of the endogenous purins originating in tissue destruction, inde- pendently of the dietary purin exchange. It was, however, early recognized that the increase of uric acid in the blood could not by any means be held fully ex- planatory of gout as an entity of disease, precisely anal- ogous increments being met in other conditions entirely different from gout. After exhibition of food rich in nucleins (as thymus) an alimentary uriccemia has been ob- served; there is a uriccemia of endogenous source in leukaemia, and, too, in pneumonia, at the time when with the exudate cellular constituents are being massively resorbed. In addition, a retention uriccemia is recognized in conditions of functional renal disturbance, as in chronic affections of the kidneys of all types and their sequel, uraemia. 3 It is to be presumed, therefore, that the increase of uric acid in the blood of gouty patients is to be ascribed either to an increased entrance or a diminished escape of the material. Question of Increase in the Formation of Uric Acid. — For a long time the first of these possibilities was the dominating one in discussions of the problem. Many agreed that the fundamental point in the disease is to be looked for in an increase in the destruction of the tissue nucleins. As a matter of fact, however, there was absolutely no basis for ' Th. Brugsch and A. Schittenhelm, Zeitschr. f . exper. Ther., 4, 438, 1907 ; B. Bloch (Med. Clinic, Basel), Zeitschr. f. physiol. Chem., 51, 472, 1907; Th. Brugsch (F. Kraus' Clinic), Berlin klin. Wochenschr., 1912, No. 34. •Literature upon the Various Forms of Uricaemia: A. Schittenhelm, Handb. d. Biochem., k' , 529-540, 1910. 172 PATHOLOGY OF PURIN METABOLISM assuming that the primary factor in the pathology of gout is to be ascribed to cellular destruction. A uniform increase of phosphorus elimination was not to be found in gout, although necessarily this should be coincident with any in- crease in the decomposition of nucleins. It cannot, of course, be denied that in the course of gout, just as in many other affections, there does at times take place an exaggerated de- struction of tissue. This is brought out in the carefully conducted studies of the protein metabolism in gout by Magnus-Levy. We frequently refer to a " toxogenic protein decomposition" in acute gouty exacerbations, and often rec- ognize in the paroxysms of gout that a period of nitrogen retention succeeds this increased protein decomposition. In gouty individuals it may often be observed that periods of nitrogen retention and of nitrogen deficit are likely to occur intermittently without any regularity ; and v. Noorden be- lieves "that in a subject of gout even in the intervals there exist (specific 1 ?) gout-substances, which exert now more, now less harmful influence upon protein decomposition just as in other chronic affections." 4 However, the author is unable to appreciate the least reason for forcing these points into the foreground of the general problem. 5 There is therefore no basis for seeking the cause of gout in an increased formation of uric acid from oxidation pro- cesses ; but there is actually less reason for assuming that it is to be met in an increased synthetic formation of uric acid, for we have seen that while this method plays an im- portant role in birds and reptiles we have no right to assert its existence in the mammalian economy. If the uric acid accumulation in the blood, character- 4 Literature upon Protein Exchange in Gout : K. v. Noorden, v. Noorden's Handb. d. Pathol, d. Stoffwechsels, 2d ed., 2, 139-143, 1907. 5 That, however, an increased cellular destruction, as induced experi- mentally, for example, by exposure to Röntgen rays, is capable of raising the hsemic content of uric acid in a gouty subject and of precipitating a gouty paroxysm, may be inferred from the observations of P. Linsen (Romberg's Clinic) : Therap. d. Gegenw., 1,9, 159, 1908. REDUCTION OF URIC ACID DECOMPOSITION 173 istically met in gout, in conformity with the above statements is not due to an increase in formation, the logical conclusion is that it must be dependent upon some interference or pro- longation in the elimination of uric acid. Question of Reduction of Uric Acid Decomposition in Gout. — The elimination of uric acid from the blood may ? doubtless, be accomplished by its destruction by oxidation. This, as has been seen, occurs physiologically in the experi- ment animals in laboratory investigation upon this point, the uric acid being changed into allantoin. This, however, is not true of man, in whom the latter process of conversion takes place only in a very minor degree. At once, then, a question which has been discussed in the previous lecture recurs. Is uric acid in man to be looked upon, as Wiechowski believes, as an end-product of metabolism; or is it, as Brugsch and Schit- tenhelm and Burian opine, subject in some degree in man to further catabolic change, perhaps to the formation of urea ? In the author's opinion Wiechowski 's arguments are entirely obvious ; and in consonance with this view he would con- clude that the basis of gout cannot be referred to a reduction in the ability of the body to decompose uric acid by oxida- tion, because this process cannot physiologically be an im- portant one in the human body. The author appreciates thoroughly that others have different opinions upon these matters ; but, as was remarked in a previous lecture, every man can observe only with his own eyes and think only with his own brain. Fortunately, every problem in the natural sciences is bound sooner or later to come to a stage where a natural end is fixed for all subjective conceptions and where the actual state of affairs objectively viewed assumes a more or less obvious aspect. If then it is correct, in accordance with the author's belief, that in gout we are not dealing with a reduction of uric acid decomposition, there is obviously only one possible explana- 174 PATHOLOGY OF PURIN METABOLISM tion remaining: that in some way and from some cause the excretion of uric acid is itself at fault. Curve of Uric Acid Excretion in Acute Gouty Exacerba- tions. — In review of the literature of gout two groups of phe- nomena (apparently beyond question of doubt) stand out prominently, which may be held to indicate that faults of excretion play an actually important part in the pathology of gout. These are the characteristic curve of urinary excre- tion of uric acid in acute gouty exacerbations and the fact of the delayed transformation of nucleins in the gouty subject. Primarily, in reference to the first of these features, Friedrich Umber may be cited as a clinical witness. This authority says in his valuable text-book upon nutrition and the diseases of metabolism: "The curve of uric acid excre- tion in the gouty subject on purin-free diet is so character- istic in the periods of exacerbation as to be of distinctly pathognomonic value. The essential endogenous curve of uric acid sinks, as His has pointed out, immediately before the paroxysm to a still lower level (which I may speak of as an anacritical stage of depression), to quickly increase as soon as the paroxysm has begun (el uric acid wave, accord- ing to E. Pfeiffer, who first observed this point), and reaches its acme on the second or third day; after which with the gradual disappearance of the gouty paroxysm it again sinks into a second or postcritical stage of depression succeeding the paroxysm. . . . This course of the endogenous purin curve may, it is true, ... be modified by frequently recurring gouty exacerbations ; but aside from this it is of decided significance as a feature in differential diagnosis. ' ' 6 Delayed Nuclein Exchange in the Gouty. — Along with this fact of the damming back of the uric acid and its break- ing through the dam in some degree in the acute paroxysms • F. Umber, Lehrb. d. Ernährung u. d. Stoffwechselkrankheiten, p. 269, Berlin and Vienna, Urban and Schwarzenberg, 1909. GOUT AND NEPHRITIS 175 of the disease, we may probably regard as one of the most important advances in our appreciation of the patholo- genesis of this anomaly of metabolism the recognition that conversion of nucleins is slow in the course of gout. The fact that the gouty individual after ingestion of purin-containing food shows a delayed excretion of uric acid has been proved by a number of investigations ; 7 he does not, as a normal person, throw off his excess of uric acid in a short time, but ' ' spreads out ' ' the excretion over a number of days. This feature is sufficiently characteristic to have come into employment in diagnosis of gouty affections, the uric acid excretion-curve being kept under observation after addition of a given quantity of nucleinic acid to the food. 8 Gout and Nephritis. — This has suggested the thought that the cause of this uric acid stagnation may reside in the kid- neys; the frequent coincidence of nephritis (particularly contracted kidneys) and gout was emphasized, and the point made that both alcoholism and lead poisoning may play an important part in the etiology of both affections. C. v. Noor- den in his valuable monograph upon gout 9 very properly says that it is not right to do violence to the facts by assum- ing in a case which presents no other evidence of nephritis a "latent nephritis" to explain the gouty uric acid stagnation. When one remembers the fact that usually the kidneys of a case of Bright 's disease perform effectually the uric acid excretion required of them, it is clearly evident that in sup- posing that the nephritis causes the gout there has been a confusion of cause and effect; actually the reverse is some- times the truth. ' Vogt, Reach, Soetbeer, Kaufmann and Mohr, L. Pollak, Brugsch, Hirsch- stein, Lesser, Bloch, Schittenhelm, Rotky and others. For Literature, consult Umber, 1. c, p. 271; A. Schittenhelm, Handb. d. Biochem., 4', pp. 531, 532, 1910. 8 H. v. Hösslin and K. Kato (Med. Clinic, Halle), Deutsche Arch. f. klin. Med., 99, 301, 1910. • C. v. Noorden, Handb. d. Pathol, d. Stoffwechsels, 2d ed., 2, pp. 164, 165, 1907. 176 PATHOLOGY OF PURIN METABOLISM Uric Acid Retention. — Umber has observed that gouty individuals may at times retain all of a given amount of uric acid which has been injected intravenously, at times may excrete it in small fractions ; while a normal individual un- der similar circumstances eliminates it completely. 10 (Simi- lar retention has been met only in individuals suffering from chronic lead poisoning and pronounced alcoholism, condi- tions which if not allied to gout are certainly allied to a disposition toward gout.) A positive statement like this is surely of more force than the negative results of v. Bene- zur, 11 who, after intramuscular injection of uric acid in a gouty case, found that it appeared promptly in the urine. Apparently, too, an individual with renal disease excretes his uric acid better on the whole than does a gouty subject with sound kidneys. 12 Observations like those of Schmoll, Magnus-Levy, Vog-t, Reach 13 and Bloch, 14 who state that after feeding thymus to gouty persons they found far less uric acid in the urine than in case of normal persons, seem to have practically but a single meaning ; and the acute out- bursts of the disease, which were repeatedly brought on by feeding thymus in cases of chronic gout, and undoubtedly are to be thought of as experiments having identical bearing, are especially impressive. Affinity of the Tissues for Uric Acid. — If the kidneys are not to be looked upon as responsible for the scanti- ness of purin excretion in gout, search must be made else- where. The author believes Umber is correct when he states that an increased affinity of the tissues for uric 10 F. Umber and H. Retzlaff (Altona), 27th Congress of Internists, Wies- baden, 1910, p. 436. u G. v. Benczur (See. Med. Clinic, Berlin), Zeitschr. f. exper. Pathol., 7, 339, 1909. 12 Cf. Tollens, Zeitschr. f. physiol. Chem., 53, 164, 1904. 13 F. Reach (F. v. Müllers Clinic, Basel), Münchener med. Wochenschr., 1902, No. 29 ; vide there Literature. "B. Bloch (Med. Clinic, Basel), Zeitschr. f. physiol. Chem., 51, 473, 1907. AFFINITY OF TISSUES FOR URIC ACID 177 acid is the real reason for the diminished urinary excre- tion of purins, for the retention of uric acid in the blood, lymph and tissues (which may lead to gross uratic deposits in the body), and for the gouty exacerbation after a purin- containing diet. "To attempt to refer the entire complex of gout completely to faults in the catabolism of nucleinic acid due to insufficiency in the enzymes concerned," says Umber, 15 ' ' as Schittenhelm and Brugsch recently declared, is not satisfactory, entirely apart from the fact that Wiechow- ski, in the course of his studies as to the possibility of uric acid decomposition in the human body, was never able to detect any uricolysis worth mentioning. If we were to refuse the idea of retention in the tissues, it would be a particularly difficult thing to understand why gouty patients do not sim- ply expel by a compensatory hyperexcretion the uric acid which is accumulated from a supposed failure of uricolysis ; precisely as in leukaemia the patient compensates simply by an exaggerated excretion of the excessive uric acid which is mobilized in the body from the excessive purin decomposi- tion. In the gouty individual there must exist some cause which makes a compensatory uric acid excretion impossible; and that is plainly a retention-affinity of the tissues, because of which the uric acid is actually held in the tissues." The impression grows on one that this hitherto little con- sidered factor, of an increased affinity of the tissues for uric acid in the gouty subject, 16 is very much closer to the real kernel of the gout problem than, for example, the question of the fixation of uric acid in the blood, about which there has been so much contention and with which of necessity we are compelled at least to some little extent to concern ourselves. 15 F. Umber, Lehrbuch der Ernährung und der Stoffwechselkrankheiten, p. 273, 1909. M Cf. F. Umber and K. Retzlaff, 1. c. 12 178 PATHOLOGY OF PURIN METABOLISM In what form does the uric acid, a substance relatively difficult of solution when free, exist when it is dissolved in the blood? Alkali Compounds of Uric Acid. — One would undoubtedly first think of the uric acid as being in some alkali combina- tion for its solution in the circulating blood. What are the known types of alkali-compounds of uric acid I First may be mentioned combinations of the type of sodium monourate (C5H.3N4O3.Na) and of sodium biurate (C 5 H 2 N 4 3 .Na 2 ) ; but it should also be noted that the readily soluble biurate of sodium cannot exist in the presence of the haemic carbonic acid. Besides these the existence of combinations of the type C 5 H 3 N 4 03Na.C 5 H 4 N 4 03 is assumed. These last have been called " quadriurates ' ' by Bence- Jones and Roberts; but the name should for logical reasons be changed to ' 'hemiurates. ' ' The assumption that these (met with in the latericious urinary sediment and in the excrement of birds and snakes) correspond structurally to a fixed molecular proportion (one atom of sodium to two molecules of uric acid) is not confirmed by recent investigations. In all prob- ability the quadriurates are in reality a mixture of primary urate and free uric acid, which is usually fairly constant under uniform external conditions, thus necessarily suggest- ing the formation of mixed-crystals (solid solutions). 17 Part Taken by Changes in Alkalescence. — In the older conceptions of the pathology of gout the hypothesis that separation of the uric acid from the blood into the tissues was due to a lowering of the alkalescence of the blood or of the tissue-juices, was maintained strongly. In spite of the fact that one of the greatest experts on gout, C. v. Noorden, 18 long since characterized all theorizations upon 17 W. E. Ringer (Physiol. Instit., Utrecht.), Zeitschr. f. physiol. Chem., 67, 332, 1910; 75, 13, 1911; R. Kohler (His's Clinic, Berlin), ibid., 70, 360. 1911; 72, 169, 1911; O. Rosenheim, ibid., 71, 272, 1911. 18 C. v. Noorden, Handb. d. Pathol, d. Stoffwechsels., 2d ed., 2, 168, 169, 1907. LACTAM AND LACTIM FORMS OF URATES 179 the interrelations of blood alkalescence, local changes of tissue alkalescence, and gouty deposits as ' ' swinging loosely in the air," it will doubtless take a very long time before physicians will stop making their patients believe that their malady is due to too much acid in the blood, and that nothing but continued drinking of this or that alkaline water (n.b., to be taken at the spring) can possibly eradicate this acid. Un- fortunately there are many instances in the chemical physi- ology of metabolism in relation with which material interests directly interfere with the acceptance of scientific facts. The opportunity should not be passed over here to point out too, how irrational it is to try to draw any sort of conclusions from the analysis of a single specimen of urine (whether from its "degree of acidity" or its "content of uric acid") for application in the diagnosis of gout or in the recognition of improvement or regression of this condition. This is pos- sible only, and then with great caution, from a long series of careful quantitative examinations during purin-free diet, or perhaps in the way that C. v. Noorden tests the limits of tolerance of his patients, by increasing dosage of purin and determining how much purin the subject can handle without manifesting a retention in the daily balance-test. When a physician allows a quantitative analysis to be made of any arbitrarily collected specimen of urine of his patient and then makes a diagnosis of presence or absence of a "gouty diathesis" after a glance at the list of data of the analysis, he is really not proving by his actions his possession of diag- nostic acumen as much as he is laying bare his total ignorance of biochemical matters. Lactam and Lactim Forms of Urates. — Among the fac- tors to be considered in reference to the solubility of uric acid in the blood and its removal therefrom, is the important point, first discovered by Gudzent, that uric acid forms two series of primary salts, and that one of these, a readily soluble, instable urate, has the tendency in its solutions to change over into the second, a less soluble (about one-third). 180 PATHOLOGY OF PURIN METABOLISM stable urate. Following the conceptions originating with Emil Fischer in relation to the tautomeric forms of uric acid, we may suppose that we have here a transformation of instable lactamurate into stable lactimurate : LACTAM FORM LACTIM FORM NH-CO N=C(OH) /I / I co c— nh > c(oh) c-nh \h-c-nh> co % n-c-nh> c (° h )- A transformation of this kind, from the instable to the stable form, seems to take place in the circulating blood. 19 It is supposed that because of the difference in solubility of these two interchangeable forms of monourate, the blood of gouty individuals must at times represent a supersaturated solution of uric acid, which can only gradually resume its equilibrium by removal of urates by crystallization. That gouty blood is not comparable, however, at least not in all conditions, to a supersaturated solution of uric acid is evi- dent from Klemperer's results, showing that the blood of a gouty subject is capable of dissolving considerable amounts more of uric acid. Apparently it were best to hold that the subject is not at present proved to be a matter of pathological importance. Nucleinic Acid Combination With Uric Acid. — From the observation that uric acid can no longer be precipi- tated either by acetic acid or by alkaline ammonio-silver- magnesia mixture from a mixed solution of uric acid and nucleinic acid, Minkowski has held that it is probable "that uric acid primarily exists in the blood and the tissue juices in combination with nucleinic acid, and that not only the conversion of the purin bases into uric acid, but also the solubility and transportation as well as the further changes of the uric acid in the living body is regulated by 18 G. Gudzent (His's Med. Clinic, Berlin), Zeitschr. f. physiol. Chem., 60, 38, 1909; 63, 455, 1909; Centralbl. f. Stoffw., 5, 289, 1910. CONDITIONS OF SOLUBILITY OF URIC ACID 181 this linking with a nucleinic acid rest. " 20 It has been sup- posed, too, that uric acid by being linked with nucleinic acid is protected to a certain degree from oxidation in the body. 21 It may be remarked with direct reference to this last state- ment, that, if Wiechowski's results are to be accepted, there is every reason to doubt whether uric acid is open to oxida- tion in the human economy ; it would therefore have no need of protection by the nucleinic acid to insure its escape from combustion. Then, too, aside from the traces found in the urine, we have not the least basis for supposing that nucleinic acid is actually present in the circulating blood. 22 Nor is the prevention of precipitation shown by uric acid in the pres- ence of nucleinic acid to be looked upon as at all remarkable ; it is not necessarily indicative of a true acid combination with nucleinic acid. 23 Such inhibition of precipitation is rather to be referred to the general group of variations of solubility which are manifested by crystalloid substances in the presence of all sorts of colloids Complex Conditions of Solubility of Uric Acid in Rela- tion to the Uric Acid Diathesis. — Complex phenomena of solubility of this type are to be seriously considered in con- nection with the uric acid of the circulating blood. The nucleinic acid is not alone important ; the general mass of blood proteins are particularly to be thought of. It has been stated that uric acid is much more soluble in serum than in pure water. 24 In the urine, again, the solubility of the uric acid is largely influenced by the presence of urea and diso- dium phosphate (Na 2 H.P0 4 ), and relation of this to monoso- sodium phosphate (NaH 2 P0 4 ). 25 There is no doubt of the importance of such interrelations, too, in the formation of 20 O. Minkowski, Die Gicht, in Nothnagel 's Handb. d. spez. Pathol., pp. 189, 190, 1903. 21 Y. Seo (Minkowski's Clinic, Greifswald), Arch. f. exper. Pathol., 58, 75, 1908. a Th. Brugsch, Zeitschr. f. exper. Pathol., 6, 278, 1909. 23 A. Schittenhelm, Zeitschr. f. exper. Pathol., 7, 110, 1910. 24 A. E. Taylor, Jour, of Biol. Chem., 1, 177, 1905. 25 Investigations of Pfeiffer, Rudel, Ritter, Strauss and others. 182 PATHOLOGY OF PURIN METABOLISM renal and cystic calculi, the "uratic diathesis," as well as in the formation of uric acid deposits in the tissues. (It may be casually added that Virchow, however, long ago expressed doubt of the existence of any close relation between gout and the genesis of urinary concretions ; and at the present time there are metabolic pathologists of wide experience, like C. v. Noorden, G. Klemperer and F. Umber, who believe that we are not justified in looking upon nephrolithiasis as the result of uric acid accumulation in the blood or in the tissues, and that it would be better to drop entirely the popular, very comprehensive and very much misused term of "the uric acid diathesis.") 2Ü Although the importance of these com- plex conditions of solubility, as they prevail among colloid and crystalloid substances in the animal juices, may be ac- cepted in relation to the formation of uric acid sediments and concretions, there is no real reason for seeking the explana- tion of gout in this sphere. We may see one sign of progress in the fact that at the present we have no appreciation of a sharp distinction between "loosely combined" and "fixed" uric acid (Pfeiffer for one tried to bring out such a differ- entiation by means of niters charged with uric acid, on which gouty urines gave up a part of their uric acid). In our modern conceptions of physico-chemical solution-interrela- tions there is no longer room for such schematic representa- tions and for different kinds of uric acid fixation. That we know but incompletely the individual physico-chemical factors which determine the "fixedness" of uric acid combination, is quite another matter. Localization of the Uric Acid Depositions. — The rea- son for localization of the uric acid deposits in certain positions of predilection as cartilages, joint capsules, ten- dons, muscles and skin is directly related with the ques- tion of the complex conditions of solubility of uric acid. w Literature upon Uratic Diathesis : F. Umber, Lehrbu. d. Ernährung und Stoffwechselkrankheiten, pp. 350-356, 1909; cf. on the contrary Neubauer, Internat. Kongr., Wiesbaden, 1911. LOCALIZATION OF URIC ACID DEPOSITIONS 183 Probably special importance for the interpretation of the general problem may be attributed to a discovery made in Hofmeister 's laboratory 27 that when thin sections of car- tilage are left for some hours in a solution of sodium urate they will take up uric acid. The degree of concentration of the urate solution is diminished ; and at the same time when the sections of cartilage are directly inspected they fre- quently show white foci and diffuse opacities of uric acid deposits. The great affinity of normal cartilage for uric acid is manifested by the fact that when large amounts are introduced into the peritoneal cavity in rabbits, the acid may often be detected by the murexide reaction in the car- tilages although not apparent in other tissues. The ac- cumulation of uric acid in cartilage in states of increase of uric acid in the blood may be explained in the same way. The well-known theory of Ebstein that the dissolved uric acid infiltrating the tissues acts primarily to produce an inflammation and that an antecedent necrosis precedes the deposition of the uric acid is no longer to be accepted in the light of the above outlined discovery. That an excess of purins in the body fluids may produce inflammatory changes is not to be denied ; confirmation may be seen in a number of observations, as that of Levinthal, 28 who, in a personal ex- periment, injected half a gram of xanthin (dissolved in piperazin) into his cubital vein, and a few days later, after a moderate strain upon the limbs from dancing, he was sud- denly seized with a fairly acute painful attack in one of his knees, attended with some swelling and local heat. Deposi- tions may form in the tissues, however, as shown by nu- merous observations upon gradually developing tophi ex- hibiting no inflammatory reactions, entirely independently of any antecedent necrosis. 27 M. Almagia (Instit. of Physiol. Chem., S'trassburg) , H'ofmeister's Beitr., 7, 4G6, 1906. 23 W. Levinthal (F. v. Mailer's Clinic, Berlin), Zeitschr. f. physiol. Chem* 77, 273, 1912. 184 PATHOLOGY OF PURIN METABOLISM Alkalinity of the Tissues in Relation to Uric Acid De- posit. — It is difficult to decide at present to what extent physical f actors, in contrast to chemical factors, are involved in increasing the absorbing power of individual types of tis- sue for uric acid. We are unquestionably here again dealing with a very complex problem of physical chemistry. It has been stated, for example, that aminoacids tend to. inhibit the absorption of uric acid by cartilage. 29 But, on the other hand, monourates are separated from uric acid solutions, and in the same way urates deposit in the tissues the more readily, apparently, the higher the proportion of sodium present. Van Loghem 30 has from this suggested that the predilection of cartilage and connective tissue for uric acid bears some relation to the large amounts of sodium contained by these tissues; and he suggested that the formation of tophi might be restricted by the use of hydrochloric acid or increased by alkalies. It is the same old story! At any rate one can derive this one point from it: The constant effort to favorably influence gout by flooding the body with " curative" alkaline waters is without the least theoretical foundation. ' ' That by the use of certain spring waters gout can be therapeutically influenced," says Umber, 31 "is at- tested by a century of practice. But it is practically proven that the essential reason for this fact does not exist in any immediate influence of the inorganic constituents of these waters upon the disturbances of purin-metabolism in gout. The importance of the thorough flushing of the body, in which even Garrod believed the pith of mineral water treatment lies, the regulated life of the patients, their isolation, and, not the least, the intelligent interest of specially trained 28 Th. Brugsch and J. Citron (F. Kraus's Clinic, Berlin), Zeitschr. f. exper. Pathol., 5, 401, 1908. 80 Van Loghem (Amsterdam), Deutsche Arch. f. klin. Med., 85, 416, 1906; Centralbl. f. Stoffw., 1907, 244; consult also Roberts, His and Paul, Gudzent, E. d'Agostino: Rendic. della Societä Chim. ItaL, 2, fasc. 6, 1910. 31 F. Umber, Lehrbu. d. Ernährung und Stoffwechselkrankheiten, p. 329, 1908. ATTEMPTS TO PRODUCE GOUT EXPERIMENTALLY 185 physicians in their little and their great complaints, — all this in the methods of a health resort for the gouty is a more important therapeutic factor than the minerals that are contained in the waters." Attempts to Produce Gout Experimentally. — Research upon the nature of gout would surely be in position to show more rapid progress, were it not that experimental attempts to produce this fault of metabolism have thus far always proved failures. The part taken by uric acid in avian metabolism (in birds the bulk of nitrogen ex- creted appears as uric acid, and in them the latter is without doubt formed synthetically) is so fundamentally different from its role in the economy of man and the mammalia, that it is decidedly difficult to recognize much of importance to the study of gout in the uric acid deposits obtained in the viscera of birds after ligation of the ureters (by Ebstein) or after exclusive meat diet (by Kionka). His has been experi- mentally able to produce tophi by local injections of rela- tively insoluble sodium monourate in dogs with coincident administration of alcohol. These coincide in detail with spontaneous gouty nodules, and confirm the view {vide sup.) that uric acid deposits may act as local irritants and cause the surrounding tissue to become the seat of an inflammatory infiltration, and produce a capsule from the granulation tis- sue thus occasioned. Spontaneous formation of gouty nodules (as by flooding the circulation with urates) has up to the present time not been successfully induced; and this is the real object to be attained. Among the different factors which favor the development of gout, as is well known, alcoholism, chronic plumbism, and long continued and excessive ingestion of foods rich in purin bodies are the most prominent. Alcoholism. — The belief in a close causal relation between chronic alcoholism and gout has been fully confirmed by thou- sands of examples, from the days when the lusty Herr von 186 PATHOLOGY OF PURIN METABOLISM Rodenstein after liquidation of his villages "had the gout in his neck from too many malmseys," down to the present time. We have no knowledge of how alcoholism disturbs the purin metabolism. According to Beebe 32 it is the exogenous fraction of the purins, according to London 33 the endogenous as well, which bears the brunt. The latter believes that de- struction of cellular nucleins is heightened by the toxic effect of the alcohol. L. Pollak, 34 in metabolism experiments in the clinic of Friedrich v. Müller, observed a retention and retarded elimination of uric acid in alcoholics, such as we customarily regard as characteristic of the uric acid meta- bolism of the gouty ; which serves to indicate the close rela- tionship existing between alcoholism and gout. Why the excretion is retarded, however, is as much a puzzle in the one case as in the other. If, as we have done before, we speak of an "increased retention effort of the tissues," we are practically throwing aside the humoral gout theories. There is, however, no occasion to pretend either to ourselves or to others that this is the same thing as a real explanation. Chronic Plumbism. — In cases of chronic lead poisoning Schittenhelm and Brugsch 35 found the endogenous uric acid reduced markedly ; while Preti 36 found the absolute quantity of excreted purin-base nitrogen above normal in three cases of chronic plumbism. In rabbits in Julius Pohl 's laboratory 37 with chronic lead poisoning there was invariably met an in- crease in the nitrogen fraction representing the purin group, this often advancing in a striking manner on increasing the intoxication. However, the uric acid manifested no such 82 S. P. Beebe (Yale Univ., New Haven), Amer. Jour, of Physiol., 12, 13, 1904. 33 A. Landau, Deutsch. Arch. f. klin. Med., 95, 280, 1909 ; cf. also Jahresber. f. Tierchem., 38, 639, 1908. 34 L. Pollak (F. v. Müller's Clinic, Munich), Deutsch. Arch. f. klin. Med., 88, 224, 1906. 35 A. Schittenhelm and Th. Brugsch, Zeitschr. f. exper. Pathol., k, 494, 1907. 36 Preti, Deutsch. Arch. f. klin. Med., 95, 411, 1909. "Rambousek (J. Pohl's Lab., Prague), Zeitschr. f. exper. Pathol., 7, 1910, S.A. EFFECT OF MEAT DIET 187 relative proportions. Here, too, therefore, the subject is open to further study. Influence of Excessive Meat Diet. — Along with alcohol- ism and chronic lead poisoning, excessive ingestion of foods rich in purin bodies, particularly meat, plays an important part in the etiology of gout. Some evidence of this is to be observed in the geographical distribution of this fault of metabolism. In Japan, China, Arabia and the tropics, where meat is but little used, the affection is apparently rare. It is not common in southern Europe ; in middle Europe it occurs more frequently, but in the countries lying about the North Sea and the Baltic it is very frequently met, in Eng- land and Holland being almost endemic. Perhaps the pref- erence which Austrians show for boiled beef, which is gener- ally disliked by the northerners, explains why gout is not very common in Austria, as the purin-rich extractives remain within the meat when it is roasted, but are removed through boiling. For this reason well-boiled meat, provided the broth is not included, presumably, may be considered as food poor in purins. Protracted Feeding of Nucleinic Acid to Dogs. — With view of determining the effect of long-continued inundation of the mammalian body with purin, the author's pupil, Hirokawa 38 has carefully studied under direction the purin metabolism of a dog fed very regularly with additions of nu- cleinic acid for months. A small dog regularly fed on mixed food was given daily five grams of sodium nucleinate for three months without manifesting any strikingly harmful result and without any loss of weight. However, a marked change in the purin exchange was noted. Whereas in the first part of the experiment out of the total purin- and allan- toin-nitrogen the results showed 98.5 per cent, as allantoin and only 1.5 per cent, as purin bases and uric acid together, "W. Hirokawa. Biochem. Zeitschr., 26, 441, 1910; under the direction of 0. v. Fürth. 188 PATHOLOGY OF PURIN METABOLISM the uric acid proportion gradually increased until after ten weeks about ten times as much was being excreted as in the first week on nucleinic acid (13 per cent, of the combined purin nitrogen and allantoin nitrogen) . Coincident increase of the purin bases was not appreciable. The results show that the metabolism of the animal was influenced by long continued flooding with the products of nucleinic acid cleav- age in such a way that the ability of the dog's economy to oxidize uric acid almost completely into allantoin was appar- ently impaired, and a larger fraction of the former appeared unchanged in excretion. However, there was no change in the transformation of the purin bases into uric acid, only a a minimal amount of the former appearing unchanged first and last. Radium Therapy of Gout. — Among the newer attempted methods of cure of gout the use of radium for the moment assumes the greatest interest; and it is undesirable to pass it over without at least brief consideration. The basis for its employment was developed in certain experiments of Gudzent, 39 in the Clinic of W. His, upon the effect of radium emanations upon the solubility of monosodium urate. As previously mentioned Gudzent assumed that the monourate of sodium in the blood may exist in two forms capable of intertransformation by intramolecular rearrangements (tautomeric forms), the lactam- and the lactim-forms, the former of which, instable and the more soluble of the two, tends to change into the relatively more insoluble and stable lactim. It is claimed that radium rays are not only capable of inhibiting this conversion but of causing the relatively more insoluble salt to revert to the more soluble form, 40 and of catabolizing the uric acid in turn 38 F. Gudzent (His's Clinic, Berlin ), Med. Klin., 1909, No. 37, p. 1381; Deutsch, med. Wochenschr., 1909, 921; 27th Intern. Kongress, Wiesbaden, 1910, p. 539; Med. Klin., 1910, No. 42; Therapie d. Gegenw., 1910, 529; Zeitschr. f. ärztliche Fortbildung, 8, 198, 1911. *°Cf. H. Bechhold and J. Ziegler, Berliner klin. Wochenschr., 1910, 712; Biochem. Zeitschr., 20, 189, 1909. RADIUM THERAPY OF GOUT 189 into more soluble substances, finally into carbonic acid and ammonia. It is said this solvent influence of the radium rays is effective in the living body and that the gouty individual is benefited by this mode of treatment, which it is claimed will destroy existing uric acid deposits in the tissues and reduce the uric acid accumulation in the blood. Yv r . His 41 regards the beneficial influence of radium upon the manifestations of gout as proved, commending its use especially by inhalation of the rays, but also advising that radiumized water be drunk, radium baths be employed and radium salts injected in the immediate neighborhood of the affected parts. His statements have had many confirmations, but have also called forth contradiction in as many instances. Incidentally, at the last Congress of Internists (Wiesbaden, 1912), the op- posing opinions were brought out in sharp contrast. Ac- ceptance of a favorable influence of radium treatment upon the symptoms of the malady aside, the procedure can with difficulty be interpreted either in the sense of increasing the solubility of the monourate of sodium or of decomposing the uric acid. From exhaustive experiments conducted by E. v. Knafn-Lenz and W. Wiechowski in the Vienna Pharma- cological Institute, 42 no effect at all from even large amounts of the rays were recognized, either in destruction or in increasing the solubility of monosodium urate. As uric acid is readily oxidized by ozone, it might perhaps be supposed that some such process is involved ; but it has been demon- strated that the quantity of ozone produced in the air by the radium preparations is incapable of producing any ap- preciable decomposition of uric acid. It is not clearly stated under what precise conditions Gudzent's contrary results were obtained. The above-named experimenters state that when an impure alkaline preparation of radium was em- 41 W. His, Internisten Kongress, 1910 u. 1912; Berliner klin. Wochenschr., 1911, 197. U E. v. Knaffl-Lenz and W. Wiechowski (H. H. Meyer's Lab., Vienna), Zeitschr. f. physiol. Chem., 77, 303, 1912. 190 PATHOLOGY OF PURIN METABOLISM ployed decomposition readily took place and suggest that the alkali given off from the glass container may also be of some import. "If there is no direct influence of the rays upon monourate of sodium," say v. Knaffl-Lenz and Wie- chowski, ' ' there may possibly be an activation of a uric acid oxidase existing in the human tissues in mere traces to explain the favorable effect of the emanation upon gout. . . . Such an influence of the rays, however, seems im- probable to us, because in almost all experiments in human metabolism the amount of uric acid excreted has been found increased. 43 In trying to explain the curative influence of radium rays in gout there is still left the supposition that the elimination of uric acid through the kidneys is made easier by the effect of the rays. Whether this is to be thought of as a direct action on the process of secretion of the uric acid, or whether it is an indirect one, as His and his pupils hold, facilitating the excretion of the uric acid by inhibiting inflammation of the organs, is a matter in which further experimentation in appropriate lines is to be desired." Since, according to Knaffl-Lenz 's observations on the effect of large amounts of radium emanations, there may not only be an influence exerted upon the respiratory apparatus but upon the central nervous system as well, caution is always requisite in the radium treatment of gout. 44 Eecent investigations in Neuberg's laboratory have given results in accord with those of Knaffl-Lenz and Wiechowski, showing that radium rays have no influence upon the solubil- ity of sodium monourate or upon its decomposition into C0 2 and ammonia. 45 43 H. Mandel (Radium in Biol., 1, 163, 1911, cited in Centralbl. f. d. ges. Biol., 12, No. 2879) found the excretion curve of uric acid absolutely unin- fluenced in cases of gout which manifested distinct improvement from the effect of radium rays. 44 E. v. Knaffl-Lenz (H. H. Meyer's Lab.), Wiener klin. Wochenschr., 25, No 12. 45 P. Lazarus, 29th Kongr. f. innere Med., Wiesbaden, Apr. 17, 1912; J. Kerb and P. Lazarus (Chem. Dept., Physiol. Instit. of the Berlin Agric. High School), Biochem. Zeitschr., ^2, 82, 1912. MEDICINAL TREATMENT OF GOUT 191 Medicinal Treatment of Gout. — So far as the medicinal treatment of gout 46 is concerned, one must confess frankly that there is little satisfactory to be said. Colchicin, the immemorially lauded poison of meadow-saffron, still has its adherents, but no one has ever been able to explain its mode of action. It is said that the much used salicylic acid and its many related substances increase uric acid elimination ; but whether its beneficial (frequently questioned) influence on gout is due to this or simply to some "antirheumatic" effect, is unknown. Quinic acid (hexahydrotetraoxybenzoic acid) with its numerous derivatives may, perhaps, be named in the same class ; but the hypotheses upon which its thera- peutic use was based were unfounded. (It was supposed that quinic acid, which is transformed in the body into benzoic acid and undergoes a hippuric acid synthesis, pre- vents glycocoll from entering into synthetic production of uric acid ; but it is now known that synthetic formation of uric acid from glycocoll does not occur in mammalia.) It is hard to say what there may be in the idea that the quinolincarboxylic acids increase uric acid elimination 47 ; the very favorable effects attributed to phenylquinolincar- boxylic acid (atophan) have recently been given consider- able prominence. 48 Efforts to increase the solubility of uric acid in the blood by administration of piperazin, substances producing formaldehyde by cleavage like hexamethylentet- ramine (urotropin), nucleinic acids, urea, alkalies and alka- line waters, have as little justification in theory as in practice. A recent conclusion goes so far (vide supra) as to declare that alkalies are not only useless in gout, but are actually *° Literature upon the Therapy of Gout: 0. Minkowski, Die Gicht, pp. 299- 322, Vienna, 1903. 47 A. Nicolaier and M. Dohrn, Deutsch. Arch. f. klin. Med., 93, 331, 1908. 48 W. Weintraud, Therapie d. Gegenwart, 1911, 97; R. Feulgen, Inaug. Dissert., Kiel, 1912, Centralbl. f. d. ges. Biol., 1912, No. 2840; B. Bauch (Weintraud's Clinic, Wiesbaden), Arch. f. Verdauungskr., 11, Ergänz. Heft, 186, 1911; E. Frank, in collaboration with Przedborski (Minkowski's Clinic, Breslau), Arch. f. exper. Pathol., 68, 349. 192 PATHOLOGY OF PURIN METABOLISM harmful, and that it is possible to obtain beneficial results from long continued use of hydrochloric acid, which is sup- posed to alter the absorption capacity of the blood and tissues for the uric acid. 49 Altogether, F. Umber 50 comes to the rather unedifying conclusion "that all the medicinal methods introduced with view of increasing uric acid elimi- nation, of determining solution of the uratic deposits or of limiting the formation of uric acid, are entirely worthless. To the present time we have not known of any means of influencing the gouty metabolism, and the good results which we are getting as our knowledge advances are clearly due to the matter of diet." Whether radium or atophan will make any difference in the force of such a statement can only be awaited with patience. The Diet in Gout. — The present view of the general sub- ject is somewhat as follows : The true nature of gout is still unknown ; but we at least know that the affection is in some way due to an accumulation of uric acid in the blood and that increase of this accumulation is an unfavorable feature. Therefore, in the dietetic management one may proceed on the idea that the ingestion of substances which form uric acid are to be limited as far as possible. Here belong particu- larly the extractives of meat, as well as those tissues which are highly nucleated and therefore contain considerable nucleinic acid, like thymus, spleen, liver, lungs and kidneys. These latter may be forbidden with propriety, as sufficient instances are known where a gouty patient has suffered an acute paroxysm directly traceable to a meal of thymus (bries). Meat should be eaten only when well boiled, not roasted. Meat broths and "whole" soups, as well as all use of meat extract, are to be prohibited. In the proscription list coffee and tea should also be placed because of the methylpurins they contain, together with alcohol in all 49 Falkenstein, Berl. klin. Wochenschr., 1906, 228; J. J. Schmidt, Mün- chener med. Wochenschr., 1911, No. 83, 1764; consult also van Loghem, 1. c. M F. Umber, Lehrbu. d. Ernährung und der Stoffwechselkr., p. 333, 1909. THE DIET IN GOUT 193 forms. While the harmful effects of the latter upon gout are not understood in a theoretical way, that they are real is proved from a practical standpoint. Finally, thorough flushing of the body with water seems a rational measure. These provisions form the theoretical fundamentals of diet in gout, as the author understands them. For the rest he would refer to the methods of practicing physicians as they appear in numerous monographs. It would be impolitic and essentially wrong to simply ignore whatever judicious objec- tive observers have found appropriate after decades of study merely because no theoretical explanation therefor has been found. It should never be forgotten that the observations of the practitioner may be true and the theories may be false, and that a judicious natural scientist generally values the former more than he does the latter. But unfortunately objective observation, especially in the treatment of chronic internal affections, is endless and difficult, and for that very reason this has been and will be at all times and among all people the favorite field for both scientific and unscientific charlatanry. 13 CHAPTER IX DIGESTION OF CARBOHYDRATES— BLOOD SUGAR— DIASTASIC FERMENTS CARBOHYDRATE DIGESTION Turning away for a time from the processes of meta- bolism which concern the proteins and nucleins, our path leads into a new field stretching out interminably before us — the field of carbohydrate metabolism. Even though the road be long, there comes a feeling of relief as we plod the way, a feeling not unlike that the mountain climber ex- periences as he reaches the tree-line on a heated day after toiling painfully up through the high forest. Let the trail before him be grievous as it will, he trudges blithely along, with freer vision, no longer shut in on every side by the dusk of the thickset woods. It really is a dusk which surrounds us in the domain of protein metabolism. How could it be otherwise'? The chemical nature of the proteins is so un- known, that in tracing their destinies in the depth of the liv- ing body there can be expected no wealth of light. In taking up the carbohydrates we at least are dealing with a chem- ically well-defined material. We may at once undertake to follow the carbohydrates in their transit through the body, beginning as in case of the proteins with their fate in the digestive tract. Ptyalin. — As is well known, the food in man and many of the animals is at once mixed in the mouth with a diastasic (carbohydrate splitting) secretion, the saliva. Because of the fact that the hydrochloric acid of the stomach inhibits the glycogenic action of ptyalin even when in relatively low concentration and destroys the ferment entirely when in higher concentration the opinion of many has been and is now that ptyalin action is of comparatively little importance and is quickly terminated when the food arrives in the stom- ach. As a matter of fact, however, this cannot be the case. 194 CARBOHYDRATE DIGESTION IN THE INTESTINE 195 It should be kept in mind that when any considerable amount of food is ingested only that first swallowed comes into direct contact with the gastric mucous membrane. The acidity need by no means prevail in the interior of the food mass, and the diastasic action of the saliva incorporated in the mass may continue for some time in the performance of its work. 1 Carbohydrate Digestion in the Stomach. — There can be no doubt of the fact that there is a carbohydrate cleavage beginning in the stomach itself, particularly as hydrochloric acid alone, without the aid of enzymes, especially at the in- cubator temperature of the stomach of the warm-blooded animal, is effective in this sense. According to the investi- gations of the Ellenberger school 2 and others it may be accepted that many mammals, as the hog, produce a special gastric diastase. And in the dog, the canine saliva being devoid of diastase, it is said the stomach is capable of form- ing a diastasic ferment, one which is active even in strongly acid reaction. Others, it is true, have reached the conclusion that the carbohydrates generally undergo no appreciable alterations in the stomach of the dog, and that a slight degree of cleavage met there may be explained by the influence of the hydrochloric acid of the gastric juice, so that to them the assumption of an amylolytic or inverting ferment seems unnecessary. 3 Carbohydrate Digestion in the Intestine. — However, it may be said that both the cleavage and the resorption of the carbohydrates for the most part reach their height primarily in the intestine, where they are subjected princi- pally to the powerful influence of the pancreatic diastase, but also to other enzymes as well (invertin, maltase, lactase). Starch unquestionably undergoes a series of catabolic 1 O. Cohnheim, Physiol, d. Verd. u. Ernährung, p. 142, 1908. 2 F. Bengla and G. Haane (Ellenberger Lab.), Pflüger's Arch., 106, 267, 286, 1904; consult there and also O. Cohnheim (1. c.) for Literature. 3 London and his associates, Zeitschr. f . physiol. Chemie., 56, 1908. 196 DIGESTION OF CARBOHYDRATES stages in the intestine. Whether this is the proper time to replace the old, much-discussed schema Starch >Erythro-dextrine >► Achroodextrine >Maltose ^Glucose with a more modern one need not be further considered here. Role of the Pancreas in the Production of Carbohydrate- splitting Ferments. — As the lion's share of the normal cleav- age of carbohydrates in the intestines is referable, as above said, to the pancreatic diastase, it is inconceivable without further knowledge how it happens that in the dog, even after exclusion of the pancreatic secretion 4 by ligation of the pan- creatic ducts, as in the experiments of Rosenberg, nine-tenths of the starchy materials may be resorbed. It is fundament- ally all the more remarkable because in the dog we are prac- tically unable to recognize any diastasic influence in the saliva, the bile and the intestinal juice, at least in normal con- ditions. If the pancreas be extirpated the resorption of carbohydrate is apparently very much disturbed (although Minkowski and Abelmann even in this state found their dogs capable of resorbing more than a half of the amylaceous ma- terial fed) . Whether we are in fact constrained to ascribe to the pancreas, in addition to its known function of internal secretion, another specialized puzzling role in resorption, as Lombroso belives, 5 is to the author's way of thinking rather doubtful. It should be kept in mind that total excision of the pancreas is a very severe interference, which "mixes up," so to say, the whole economy. Why should it be expected to leave the carbohydrate resorption completely without dis- turbance f Pawlow has insisted, it may be remembered, upon his doctrine of the adaptation of the digestive fluids to the par- ticular quality of food ingested according to the require- ments. Weinland 6 and others 7 have concluded, in special * Cf . inclusive Literature : J. Munk, Ergebn. d. Physiol., 1, 308, 1902. W. Lombroso (Turin), H'ofmeister's Beitr., 8, 51, 1906. FATE OF THE DISACCHARIDES 197 reference to the milk-sugar splitting function of the pan- creas, that it is greatly increased or primarily induced by milk diet in dogs and newly born human beings ; but other investigations failed to adduce any confirmation of such statements. 8 In view of the fact (to be discussed later) that the pan- creatic fat-splitting ferment is very materially increased in its effectiveness by the access of bile, it is not without inter- est that the bile also favorably influences the digestion of starch, probably because the bile salts reduce the surface tension of the starch paste. 9 Fate of the Disaccharides. — In the resorption processes in the intestine it is particularly important to note that the intestinal wall is strikingly less permeable not only for the high-molecular colloids, but also for the disaccharides than for the monosaccharides. It is correct to say that the bowel- wall apparently allows only those sugars to pass readily which can easily be used by the tissue cells. 10 The cells are not adapted to deal with the majority of disaccharides, as may be recognized in the fact that if cane-sugar or lactose be introduced parenterally, that is, subcutaneously or intraven- ously, they are simply excreted without change. Although this is not true for maltose there is a special reason, in that the blood contains a ferment, "maltase," which is capable of splitting the parenterally introduced sugar, after it has entered the circulation, into glucose. But when we consider the fact that a man can take up from the intestine large amounts of cane sugar (three hundred grams and more) without any appearing in the urine, it is obvious that double 6 E. Weinland (Munich), Zeitschr. f. Biol., 38, 607, 1899; 40, 386, 1900. 7 F. A. Bainbridge (Univ. College, London), Jour, of Physiol., 31, 98, 1905; P. Sioto (Fano's Lab.), Arch d. Fisiol., 4, 116, 1907; O. Martinelli (Bologna), Centralbl. f. Stoffwechselkr., 8, 481, 1907. *R. Aders Plimmer, Jour, of Physiol., 34, 93, 1906; 35, 20, 1906-07; J. Ibrahim and L. Kaumheimer, Zeitschr. f. physiol. Chem., 62, 1909. 'G. Buglia (Bottazzi's Lab., Naples), Biochem. Zeitschr., 25, 239, 1910. 10 Cf. E. H. Starling, Handb. d. Biochem., 3", 241, 242, 1909. 198 DIGESTION OF CARBOHYDRATES sugars generally and certainly the high-molecular carbohy- drates undergo complete cleavage before they gain access to the blood-stream. 11 This rule is not invalidated by the fact that after a diet rich in carbohydrates, as pointed out by v. Mering, Otto and others, dextrin-like carbohydrates may gain access in the portal blood, 12 and that they are recognizable in small amount in urine normally, but to a much greater extent in diabetes. 13 Dilution of a Sugar Solution in the Intestine. — According to the investigations of London carbohydrate absorption in the stomach is of little importance. If a concentrated sugar solution be introduced into the small intestine sugar is absorbed, and at the same time water is given off into the intestinal lumen until the degree of sugar concentration in the latter is reduced to about 6 or 8 per cent., after which at this dilution the resorption proceeds rapidly. 14 From researches published from Röhmann's laboratory, the re- sorption of glucose from the intestine reaches its relative maximum at a concentration corresponding with the osmotic pressure of the blood serum. 15 The author would next pass to the consideration of one of the problems relating to the fate of carbohydrates in the di- gestive tract, which is at present of special interest to him, the problem of digestion of cellulose. Disappearance of Coarse Vegetable Fibres from the Digestive Tract. — In view of the great quantities of cellulose which vegetarians ingest with the food the question naturally presents itself as to whether and how this can physiologically be made use of in the body. Even the early investigations of 11 H. Bierry, Biochem. Zeitschr., U, 402, 405, 426, 1912. 12 J. Munk, Ergebn. d. Physiol., 1, 306, 1902. 13 K. v. Alfthan, Ueber dextrinartige Substanzen im diabetischen Harne, Helsingfors, 1904. 14 E. S. London and W. W. Polowzowa, Zeitschr. f. physiol. Chem., 56, 513, 1908; 57, 529, 1908.. 15 K. Omi (Röhmann's Lab., Breslau), Pflüger's Arch., 126, 428, 1909. DISAPPEARANCE OF VEGETABLE FIBRES 199 Haubner, Henneberg and Stohmann, v. Hofmeister, Weiske, Knieriem and others left no doubt that in vegetarians a portion of the ingested "coarse vegetable fibre" (a mixture of celluloses, hemicelluloses, pentosans, lignin and similar substances remaining after exhausting vegetable food with dilute acids, caustic soda, alcohol and ether) actually disap- pears in the intestine. 16 We are not dealing here with a subtile matter, but on the contrary with decidedly gross relations in that the fraction of the ingested cellulose which vanishes within the digestive tract and fails to appear in the excrement in the herbivorous domestic animals is estimated to amount to from 30 up to 70 per cent. The degree to which it can be used in the body depends upon the character of the cellulose. That of hay and still more that of tender young plants is more readily handled than, for example, is that of the bran of oat-seed and of barley, which is practically or entirely indigestible. Surprisingly, birds, even the typical graminivorous species, seem entirely unable to digest cel- lulose; W. Biedermann 17 believes this is perhaps explicable by the thought that, birds which live upon vegetable food generally possess a powerfully developed muscular stomach, by which they are mechanically able to break up into fine bits the seeds they swallow without the need of chemical solvents for the cellulose of the hulls. The ability to digest cellulose is entirely absent from the carnivorous animal, especially the dog ; this may be looked upon as settled by the studies of Scheunert and others after much controversy. 18 Man is apparently to be classed among the vegetarian animals from his position in relation to cellulose. It is said that about 50 per cent, of the cellulose and hemi- 18 Literature upon Cellulose Digestion : A Scheunert, Handb. d. Biochem., 3", 134-138, 1909; W. Biedermann, Handb. d. vergleich. Physiol., 2' 1314-1344, 1911. " W. Biedermann, 1. c, p. 1314. 13 A. Scheunert and E. Lötsch, Berl. tierärztl. Wochenschr., 1909, No. 47 ; Zeitschr. f. physiol. Chem., 65, 219, 1910; Biochem. Zeitschr., 20, 10, 1910; H. Lohrisch, Zeitschr. f. physiol. Chem., 69, 143, 1910; H. v. Hösslin (Med. Clinic, Halle), Zeitschr. f. Biol., 54, 395, 1910. 200 DIGESTION OF CARBOHYDRATES cellulose ingested in vegetables and fruits is digested in the human canal, in habitual constipation as much as 80 per cent. The proposal to use it as a substitute for the ordinary, readily-resorbed carbohydrates in cases of severe diabetes is related to the assumption (which, as we will see later, is not yet satisfactorily proven) that cellulose undergoes a cleavage into sugars in full analogy to starch, but much more slowly than the latter. 19 Determination of Cellulose. — The differences of opinion upon the availability of cellulose in the body are partly to be ascribed to theunsatisfactoriness of the methods used for its quantitative estimation. The commonly used method of Lange is based upon the unconfirmed proposition that cel- lulose is not acted upon by caustic alkali. The practice of Simon and Lohrisch, in which the material under examina- tion is heated with 50 per cent, solution of caustic soda and then decolorized with peroxide of hydrogen is attended by great losses, according to Scheunert. 20 The latter author recommends that the substance to be examined be first heated with a highly concentrated sodium hydrate solution and the undissolved residue washed well on a hardened filter, and finally weighed. The ash of the cellulose thus obtained must eventually be taken into consideration. Cytases. — How is the digestion of cellulose accomplished? One naturally at once assumes that the organism of the vege- tarian can furnish ferments especially adapted for the cleavage of cellulose just as it produces ferments for the splitting of protein, sugar and fat. And for the lower forms of animals an experimental foundation has been found for this assumption. The brilliant researches of Biedermann 21 19 H. Lohrisch (Med. Clinic, Halle), Zeitschr. f. exper. Pathol., 5, 478, 1909; F. Moeller (Med. Clinic, Halle), Inaug. Dissert., Halle, 1911, and Intern. Beitr. z. Pathol, u. Therap. d. Ernährungsstörungen, 1, 325, 1910; F. Schilling, Arch, f. Verdauungskr., 16, 720, 1910; W. Biedermann, Handb. d. vergl. Physiol., 2', 1315, 1911. "A. Scheunert, Handb. d. biochem. Arbeitsmeth., 3, 277-280, 1910; W. Grimmer and A. Scheunert, Berlin, tierärztl. Wochenschr., 1910, No. 7. 21 W. Biedermann and P. Moritz, Pflüger's Arch., 73, 219, 1898. CYTASES 201 have established the fact that the hepatic secretion of certain molluscs and crustaceans contains a very effective cellulose- solving ferment, a "cytase." If, for instance, the liver secretion of a Weinberg snail is allowed to act on a thin section of the starchy endosperm of a grain of wheat it will be apparent that the cell membranes are rapidly dissolved even before the enclosed starch granules are visibly affected. The energy with which the gastric secretion of the snail dis- solves the thick and very resistive cell walls of date seeds, ivory nuts or coffee-beans is still more striking. It has been ascertained, moreover, that the various celluloses and hemi- celluloses are broken down into the same cleavage products (glucose, mannose, galactose, pentoses, etc.) by the action of cytase, as occur from the cleavage from boiling with mineral acids. There is, therefore, a real hydrolytic cleavage in operation. The statements of Biedermann have been fully confirmed by a series of control tests, 22 especially by French authors. 23 The expectation that an analogous ferment might be found in the intestine of vegetable eating animals has not been realized. The mammalian intestinal contents, carefully sterilized by being passed through a Berkefeld or similar filter, has always been found inactive for cellulose. 24 If the cellulose be protected by the addition of sugar which many bacteria, particularly anaerobic forms, prefer as their source of energy above any other material, the cellulose does not undergo cleavage, — a result which would scarcely be ex- pected if we were actually dealing with a hydrolytic cleavage from cytases. 23 22 E. Müller, Pflüger's Arch., 83, 619, 1901. 25 H. Bierry, J. Giaja, M. Pacaut, G. Seillere and others in C. R. Soc. de Biol.; H. Bierry and J. Giaja (Sorbonne, Paris), Biochem, Zeitschr., J f 0, 370, 1912. 24 A. Scheunert, Zeitschr. f. physiol. Chem., 48, 9, 1906; and the experi- ments of Ellenherger, V. Hofmeister, Holdefleiss and H. T. Brown, cited by Scheunert in Handb. d. Biochem., 3" 135, 1909. 25 H. v. Hössiin and E. J. Lesser (Physiol. Inst, and Med. Clinic, Halle), Zeitschr. f. Biol., 64, 47, 1910. 202 DIGESTION OF CARBOHYDRATES Part Taken in Digestion by Enzymes Contained in the Food. — A number of years ago Ellenberger 26 showed that enzymes contained in vegetable food itself (especially those capable of reducing sugars and proteolytic enzymes) may become active when the food is in course of digestion, and may aid the digestive juices contributed by the animal body. It was thought this was especially true of the cytases of the food, 27 although apparently this has proved incorrect. The fact that in autolysis of wheat seeds no diminution is mani- fest in the cellulose may be taken to indicate, as Scheunert believes, 28 that these vegetable cytases are of no practical importance in the digestion of cellulose. Importance of Symbiotic Microorganisms. — There is nothing left, therefore, but to accept the view that the digestion of cellulose is effected by the microorganisms of the digestive tract. Eeference has previously been made (Vol. I of this series, p. 40, Chemistry of the Tissues) to the biological importance of the enormous quantities of minute organisms inhabiting the intestine. "It is of the greatest interest, and, in my own opinion, scarcely sufficiently emphasized," says W. Biedermann, 29 "that we are here dealing with a typical instance of symbiosis, ir which foreign microorganisms, originating from the external world, by their vital processes not only facilitate and promote the thorough utilization of ingested foodstuffs, but actually are more capable of effecting this result than any other agents." Marsh-gas Fermentation. — What do we actually know of the mechanism of this process of utilization? In the first place marsh-gas fermentation (cleared up especially by the studies of Popoff, Zuntz, Hoppe-Seyler, Tappeiner and Omeliansky) should be mentioned, a process which the cellu- 26 W. Ellenberger, Skandin. Arch. f. Physiol., 18, 306, 1906; and earlier works. "P. Bergmann (I. Bang's Lab., Lund), Skandin. Arch. f. Physiol., 18, 119, 1906. 28 A. Scheunert and W. Grimmer, Zeitschr. f. physiol. Chem., 48, 27, 1906. 29 W. Biedermann, Handb. d. vergl. Physiol., %' 1330, 1911. FOOD VALUE OF CELLULOSE 203 lose undergoes in the intestine and in which it is broken down into CH 4 , C0 2 and volatile fatty acids (acetic acid, isobutyric acid, valerianic acid). Hitherto marsh-gas fermentation of cellulose has been usually regarded as about equalled by its hydrogen fermentation. From recent investigations, ema- nating from the laboratory of N. Zuntz 39 (in which the gases of fermentation were obtained directly by puncture from the digestive apparatus of a goat, in which the paunch was sutured into a wound of the abdominal wall) it is apparent, however, that the hydrogen of the total fermentation gases never amounts to more than 10 per cent, of the methane found with it. Under normal circumstances it would appear that the CH 4 fermentation preponderates to a marked de- gree, at least as long as the reaction of the fermenting mix- ture is acid. A simple chemical formulation of this fermen- tation process is at the present out of the range of possibility. The part of the tract in which food rich in cellulose is prin- cipally broken up is in ruminants in the proventricle (paunch) ; in other herbivora with single-chambered stom- ach, as the horse and rabbit, an analogous role is to be as- signed probably to the highly developed caecum in handling cellulose. 31 In man the colon seems to be the part concerned in cellulose digestion. 32 Food Value of Cellulose. — This brings us to the main point at issue — has cellulose or its fermentation products a real value as a nutrient? 33 A number of physiologists especially interested in metabolism have absolutely denied this view, as Weiske, who experimented upon a sheep, and E. Wolff, who worked on a horse ; while others, as Knieriem, Kellner, 34 and recently Ellenberger and Scheunert, take the 30 J. Markoff (N. Zuutz's Lab., Berlin), Biochem. Zeitschr., 3> f , 211, 1911. a N. Zuntz, Verh. d. Berlin, physiol. Ges., March 10, 1905; Centralbl. f. Physiol., 19, 581, 1905; W. Ustjanzew (Zuntz'3 Lab.), Biochem. Zeitschr., //, 154, 1907. 32 F. Schilling, Arch. f. Verdauungskr., 16, 720, 1910. 33 Cf. W. Biedermann, Handb. d. vergl. Physiol., p. 1336, 1911. 34 O. Kellner, Die Ernährung d. landwirtschaftl. Nutztiere, Berlin, Parey, 1906. 204 DIGESTION OF CARBOHYDRATES position that cellulose, especially if no other more easily utilizable foods are readily available, may be drawn on as a food, and that its nutritive value is to be ascribed as equiva- lent to that of starch under proper circumstances. 35 How are we to interpret the situation? Apart from the fact that cellulose fermentation extracorporeal^ (by in- oculating a suspension of cellulose in meat extract with a bit of intestinal contents) proceeds only very slowly, while in the animal body large amounts of cellulose disappear rela- tively quickly, in its fermentation there arise products (like methane, acetic and butyric acids) which either cannot be handled at all or only with considerable difficulty by the economy. 36 Here is an open break in our knowledge. In case cellulose really has an important value as a food (al- though this does not seem so thoroughly determined as to make further investigation undesirable) there must be hid- den back of the method of its utilization some process of which we are entirely in the dark. Certain recent investigations by Pringsheim 37 are prob- ably of importance in this connection. If the full fermenta- tive power by cellulose-splitting microorganisms, which nor- mally produce only methane, hydrogen, carbonic acid, lactic acid and the lower fatty acids, be inhibited by means of anti- septics, or (in case of thermophilic microorganisms) by re- duction of temperature, one may without difficulty find in the cultures in the course of a few days glucose and cel- lobiose (a disaccharid). Then, too, it must be remembered that lactic acid is also readily oxydized by the body and may be utilized by it. Importance of Infusoria in Cellulose Digestion. — Per- haps, too, the very reasonable hypothesis suggested by 25 W. Ellenberger and A. Scheunert, Lehrb. d. vergl. Physiol, d. Haus- säugetiere, p. 354, Berlin, Parey, 1910. a6 Cf . S. Fränkel, Dynamische Biochem., p. 37, et seq., 1911. " H. Pringsheim (Chem. Instit., Berlin), Zeitschr. f. physiol. Chem., 78, 266, 1912. ESTIMATING SUGAR IN BLOOD 205 Eberlein may afford some aid in solving the puzzle. In the digestive tract of vegetarian animals, as in the proven- tricle of ruminants, besides bacteria there are always great numbers of infusoria of different kinds which have been introduced with the hay and grasses, which increase there tremendously and may possibly be of importance in the nor- mal process of cellulose digestion. We cannot overlook the possibility that these microorganisms may relieve herbiv- orous animals of part of the labor of digestion by trans- forming the cellulose into sugar by means of cytases for their own needs and utilizing it for building up their own corporeal structure; then later on when they die and are in turn digested, the material they have assimilated may indirectly come to be of use to the host animal. The proof of this suggestion is somewhat difficult because thus far the parasites of the stomach of ruminants have apparently never been artificially cultivated, although this should not be very difficult of accomplishment. 38 BLOOD SUGAR In continuation of the study of the passage of the carbo- hydrates through the living organism the next question to be considered is that of the form in which the sugar, when resorbed from the intestine, circulates in the blood. Teclmic of Estimating the Sugar in the Blood. — Prog- ress in the problem of blood sugar depends upon our ability to recover the minute amounts of the sugar unchanged from the great quantities of albumenoid blood colloids in which it may be said to be buried. The technic of analysis of haßmic sugar 39 is therefore closely connected with processes of removal of albumin. An important and very welcome improvement in this connection is the method proposed by 38 R. Eberlein, Zeitschr. f. wissensch. Zoöl., 49, 233, 1895; A. Scheunert, Berlin, tierärztl. Wochenschr.. 1909, No. 45; E. Liebetanz, Arch. f. Protistenk., 19, 19, 1910; W. Biedermann, Handb. d. vergl. Physiol., 2' pp. 1337-1344, 1911. 39 Cf. 0. Schumann and C. Hegler, Mitt. a. d. Hamburger Staatskrankenan- stalten, 12, 429, 1911 ; cited in Centralbl. f. d. ges. Biol., 1911, No. 1773. 206 BLOOD SUGAR Michaelis and Eona for the removal of albumin by shaking with a colloidal iron solution, based on the physical-chemical principle that two colloids of different content will precipi- tate each other 40 ; (liquor ferri oxydati dialysati is not in- appropriate for the purpose). The same principle is em- ployed by Möckel and Frank 41 in their method of estimation of sugar in the blood. Ivar Bang and his collaborators 42 remove the albumin with alcohol, and get rid of the protein residue by shaking with blood charcoal in the presence of hydrochloric acid. The older approved methods of remov- ing albumin by precipitation by mercuric chloride and mercuric nitrate or phosphotungstic acid are in comparison unquestionably valuable methods 43 ; the author confesses he could not well dispense with them. When the albumin has been separated, there is no further difficulty in the actual es- timation of the sugar in the highly concentrated fluid by some one of the reduction methods, or by polarization or fermenta- tion in the usual way. Comparison of these procedures show that the old Fehling's method and the reduction meth- ods of Bertrand and Kumagawa-Suto give practically the same results, while the hydroxylamine method of Bang yields higher figures. 44 Tachau has proposed a modification of Knapp 's method, consisting of adding excess of cyanide of mercury, separating the reduced mercury, then precipitating the mercury which remains in solution and weighing it. 45 Attempts have also been made to use various color re- 40 Cf. P. Rona and L. Michaelis, Biochem. Zeitschr., 16, 60, 1909. 41 K. Möckel and E. Frank (Wiesbaden), Zeitschr. f. physiol. Chem., 65, 323, 1910; 69, 85, 1910. 42 1. Bang, H. Lyttkens and J. Sandgren (Lund) , Zeit3chr. f. physiol. Chem., 65, 497, 1910. 48 B. Oppler, Zeitschr. f. physiol. Chem., 6k, 393, 1910; J. J. R. Macleod, Jour, of Biol. Chem., 5, 443, 1909 ; H. Bierry and Portier, C. R. Soc. de Biol., 66, 577, 1909. 44 Cf. I. Bang, Biochem. Zeitschr., 7, 327, 1908; D. Takahaschi, Biochem. Zeitschr., 37, 30, 1911. 46 H. Tachau ( Schwenkenbecher's Clinic and Embden's Lab., Frankfurt a. M.), Deutsch. Arch. f. klin. Med., 102, 597, 1911. EXISTENCE OF FREE SUGAR IN BLOOD 207 actions peculiar to the carbohydrates in colorimetric estima- tion of the blood sugar. Wacker has elaborated a method 46 which utilizes a sensitive red color reaction given by p-phenylhydrazinsulphonic acid with carbohydrates in presence of alkali (also with a number of alcohols and aldehydes.) By comparing the red color with a color scale worked out from a standard sugar solution the method is said to successfully differentiate as small amounts as 0.05 mg. of glucose and too requires only small quantities of blood (about 10 to 15 drops). In view of certain inherent sources of error 47 , the method is of doubtful availability. Existence of Free Sugar in the Blood. — It has been fre- quently asked whether the blood sugar is present in the blood free or in colloidal combination, as many authors have concluded from a variety of physiological and analytical observations (particularly from the fact that sugar, which diffuses readily, does not normally pass through the renal filter). Primarily F. Schenk, in connection with his pioneer work on the sugar of the blood, concluded because of its diffusibility that the sugar is not combined in any way with albuminoid substances, but that it exists free in the blood. Further observations in the same line have been published by Arthus, and, too, by Asher, 48 who has been able to prove that diffusion of sugar takes place when, instead of water, a like sample of blood, but previously freed from sugar by fermentation, is used as the outer fluid. Finally, Michaelis and Bona 49 have shown in detail how the sugar proportions in an isotonic salt solution may be prepared so that in 46 L. Wacker (Würzburg), Zeitschr. f. physiol. Chem., 67, 197, 1910; L. Wacker and F. Poly, Deutsch. Arch. f. klin. Med., 100, 567, 1910; 102, 597, 1911. 47 Forschbach and S'everin (Minkowski's Clinic, Breslau), Centralbl. f. Stoffwechselkr., 6, 54, 1911. 4S L. Asher and R. Rosenfeld (Berne), Biochem. Zeitschr., 8, 351-358, 1907; consult therein Literature; consult also the objections of E. Pflüger, Pflüger's Arch., Ill, 217, 1907. 4 * L. Michaelis and P. Rona, Biochem. Zeitschr., 14, 476, 1908, and earlier contributions. 208 BLOOD SUGAR dialysis against blood, sugar will not enter the blood or diffuse from it ; and in the author 's opinion have, by this "method of osmotic compensation," furnished the final proof that the sugar exists free in the blood or at least in a condition to become free spontaneously. "Sucre Immediat" and "Sucre Virtuel." — A second question, however, presents itself : whether we may assume that the total amount of sugar in the blood is in free form. From this point of view the writer regards the results of Le- pine and Boulud, 50 after a long series of careful studies, as apropos. These authors differentiate in the blood between " sucre immediat" and "sucre virtuel." The first is de- termined by catching the blood directly in an acid solution of mercuric nitrate, filtering, pressing the blood cake dry and determining the sugar in the collected filtrate after separa- tion of the mercury. The virtual sugar, supposed to be combined in the blood as glucosides, is determined from the increase of reduction power observed in the blood after keeping it for an hour in the incubator with added invertin or emulsin before estimating the sugar. Apparently, however, the conversion of the virtual sugar into the de- terminable form will take place spontaneously if it be al- lowed to stand for a quarter of an hour; and it has been proposed to always allow fifteen minutes to elapse after withdrawal of the blood before beginning the sugar estima- tion. 51 It is obvious from this that in the above noted diffusion experiments the virtual sugar must in reality appear as free sugar. Does the Blood Contain Other Carbohydrates in Addition to Glucose? — It may, therefore, be asked what significance is to be ascribed to the "sucre virtuel"? This is certainly not a simple matter. The French authors, for example, con- tinue to speak of "sucre virtuel" even after heating the 00 R. Lupine and Boulud, Jour, de Physiol., 11, 12, 557, 1909; 13, 178, 1911; and a number of other contributions in C. R. and C. R. Soc. de Biol. 11 E. Frank (Wiesbaden), Zeitschr. f. physiol. Chem., 70, 129, 1910. DOES BLOOD CONTAIN OTHER CARBOHYDRATES ? 209 blood cake for twenty-four hours with hydrofluoric acid. They must in this case certainly be dealing with some glucos- amine compounds, connected with the structural composition of the blood proteids (and too, in that of other proteins) separable by hydrolytic cleavage from the latter; these are not, however, a part of the true blood sugar. 52 Contrary to the statement that a fourth or more of the blood sugar which is concerned in normal reduction experi- ments is in the form of a non-fermentescible rest-sugar, 53 no such substance could be recognized by estimating the reduc- ing power of blood by Bertrand's method after fermentation either in the blood or in the serum. 54 It will be readily realized that the blood at times may con- tain not inconsiderable quantities of maltose and glycogen (carbohydrates which may decidedly increase the reducing ability of the blood after fermentative cleavage) . 55 It seems doubtful, however, whether these substances are sufficient to fully explain the observations recorded in reference to " virtual sugar." In the author's opinion it is more prob- able that in this we have to do with phenomena of a physical- chemical nature, which we know very well are able to markedly influence the conditions of solubility of sugar. Occasion has been taken in a previous lecture (v. Vol. I of this series, p. 170, Chemistry of the Tissues) to call atten- tion to the enveloping phenomena which gave rise to the mistaken assumption of a chemical combination between lecithin and glucose ("jecorin"). 50 One may easily im- agine how a part of the sugar in the blood in some such "Literature upon the Carbohydrate Groups of Proteins: L. Langstein, Ergebn. d. Physiol., 1, 91-98, 1902; Monatshefte f. Chem., 26, 531, 1905. 63 J. G. Otto, Pflüger's Arch., 85, 474, 1885; N. Anderson ( Lund ) , Biochem. Zeitschr., 12, 1, 1908. M E. Frank and A. Brettschneider, Zeitschr. f. physiol. Chem., 71, 157, 1911. 55 Literature: A. Magnus-Levy, Handb. d. Biochem., V 318-319, 1909; E. Frank and Brettschneider, Zeitschr. f. physiol. Chem., 76, 226, 1911; F. Spallitta (Palermo), Arch. ital. de Biol., 53, 356. 1910. 63 Note the doubt expressed by P. Mayer (Salkowski's Lab.), Biochem. Zeitschr., Jf, 545, 1908, as to the chemical individuality of jecorin. 14 210 BLOOD SUGAR colloidal investment might be carried along in the precipita- tion of the other blood colloids and thus escape estimation; while in the sense of " virtual sugar" it would be recognized as soon as freed from its investment in the lecithid by any suitable separating agent. Whether this, in a general way, will satisfactorily explain the activity of glucoside-splitting ferments, as understood by Lepine and Boulud, must be left unanswered. But at any rate we cannot insist specifi- cally upon lecithids as the enveloping factors. Sugar Content of the Red Blood Cells. — In the last few years the question whether glucose is found in the red blood corpuscles as well as in serum has been freely agitated. The statement that these cells contain, not glucose, but only a non- fermentescible carbohydrate, a polysaccharide, 57 is appar- ently contradicted by numerous investigations. There is suf- ficient positive evidence 58 that the red cells of fresh blood (in contrast with washed cells, which vary) 59 are permeable for glucose and actually absorb it (in addition, the corpuscles, as well as the plasma, contain variable amounts of a complex carbohydrate which is converted into fermentescible sugar by hydrolysis). The sugar is not always in proportionate distribution in the plasma and cellular elements ; sometimes, especially in hyperglycemias, very striking differences have been found in the proportionate amounts found within and outside the corpuscles, which has been taken to indicate that the blood cells play an independent role in sugar metab- 67 H. Lyttkens and J. Sandgren (Instit. Med. Chem., Lund), Biochem. Zeitschr., 26, 382, 1910; Si, 153, 1911; 36,261,1911; S. E. Edie and D. Spence (Liverpool), Biochem. Jour., 2, 103, 1907. M L. Michaelis and P. Rona, Biochem. Zeitschr., 16, 60, 1909; 18, 375, 514, 1909; 37, 47, 1911; P. Bona and A. Döblin, ibid., 31, 215, 1911; R. Lepine and Boulud, ibid., 32, 287, 1911, and earlier contributions; A. Hollinger (Frankfurt a. M.), ibid., 17, 1, 1909; D. Takahaschi (Rona's Lab.), ibid., 37, 30, 1911; E. Frank and A. Brettschneider (Wiesbaden), Zeitschr. f. physiol. Chem., 76, 226, 1911. 6 * Consult the studies of Hamburger, Gryns, Koeppe, Hedin and others: Literature upon the Permeability of Red Blood Cells: E. Overton, Nagel's Handb. der Physiol., 2, 828-839, 1907. CARBOHYDRATE-SPLITTING FERMENTS 211 olism. 60 Fundamentally, the fact that red blood cells con- tain sugar is, in the writer's opinion, a self-evident one; and he cannot suppress feelings of regret for the loss of valuable time devoted by so many noted investigators to this matter. We have absolutely no reason to doubt that sugar may penetrate into every living cell in the system, to become, as it were, the ready cash for defraying the expenses of the vital combustion processes. Why should the red blood cells especially be held different from the rest ? Sugar in the Aqueous Humor. — In conclusion a new method, devised by E. H. Kahn, 61 should be mentioned for rapid and simple reckoning of the proportion of blood sugar in experiment animals. The anterior chamber of the eye is punctured with a sharp hypodermic needle and the fluid from the chamber caught in a tube. It is best to punc- ture one eye before the experiment, and the other after the conclusion of the experiment, and to determine the differ- ence in the fluid from the separate eyes. If care be taken not to wound the iris this interference is likely to cause no more than a temporary disturbance of vision in the animal, as the anterior chamber is very soon filled again. A hyper- glycemia, as that brought on by puncture in the fourth ventricle (sugar puncture), or by adrenin, or phloridzin becomes readily appreciable by an increased reducing power of the fluid. CARBOHYDRATE-SPLITTING FERMENTS Another large group of phenomena, which next demand our attention, is that due to the ferments which induce cleav- age of carbohydrates, including the diastases, maltases, invertases, lactases, etc. It must suffice to bring out here only a few points of physical and pathological significance, and for the rest, especially for all those questions which 60 R. Höber (Kiel), Biochem. Zeitschr., 4-5, 207, 1912. "R. H. Kahn (Prague), Centralbl. f. Physiol., 25, No. 3, 1911. 212 CARBOHYDRATE-SPLITTING FERMENTS relate to the kinetics of fermentation, to make reference to the comprehensive monographs which have appeared. 62 Quantitative Determination of Diastase. — Each advance of onr knowledge of the physiological role and importance of the "carbohydrases," especially of the diastases, presup- poses the possibility of their being quantitatively estimated with reasonable exactness in animal fluids and tissues. In a fluid this is not particularly complicated, aside from the difficulty, which obtains in all fermentation investigations, that it is impossible to estimate the ferments directly, but only possible to determine them in terms of their active strength. If a fluid containing diastase be treated with an excess of soluble starch or glycogen solution, and after a time the quantity of newly formed reducing sugar calculated, an applicable measure of the activity of the ferment is obtained. Because of the difficulties of detail attendant in such a method there is need of methods capable of affording a quicker means of orientation. The method of delle pro- duction on a starch-paste plate, for example, has proved a simple aid for this purpose in the study of diastasic fer- ments. 63 In Pawlow's Institute, in imitation of the well- known Mett method of estimating pepsin, the shortening of starch cylinders (made by packing the starch in glass tubes) has been employed for the same purpose. 64 A colorimetric method devised by ^Wohlgemuth has, however, proved to be the most useful of these measures; being based upon the determination of the amount of ferment solution re- quired to so change a known solution of starch, at given temperature and length of time, that further bluing of the solution will not follow when iodine is added. The tints ap- pearing in serially arranged tests when iodine is added 62 Literature upon Carbohydrate-splitting Ferments: F. Samuely, Handb. d. Biochem., 1, 537-546, 1909 ; C. Oppenheimer, Die Fermente, 3d ed. ; II, 66- 108, 1910; H. M. Vernon, Ergebn. d. Physiol., 9, 183-193, 1910; W. Biedermann, Handb. d. vergl. Physiol., 2' 1397-1402, 1911. 63 E. Müller, Centralbl. f. innere Med., 1908, 386. 64 Walter, cf. Pawlow, Arbeit, der Verdauungsdrüsen, Wiesbaden, 1898. WIECHOWSKI'S TISSUE-POWDER METHOD 213 (blue, violet, red, yellow), representing the progressive dis- solution of the starch, are sufficiently characteristic to make a convenient estimation possible from them. W. Schles- inger has also proposed a method based on the same principle. 65 It becomes a much more difficult matter when it is desir- able to estimate the diastases, not in a fluid, but in a tissue. A process may be employed on the lines followed by F. Kisch (in connection with an investigation made at the author's suggestion upon the gradual loss of glycogen in the muscles after death) by adding a large excess of a glycogen solution to a mushy mass of the tissue, and then determining the amount of newly formed sugar after lapse of a given time. 66 An undesirable uncertainty is inherent to methods of this general type, however, because there is no guarantee that there is not some ferment included in tissue pulp which may fail to come in contact with the available carbohydrate. The author looks upon a method devised by Wiechowski for quan- titative ferment estimation as a very important advance in technique, but insufficiently appreciated as a general thing, and as having first made possible a precise treatment of a large number of important problems of the physiology and pathology of metabolism : Wiechowski' s Tissue-Powder Method. — Wiechowski 's method 67 is carried out as follows: Tissue obtained from a freshly killed animal, is perfused with normal salt solu- tion until free from blood ; is then chopped into small pieces and passed through a fine sieve. The pulp is then spread out in thin layers upon large glass plates and dried by a strong stream of air from a special ventilator constructed in properly adapted form. The tissue powder is next treated 05 J. Wohlgemuth, Biochem. Zeitschr., 9, 1, 1908; W. Schlesinger, Deutsch, med. Wochenschr., 1908, 593. «•F. Kisch, Hofmeister's Beitr., 8, 210, 1906; under direction of O. v. Fürth. 6T W. Wiechowski, Hofmeister's Beitr., 9, 232, 1907; Handb. d. biochem. Arbeitsmethoden, 3', 282, 1909. 214 CARBOHYDRATE-SPLITTING FERMENTS with toluol in a special extraction apparatus in the cold, and thus freed of fats and lipoids. At this stage the tissue con- sists of a fine pulverulent mass which includes proteins and ferments in soluble and well preserved form, and affords an excellent and well adapted supply of material for quantita- tive fermentation studies. If a weighed amount of the pow- dered tissue be rubbed up with physiological salt solution in a mortar, a very fine emulsion is obtained which sediments only very gradually, and may be subdivided with graduated pipettes; and is very well suited for employment in com- parative study with similarly prepared powdered tissue from other source. In applying this method to determination of the diastases in tissues, Starkenstein, 68 in the laboratory of J. Pohl, found it essential to keep up a continuous agitation so as to insure adequate contact of the ferment and the substrate, lest the tissue protein, quickly coagulating at room temperature, take up by mechanical adsorption both starch and ferment, and in this way be sure to occasion too low a result in the quantity of the latter. Thereafter the actual estimation may be conducted by the method of Wohlgemuth. From what has been said we may be satisfied that many of the older statements as to the effect of various physio- logical and pathological factors upon the quantity of diastase in the tissues have but doubtful value. Hepatic Diastase. — As for the liver, it can be finally said that the constantly recurring doubt as to whether its vital diastasic activity is really due to an enzyme, has been con- clusively settled. 69 The present attitude is to regard the quantity of diastase in the liver as subject to a nervous regu- lation ; and the tendency is to relate starvation diabetes of the dog, as well as the glycosurias following brain injuries, Claude Bernard's puncture, or administration of adrenin, 68 E. Starkenstein (J. Pohl's Lab.), Biochem. Zeitschr., 24, 191, 1910. 69 Consult Literature in F. Pick, Hofmeister's Beitr., 3, 163, 1902. MUSCLE DIASTASE 215 phloridzin, phloretin, morphin, strichnin, etc., with a sup- posed increase of hepatic diastase. It is claimed, too, that section of the vagus is followed by marked increase in the hepatic diastase. 70 In view of all such statements it must be regarded as very important that Starkenstein, 7 1 employ- ing a technical method said to be highly perfected, has been unable to recognize any important increase of diastasic power of the liver, either in case of Bernard's puncture or after injections of adrenin. Wohlgemuth, and, too Möckel and Rost have also failed in case of adrenin glycosuria and puncture glycosuria to find any increase of diastase in the blood and tissues. 72 It therefore seems highly improbable that the exaggerated output of glycogen from the liver, pro- duced by various physiological and toxic agencies, is really an expression of an activation of the hepatic diastase. Muscle Diastase. — In view of the great physiological im- portance of muscle diastase the author assigned to F. Kisch 73 inquiry into the question whether muscle is able to accommodate itself to the varying requirement of the body for mobile sugar, perhaps by changing its diastasic power, possibly by forming fresh diastase from an inactive proferment whenever a need for increased sugar arises in states of hunger or fatigue. No apparent difference was found, however, in diastatic ability of the muscles of a given animal whether at rest or after excessive functional effort, whether after full feeding or in starvation. We cannot but assume, therefore, that the body has recourse to other means than the production of diastase to mobilize its carbohydrate supply at the precise time when it is needed. ,0 1. Bang, M. Ljungdahl and V. Böhm, Hofmeister's Beitr., 9, 408, 1907 ; 10, 1, 312, 1907; P. Zegla, Biochem. Zeitschr., 16, 111, 1909. "1. c. "J. Wohlgemuth, Biochem. Zeitschr., 22, 381, 460, 1909; K. Möckel and F. Rost, Zeitschr. f. physiol. Chem., 67, 433, 1910. 73 F. Kisch, Hofmeister's Beitr., 8, 210, 1906, under direction of O. v. Fürth; cf. therein Literature upon Postmortem Loss of Glycogen in Muscles; cf. also Z. Gatin-Gruzewska, Jour, de Physiol., 1907. 216 CARBOHYDRATE-SPLITTING FERMENTS It is impossible to say at this time why the cardiac muscle has so much higher diastasic ability than the skeletal muscles of the same animal 74 ; the marked access of diastasic power, noted by Kisch, in a tissue pulp transfused with oxygen in the presence of blood may possibly have some relation with retardation of postmortem lactic acid production. Diastases in Embryos. — The observation of Alice Stauber in Exner's Institute is not without interest, showing that at an early stage of embryonal development diastase exists in the parotid gland and in the pancreas (at a time long before any digestive activity of the alimentary apparatus could possibly be considered) ; the embryonal thymus, too, is scarcely inferior to the pancreas and parotid (organs naturally destined to secrete diastasic ferments) in its diastasic ability. 75 The diastasic ability of muscle at an early stage of embryonal life exceeds that of the liver, as shown by the careful quantitative examinations of Lafayette Mendel. 76 Origin of Diastase. — L. Haberlandt has determined in Zoth's Institute that the ability to form diastasic ferment is a property of leucocytes (particularly of the polymorpho- nuclear leucocytes), the enzyme partly remaining within these cells, partly passing into the surrounding fluid. In massive collections of leucocytes in the subcutaneous con- nective tissue (as induced by some local irritant) the diastasic power of the tissue of such a part becomes increased and Haberlandt believes the assumption justified that at least in part the diastatic ferment of the blood-serum has its origin in the leucocytes. 77 This introduces the important subject of the relation of 74 Boruttau, Kisch, 1. c. 76 A. Stauber (S. Exner's Lab., Vienna), Pflüger's Arch., UJ h 619, 1906. 76 L. B. Mendel, with P. H. Mitchell and Saiki (Yale Univ.), Amer. Jour, of Physiol., 20, 81, 1907; 21, 64, 1908. 77 L. Haberlandt (Zoth's Lab., Gratz), Pflüger's Arch., 132, 175, 1910. ROLE OF THE PANCREAS 217 the tissue diastase and blood diastase and the source of the latter. Role of the Pancreas in the Production of the Blood Dias- tase. — Observations showing that the amount of diastase in milk in the first part of lactation may be from one to two hundred times greater than that of the blood, 7 7a clearly indi- cate that the ferment, for the most part at least, cannot in such cases come from the blood but rather from the tissues themselves. Comparative physiology, too, would suggest that diastases are to be classed among the enzymes common to the general tisues and found in all forms of life from the lowest to the highest. 78 For this reason especially the author has never had any sympathy with ideas referring the production of the diastase of the blood and other tissues solely to one or more definite organs, as the pancreas and salivary glands. Yet it does not in itself seem implausible that diastase may be resorbed into the circulation from the pancreas and eventually pass into the urine; but statistics as to the quantity of diastase in the blood of the pancreatic veins are in general contradictory to this idea. However, extirpation of the pancreas seems sometimes to diminish, for a time at least, the amount of diastase in the blood ; while conversely, after ligation of the pancreatic duct, the ferment dammed up in the gland may pass in decidedly increased amount into the blood and thence into the urine. For this reason examination of the urine for its diastase has been recommended as a diagnostic test of the function of the pan- creas. 79 Recent observations from Carlson's laboratory "a J. Wohlgemuth and M. Strich, Sitzungsber. d. preuss. Arcad., 1910, 520. 78 Cf. Literature in 0. v. Fürth, Vergl. chem. Physiol, d. niederen Tiere, Jena, 1903. '• W. Schlesinger, Deutsch, med. Wochenschr., 190S, 593 ; A. J. Carlson and A. B. Luckhardt (Chicago), Amer. Jour, of Physiol., 23, 148, 1908; J. Wohlgemuth and collaborators, Biochem. Zeitschr., 21, 381, 422, 432, 447, 460, 1909; K. Möckel and F. Rost, Zeitschr. f. physiol. Chem., 67, 433, 1910; J. J. R. Macleod and R. G. Pearce (Cleveland), Amer. Jour, of Physiol., 25, 255, 1910; G. Hirata, Biochem. Zeitschr., 27, 23, 1910; E. Marino (M. Jacoby's Lab.), Deutsch. Arch. f. klin. Med., 108, 325, 1911; J. v. Benczur, Wiener klin. Wochenschr., 28, 890, 1910. 218 CARBOHYDRATE-SPLITTING FERMENTS prove beyond doubt that the pancreas cannot possibly be the only source of the blood diastase. They show that the curve of the blood diastase, which is deeply depressed after pan- creatic extirpation, rises again after a few days, without, however, fully regaining its normal level. 80 It is not im- probable that the liver may also give off diastasic ferment into the blood, 81 more plausible in fact than the reverse hypothesis that the hepatic ferment should be derived from the blood. Maltases, Invertases and Gluceses in the Blood-serum. — Besides diastase maltase also is met in the blood serum. This has been recently investigated closely in F. Röhmann's laboratory. In connection with these studies brief inquiry was made as to whether the maltase of the blood serum is capable in concentrated solutions (as proved by Croft-Hill for yeast maltase) of building disaccharides and poly- saccharides from glucose molecules; points were noted fundamentally corroborative of the conception of such synthetic fermentation. Following a terminology proposed by Euler we would here have to recognize the existence of a "glucese" in the blood serum. 82 There is, too, much of interest in the recognition by Abderhalden that in the blood serum after parenteral intro- duction of a group of carbohydrates which do not normally enter the circulation (as cane sugar, milk sugar or starch) ferments may be found which are capable of inducing cleav- age of such substances. 83 80 H. Otten and T. C. Galloway ( Carlson's Lab., Chicago ) , Amer. Jour, of Physiol., 26, 347, 1910; cf. also S. van de Erve (Carlson's Lab.), ibid., 29, 182, 1911. 41 A. Pugliese, Arch, di Farmacol., 12, 1, cited in Centralbl. f . Physiol., 20, 827, 1906. 89 CI. Kusumoto, L. Doxiades (F. Röhmann's Lab.), Biochem. Zeitschr., U, 217, 1908; 32, 410, 1911; 38, 306, 1912. 83 E. Abderhalden, with C. Brahm, G. Kapfberger and E. Rathsmann, Zeitschr. f. physiol. Chem., 64, 429, 1910; 69, 23, 1910; 71, 367, 1911. Litera- ture upon Animal Invertases: C. Oppenheimer, Die Fermente, 3d ed., II, p. 40, 1910. ACTION OF DIASTASE ON STARCH 219 Action of Diastase on Various Forms of Starch. — The utilization of complex carbohydrates by the body is clearly a very complicated process as was believed. Sigmund Lang confirmed this in his comparative studies upon the influence of pancreatic diastase upon starches from different sources, showing that oats starch, which is highly resistive to catabol- ism into products which do not give a color reaction with iodine, is very readily converted into sugar; that potato starch, conversely, while very readily decomposed into achroödextrin, is converted into sugar only very slowly. This behavior of oats starch to diastase is scarcely con- sonant with the strikingly favorable results obtained by the ' ' oats treatment ' ' employed by v. Noorden for diabetes. On the other hand, it is questionable whether it is really proper, as is usually done at the present time, to make the disappear- ance of the iodine reaction the basis of quantitative de- termination of the effect of diastase, and whether it would not be better, as Lang proposes, to estimate the effectiveness of diastasic ferments from the amount of end-product, glucose, than from such arbitrary intermediate product. In the end it is undoubtedly the end-product which concerns the requirements of the body. 84 It is well to recall here besides that the individual phases of starch cleavage are by no means well known, and that the "dual enzyme theory" (strongly opposed by plant physiologists) is still a matter of dispute (this theory assuming that diastase is not a single ferment but is composed of two different enzymes, maltase and dextrinase). 85 Attempts to "isolate" diastase have failed of satisfac- tory result, precisely as in the case of other ferments. 86 In contrast, the physical-chemical behavior of diastase is ap- parently an inexhaustible mine of notable observations. M S. Lang (F. Kraus's Med. Clinic, Berlin), Zeitschr. f. exper. Pathol, und Ther., 8, 1910, s. a. 85 Literature : C. Oppenheimer, Die Fermente, 3d ed., Ill, pp. 84-86, 1910. 86 S. Fränkel and M. Hamburg, Hofmeister's Beitr., 8, 3S9, 1906; E. Pribram (Fränkel's Lab.) , Biochem. Zeitschr., U, 293, 1912. 220 CARBOHYDRATE-SPLITTING FERMENTS Thus it has been noticed that diastases are at least partly inactivated by prolonged dialysis and may be reactivated by the addition of certain salts 87 ; that the blood serum contains a substance which is resistive to boiling and is soluble in alcohol, which apparently augments the diastases 88 ; that hydrolysis of starch is strengthened by alternating currents of low intensity 89 ; that there is a hydrolyzing influence in the ultra-violet rays of a mercury quartz lamp, 90 etc. For the most part, however, these matters fall within the sphere of physical-chemistry; and are beyond the limitations set for these lectures. 87 H. Bierry and J. Giaja, C. R. Soc. de Biol., 60, 749, 1131, 1906; 62, 432, 1907; C. R., 143, 300, 1906; L. Preti, Biochem. Zeitsclir., //, 1, 1909; I. Bang, ibid., 32, 417, 1911. 88 J. Wohlgemuth, Biochem. Zeitschr., 33, 303, 1911. 89 A. Lebedew (Moscow), Biochem. Zeitschr., 9, 392, 1908. 80 H. Bierry, V. Henri and A. Ranc, Jour, de Physiol., 13, 700, 1911; L. Massed, C. R., 152, 902. 1911 ; J. Giaja, C. R. Soc. de Biol., 12, 2, 1912. CHAPTER X GLYCOGEN. FORMATION OF SUGAR FROM PROTEIN AND FAT GLYCOGEN In the present lecture we have first to take up the subject of glycogen. This is a reserve form of carbohydrate which plays very much the same role in animal metabolism as starch does in plant life, and its lot stands in very close con- nection with the general questions of carbohydrate metab- olism. One need not wonder, therefore, that the literature upon the subject is a very extensive one, so bulky in fact that there would be occasion for doubt whether it would be possible to properly systematize even its principal features, were it not for the fact that two scientists, Edward Pflüger and Max Cremer, had devoted themselves to the laudable task of sifting and arranging it. Thanks to their work it is no longer a matter of great difficulty to review with a reason- able degree of clarity the problems which glycogen research faces for the immediate future. 1 Quantitative Determination of Glycogen. — Our ability to estimate with precision the glycogen supply accumulated in the tissues has been of the greatest importance for research in carbohydrate metabolism. In some of the later years of his activity Edward Pflüger performed a service of lasting value by devoting his attention to the problem of quantitative es- timation of glycogen, applying to it his phenomenal ability and thorough critical acumen. It will not be easy for pos- terity to replace Pflüger 's method with a better. This process consists in dissolving the tissues in a highly con- centrated solution of caustic potash, precipitating the glyco- 1 Literature upon the Physiology of Glycogen: E. Pflüger, Das Glycogen, 2d ed., Bonn, 1905, and Pflüger's Arch., 96, 1-398, 1903; M. Cremer, Ergebn. d. Physiol., 1', 803-909, 1902; E. Pflüger, Pflüger's Arch., 96, 55-127, 1903. 221 222 GLYCOGEN gen from solution by alcohol, converting it by hydrolysis into sugar and determining the latter directly. 2 Chemistry of Glycogen. — The chemical study of glycogen is almost stationary. It is true, Madame Gatin-Gruzewska 3 seems to have succeeded once in Pflüger 's laboratory in ob- taining it in the form of minute prismatic crystals; the method pursued consisting in adding alcohol to a weak solution of glycogen until turbidity began to appear, dissolv- ing the precipitate in water and allowing this solution to stand in the refrigerator. Little further has been heard of the procedure. The molecular weight of glycogen is un- doubtedly very great. Zdenko Skraup, in collaboration with E. v. Knaffl-Lenz, has attempted its determination, treating the glycogen with acetic acid anhydride saturated with hydrochloric acid. With polysaccharides it is possible in this way to obtain chlor acetyl products, the chlorine of which may then be used in deduction of the molecular weight of the original substances; in case of glycogen the figure obtained was about 24000. Really, however, the molecule is probably even larger. 4 The well-known opalescence of glycogen solutions depends upon ultramicroscopic particles suspended in it, as observed by Rählmann and others, these particles being capable of uniting to form granules of larger size, and determining the physical-chemical characteristics of the solutions. 5 It goes without saying that under these circumstances it is difficult to expect much from cryoscopic determinations. 6 2 Literature upon the Quantitative Estimation of Glycogen : E. Salkowski, Biochem. Zeitschr., 1, 337, 1903; K. Grube, Handb. d. Biochem., 2, 159-166, 1910; cf. also W. Grebe, Pflüger's Arch., 121, 602, 1908; B. Schöndorff, P. Junkersdorf and G. Franke, ibid., 126, 578, 582, 1909; 121, 277, 1909. 3 Z. Gatin-Gruzewska, Pflüger's Arch., 102, 569, 1904. 4 Zd. H. Skraup, Monatsh. f. Chem., 26, 1415, 1905; E. v. Knaffl-Lenz (Chem. Instit., Gratz), Zeitschr. f. physiol. Chem., 46, 293, 1905. B Cf. Z. Gatin-Gruzewska and W. Biltz, Pflüger's Arch., 105, 115, 1904; F. Bottazzi and G. d'Errico (Naples), ibid., 115, 359, 1906. "Literature upon the Chemistry of Glycogen: C. Neuberg and B. Rewald, Biochem. Handlexikon, 2, 255-264, 1911. CHEMISTRY OF GLYCOGEN 223 One can readily understand how a colloidal substance like glycogen, distributed as it is in another colloidal sub- strate, the cellular protoplasm, often comes to show an atypical behavior. Thus the microchemical identification of glycogen by iodine-iodide of potassium (as in a frog's ovary) may be attended with difficulties even though glycogen is abundantly present ; but if the combination be- tween glycogen and the tissue be released by repeated freez- ing and thawing of the latter, the reaction readily takes place. 7 We are manifestly here dealing with a phenomenon of physical chemistry ; as we have no foundation at present for assuming a chemical combination of the glycogen within the tissue. 8 Moreover, R. Türkei (in opposition to the state- ments of Seegen and others) was unable to convince himself that liver-extracts, freed of their glycogen, fermentescible sugar and albumen, contain any appreciable quantities of a substance which may be precipitated by alcohol or which will yield sugar by hydrolytic cleavage. 9 That the tremendous application of work devoted during the past decades to experimental investigation of the physi- ology of glycogen, the details of which obviously cannot here be discussed, has not been without result, may be realized when it is stated that we are able even now to indicate con- cisely the conditions under which the body draws upon its glycogen supply. 10 (We have more definite information about this phase of the subject than in regard to the manu- facture of glycogen in the body, a feature undoubtedly related with the important and very imperfectly understood questions of formation of sugar from proteins and fats.) T M. Bleibtreu, K. Kato, Pflüger'a Arch., 127, 118, 125, 1909. *H. Loeschke (Pfliiger's Lab.), Pfluger's Arch., 102, 592, 1904. »R. Türkei, Hofmeister's Beitr., 9, 89, 1906; cf. therein Literature. 10 Literature upon Physiological and Pathological Catabolism of Glycogen in the Body: 0. v. Fürth, Ergebn. d. Physiol., 2, 584-589, 1902; R. Tigerstedt, Nagel's Handb. d. Physiol., 1, 495-502, 1905 ; E. Weinland, ibid., 2, 430, 1907 ; A. Magnus-Levy, Handb. d. Biochem., 4' 311-316, 1909; J. Wohlgemuth, ibid., 8', 160-161, 168-174, 1910. 224 GLYCOGEN Relation Between the Consumption of Sugar by the Muscles and the Disappearance of Glycogen of the Liver. — It is commonly accepted that glycogen is one of the generally distributed tissue constituents. Its distribution in the econ- omy is, however, by no means uniform, by far the greatest proportions being accumulated in muscle and the liver. The extent of its accretion in these latter structures is illustrated by the fact that in a frog's liver glycogen may actually con- stitute more than half of the dried substance. 11 In view of the unquestioned importance of sugar as a source of mus- cular power it may be appreciated why the living body should generally show a more constant fixation of muscle glycogen than of liver glycogen; and among the different muscles why the heart should give up its glycogen least readily. By long-continued strychnine convulsions, which is one of the most effective means of inducing the body to use up its supply of glycogen, P. Jensen ultimately suc- ceeded in getting the frog's heart free of glycogen and show- ing that even then it is capable of continuing its activity. As long as the body has a carbohydrate supply at its disposal, however, the functionating muscles are sure to find some means and some way to obtain it; but how they actually accomplish this is, it must be confessed, at present unknown. There has been a hypothesis that in muscular contraction the intramuscular nerve-endings are stimulated and transmit, as it were, telegraphic information to the great central sup- ply office in the liver to provide additional fresh nutrient material; but as a matter of fact no one has proved such relationship. It is quite possible that nervous telegraphic lines have nothing whatever to do with notifying the liver and other organs that assistance is needed for the muscles. It may very well be that the circulating blood enacts this role of messenger automatically when its level of sugar is lowered. There is no doubt that in a general way the 11 M. Bleibtreu, Mitt. a. d. Naturwiss. Vereinigung für Neuostpommern und Rügen, 1907, cited in Centralbl. f. Physiol., 22, 448, 1908. GLYCOGEN IMPOVERISHMENT 225 mobilization of sugar in the liver is presided over by nervous influences, following the evidence given by the gifted Claude Bernard when he announced his "sugar puncture" and showed that injury of the floor of the fourth ventricle is followed by glycosuria. The same thing has since then been noted after divers wounds in the general field of the nervous system; and the present belief is that the function of the liver in carbohydrate metabolism is under the regulating influence of a "sugar centre" in the medulla oblongata, the vagus nerves carrying centripetal impulses and the splanch- nics centrifugal. According to Ernst Freund and H. Pop- per in the dog an abundant deposit of glycogen can be obtained in the liver from intravenous injections of sugar, only if all cerebral stimulation is eliminated, either by narcosis or by interruption of the centrifugal nervous paths. 12 Production of Glycogen Impoverishment in the Body. — Besides muscular activity there are a great number of other physiological and pathological factors known which may pro- duce reduction in the body supply of glycogen. All forms of inanition should be prominently named in this connection in which loss of sugar from the economy as in pancreatic, phloridzin or adrenin diabetes may give rise to such serious exaggeration as to impoverish the system of its carbohy- drates. Here, too, should be classed increased heat produc- tion, as seen in fever and, too, in exposure to cold; and, finally the influence of local hepatic lesions (as from ligation of the hepatic duct or from injection of acid into the bile duct) and of intoxications (as from phosphorus, arsenic, chloroform, amyl nitrite and many others) should be added. Among the poisons which cause degeneration of the liver parenchyma and disturb its glycogen function, according to Asher, 13 lymphagogues, like peptone, extract of crab-muscle and leech-extract, are to be included. B E. Freund and H. Popper, Biochem. Zeitschr., 41, 56, 1912. 13 L. Asher and Kusmine (Berne), Zeitschr. f. Biol., ^6, 554, 1905. 15 226 GLYCOGEN There is little occasion, however, for lingering over these essentially obvious features, which take up a large part of the literature of the subject. Briefly stated there is scarcely any form of pathological change in the body which may not, at times, come to involve the reserve supplies of carbo- hydrate which have been stored up when times were good ; but the author confesses he cannot bring very much interest to bear upon the details of these processes. Attention should here be called to the fact that the im- portance of the liver as the place of greatest deposit of glycogen has been much over-estimated in connection with the normal course of carbohydrate metabolism. Dogs can undoubtedly readily assimilate and consume large quantities of carbohydrates, even after the liver has been excluded from the portal circulation, as by the establishment of an Eck's fistula or by transient compression of the portal vein (by a suture carried about the vein and through the abdominal wall) 14 (Cf. Vol. I of this series, p. 296, Chemistry of the Tissues). Formation of Glycogen in the Perfused Liver. — Turning next to the manner in which the body builds up its glycogen it may be said that conclusive information upon the formation of glycogen was reasonably to be expected from perfusion experiments, in which substances believed capable of con- tributing the material for its construction are introduced into the blood used in perfusing the living, excised liver. While at first the procedure required the determination of the glycogen in a portion of the liver before the inception of the experiment and thereafter the perfusion of another part of the organ, Grube 15 has devised a method (based on 14 F. de Filippi (Rome), Zeitsehr. f. Biol., 50. 38, 1908; F. Verzär (Tangl's Lab.), Biochem. Zeitsehr., 34, 52, 63, 1911; E. Wehrle (Basel), ibid., 34, 233, 1911; N. Burdenko (Dorpath), Internat. Beitr. z. Pathol, u. Tlier. d. Ernährungsstörungen, 4, 93, 1912. 15 K. Grube (Pflüger's Lab.), Pflüger's Arch., 107, 483, 1905; cf. opp. H. Serege (Bordeaux) , C. E,. Sbc. de Biol., 57, 000, 1904. 18 K. Grube ( Haliburton's Lab., London), Pflüger's Arch., 101, 590, 1905. FORMATION OF GLYCOGEN 227 the fact 16 that glycogen is uniformly distributed in the hepatic parenchyma proper, and that any slight analytical differences are related with the variations in amount of connective tissue in the portions under examination), which permits in the turtle artificial circulation to be maintained for two completely separated portions of the liver. J. de Meyer 17 has since succeeded in applying an analogous method to the mammalian liver. It is thus possible to per- fuse one lobe of the liver with blood containing sugar and a second with blood free from sugar. We have learned from observations of this type, that a marked new-formation of glycogen takes place in a liver when perfused with blood containing sugar; and the assertion 1S that the liver is able to form glycogen from glucose only after the latter has un- dergone a preparatory polymerization in the course of its resorption in the bowel has been thoroughly disproved. 19 We may go a step further and take up the question of what must be the constitution of a sugar to fit it as material for the formation of glycogen. Based upon an extensive literature, the details of which cannot be taken up here, this question would be answered somewhat as follows at the present. 20 Formation of Glycogen from Glucose, Fructose and Galactose. — Besides glucose, which has naturally occupied the central place in the whole carbohydrate problem, there are two hexoses which are undoubted glycogen-formers r fructose and galactose, the stereo-chemical configurations of which closely approach that of glucose. The capability of a 17 J. de Meyer ( Solvay Instit., Brussels ) , Arch, intern, de Physiol., 8, 204, 1909. 18 A. C. Croftan (Chicago), Pfliiger's Arch., 126, 407, 1909. 19 E. Pflüger, Pflüger's Arch., 126, 416, 1909; K. Grube (Pflüger's Lab.), Pflüger's Arch., 121, 529, 1909. 20 Literature upon Glycogen Formation from Sugars and Related Sub- stances: M. Cremer, Ergebn. d. Physiol., 1' 896-901, 1902; R. Tigerstedt, Nagel's Handb. d. Physiol., 1, 502-503, 1905; E. Weinland, ibid., 2, 433-499, 1907; J. Wohlgemuth, Handb. d. Biochem.. 8', 160-164, 1910; A. Magnus-Levy, ibid., J t ' 323-326, 352-353, 1909; H. Haffmanns, Inaug. Diss., Univ. Berne, 1910, cited in Jahresber. f. Tierchem., 40, 414. 228 GLYCOGEN number of other hexoses, as the mannoses, sorbose and chi- tose, to form glycogen seems, to say the least, questionable. Glycogen formation from the first-named types of sugar was proved even as early as C. v. Voit and his school. They are by no means equally capable, galactose normally being fixed in the form of the reserve carbohydrate with more difficulty than are glucose and laevulose. A fasting, healthy human being can take up as much as 100. to 150. grams of glucose from the stomach at one time without excreting sugar in the urine; the "limit of assimilation" for galactose is very much lower, about 30. to 40. grams. Galactose is assimilated with especial difficulty by Carnivora ; canine urine showing reducing power even when the animal is merely kept on a milk diet, as observed many years ago by F. Hofmeister. When Grube perfused the living excised turtle-liver with Ringer's solution to which was added sugar of different kinds, he noted that large amounts of glycogen were pro- duced from glucose and from fruit sugar but far less from galactose. It is a strange thing that galactose is better assimilated by the human diabetic, according to F. v. Voit, than is glucose, the diabetic patient having lost the capacity of utilizing the latter. The same is true of laevulose, as shown by Minkowski in case of pancreatic diabetes and by L. Pollak 21 in case of adrenin diabetes. From a further in- vestigation (in the Vienna Pharmacological Institute) 22 it would seem that in phosphorus-poisoning the liver, although it has lost its power of forming glycogen from glucose, is still able to freely construct glycogen when laevulose is exhibited. It is impossible thus far to explain this remarkable peculiarity. It might be conjectured that the glycogens formed from laevulose or galactose are in some way different from the form of glycogen from dextrose; that perhaps the glycogen from laevulose would hydrolyse, a L. Pollak (Pharmacol. Instit., Vienna), Arch. f. exper. Pathol., 61, 149, 1909. 22 E. Neubauer (Pharmacol. Instit., Vienna), Arch. f. exper. Pathol., 61, 174, 1909. DISACCHARIDES AND POLYSACCHARIDES 229 not into grape-sugar, but into fruit-sugar. However, a series of special investigations conducted by Pflüger, 23 with this point in view and using glycogen from animals after a diet rich in lasvulose, have failed to give the least foundation for such assumption. The liver and the other organs as well must be looked upon as capable of inverting the polarizing properties of sugars introduced. Behavior of Disaccharides and Polysaccharides. — The facts concerning the disaccharides are fairly well known. Cane-sugar and milk-sugar can be completely assimilated only if they enter the blood from the intestine, that is, after undergoing full fermentation cleavage. When introduced parenterally they pass for the most part unchanged into the urine. Nor are they found capable of forming glycogen when directly perfused through the living, excised liver (vide sup., p. 228). Maltose, however, does not follow the same rule, as the blood and tissues contain "maltases," ferments capable of splitting this disaccharide when introduced parenterally into the circulation; for which reason it is readily assimilable. (Because of the readiness with which maltose undergoes cleavage into dextrose, Murschhausen's statement 24 that this sugar produces much less glycogen than grape-sugar, fruit-sugar and cane-sugar cannot well be understood.) It is not to be understood that the normal body has absolutely no power of splitting parenterally intro- duced cane-sugar. Small amounts of this carbohydrate (one or two grams per kilo) introduced subcutaneously or intravenously into a dog or a cat (as Lafayette Mendell has recently discovered) 25 are not entirely excreted in the urine. Ernst Weinland 26 observed that when cane-sugar was in- jected in large amounts subcutaneously into grown dogs it was usually entirely excreted; but when solutions of this same sugar were injected into young dogs in increasing 23 E. Pflüger, Pfluger's Arch., 121, 559, 1908. 24 Pfluger's Arch., 1S9, 255, 1911. 25 L. B. Mendel and J. S. Kleiner, Amer. Jour, of Physiol., 26, 396, 1910. 30 E. Weinland, Zeitschr. f. Biol., ^7, 279, 1906. 230 GLYCOGEN doses over a longer period of time, the serum would take on inverting properties. This is evidently nothing more than an exaggeration of a property which is already normally possessed, perhaps in analogy to the phenomena noted in the observations of Abderhalden already mentioned (vide sup., p. 218). The same thing is indicated by the studies of Hohlweg and Voit, 27 who noted almost complete elimination after subcutaneous injection of twenty grams of cane-sugar into normal rabbits ; while in animals with their metabolic processes increased by overheating a loss of about twenty per cent, was obtained. That polysaccharides like starch and inulin, which under- go cleavage into glucose or laevulose in the intestine, con- tribute to glycogen formation is self-explanatory. As re- sorption of these substances can take place only after com- plete cleavage has occurred, large amounts may be ingested by human beings without necessarily inducing excessive presence of sugar in the system and without consequent alimentary glycosuria. Other Substances of the Sugar Series. — Statements as to the glycogen forming properties of the pentoses are more or less confusing 28 ; off-hand it must be regarded as very questionable whether they possess such properties. Con- version of the pentoses into glycogen, as a matter of fact, would be possible only in a very round-about way (decompo- sition into molecular groups with two or three carbon atoms). It is rather singular that glucosamine cannot be classed among the glycogen producers when one thinks how close it stands to grape-sugar stereochemically 29 ; the body is evi- "H. Hohlweg and F. Voit (Giessen), Zeitschr. f. Biol., 51, 491, 1908. 28 M. Cremer, Salkowski, Frentzel, Neuberg and Wohlgemuth ; cf . critical review of the Literature; M. Cremer, Ergebn. d. Phyäiol., 1' 898-899, 1902; cf. also L. B. Stookey and A. H. Jones, Proc. Soc. Exper. Biol., 5, 123; cited in Jahresber. f. Tierchem., 38, 446, 1908. 28 Fabian, S. Fränkel and Offer, Cathcart, Bial, Forschbach, K. Meyer, Hofmeister's Beitr., 9, 134, 1907; F. Rogozinski, C. R., 153, 211, 1911. FORMALDEHYDE 231 dently unable to replace its amine group by a hydroxyl group and thus effect its transformation. It is a matter of importance in connection with the question of sugar forma- tion from protein that the amidized sugar group in the protein molecule is not capable of direct and immediate transformation into grape-sugar. We have no proof that any of the alcohols or of the acids of the sugar group (glyconic acid, saccharic acid, glycuronic acid) take part in glycogen formation, which may be easily appreciated when it is recalled that their transformation into sugar presupposes complicated oxidation or reduction processes. Formation of Glycogen from Formaldehyde. — Nor has TIT Grube 's statement 30 to the effect that formaldehyde, | COH, which to all appearances plays an important role in the light- synthesis of sugar in plants, is formed into glycogen when perfused through the surviving liver, remained without contradiction. 31 A synthetic process of this sort would not be very hard to imagine. According to the latest studies of R. Przibram and A. Franke, 32 the action of ultraviolet light rays upon an aqueous solution of formalydehyde is sufficient to produce the " simplest sugar," glycolaldehyde, as well as higher condensation products. The process may be sup- posed to follow the adjacent schema with possible eventual formation of sugar : FORMALDEHYDE GLYCOLALDEHYDE GLYCERINALDEHYDE H | CH 2 .OH COH CH 2 .OH | + — > | > CH.OH ► H COH H | I + I COH + COH COH Limit of Saturation and Utilization. — The subject of as- 30 K. Grube (Bonn), Pntiger's Arch., 121, 636, 1908; 126, 585, 1909; 139, 428, 1911. 81 B. Schöndorff and F. Grebe (Bonn), Pflüger's Arch., 138, 525, 1911. 32 R. Przibram and A. Franke, Sitzungsber. d. Wiener Akad., Mathem. Naturw. Klasse, 71, IIb, Feb., 1912. 232 GLYCOGEN similation limit of the various sugars has attained consider- able importance from studies which have been conducted in F. Hofmeister 's laboratory. 33 Using the same rabbit in a series of injections of varying dosage of sugar into the auricular vein one may determine the dose which the animal can take without glycosuria ensuing in the course of a few minutes. This amount, determined by F. Blumenthal as 1.8 to 2.8 grams for a rabbit, proved repeatedly for the individ- ual animal remarkably constant (within 0.1 gram). These figures, an expression of the ability of the body to take up the sugar and its conversion products from the circulation to a point of saturation, is known as the ' ' saturation limit. ' ' It furnishes a useful means of measuring the actual appropri- ating power of the body for any given time. It is not quite the same thing as the "utilization limit," which is de- termined by finding out by intermittent, graded sugar injec- tions the largest amount which can be borne continuously when introduced regularly at short intervals without ensuing glycosuria. Apparently when the saturation limit has been once reached by a large administration of sugar, a very small continued additional amount is sufficient to maintain a glycosuria. It may be readily seen that an alimentary glycosuria 33a is more likely to occur in an individual whose glycogen de- posits are excessive from previous indulgence in carbo- hydrate food, than in one poor in glycogen; and that muscular labor 34 and over exposure to heat, 35 by increasing the requirement of sugar, raise the limit of assimilation. A number of other agencies which are apt to modify the assimilation limit will be taken up later. 83 F. Blumenthal (F. Hofmeister's Lab., Strassburg), Hofmeister's Beitr., 6, 329, 1905. 33 a Literature upon Alimentary Glycosuria: A. Magnus-Levy, Handb. d. Biochem., //', 323-327, 1909. 34 G. Comessati (F. Hofmeister's Lab., Strassburg), Hofmeister's Beitr., 9, 66, 1907; Grober (Stintzing Clinic, Jena), Deutsch. Arch. f. klin. Med., 95, 137, 1909. 35 H. Hohlweg and F. Voit (Giessen), Zeitschr. f. Biol., 51, 491, 1908. FORMATION OF SUGAR FROM PROTEIN 233 FORMATION OF SUGAR FROM PROTEIN Coming next to one of the great fundamental problems of the study of metabolism, one upon which biologists from the olden times of Claude Bernard to the present have time and again tested their acumen, we have to consider the question of the formation of sugar from protein. Carbohydrate Group in Protein Molecule. — Long after the possibility of separation of a reducing compound from mucinous substances was known, the discovery that a carbo- hydrate group is a natural component of the proteins was made by Pavy. Friedrich v. Müller, the clinician, and his pupils contributed mainly to establish the fact that protein sugar is not identical with glucose or any of the other typi- cal hexoses, but is rather an amidized sugar, glucosamine, CH 2 (OH) - CH(OH) - CH(OH) - CH(OH) - CH(NH 2 ) - COH, obtained also by Ledderhose by cleavage of chitin. 36 While mucins and many mucoids (as the egg envelopes of the frog and of cephalopods) 37 are about one-third made up of glycosamine, and the amount of this material in ovalbu- min, which is especially rich in sugar, is commonly estimated at about ten per cent., in other true proteids a low percentage or only a fraction of one per cent, is present. Many, as case- in, contain no carbohydrate at all. (It is not difficult to de- termine the amount of carbohydrate of a protein if the latter is hydrolyzed by boiling with a mineral acid, by separating from the mixture the substances which can be precipitated by phosphotungstic acid, and estimating the amount of sugar in the filtrate in the usual manner.) The expectation of finding in the carbohydrate group of the protein molecule the key to the problem of forming sugar from protein soon proved fruitless. We were forced to recognize very soon that the quantity of proteid sugar cannot by any means 36 Literature upon the Carbohydrate Group in Protein: L. Langstein, Ergebn. d. Physiol., V , 77-99, 1902; 3', 456-467, 1904; O. Cohnheim, Chemie der Eiweisskörper, 4th ed., 82-89, 1911. "0. v. Fürth (F. Hofmeisters Lab., Strassburg, and Zoological Station at Naples), Hofmeister's Beitr., 1, 252, 1901. 234 FORMATION OF SUGAR FROM PROTEIN suffice to cover even a small fraction of the large amount of sugar which the body under certain circumstances is capable of producing from protein. Before long, too, the previously mentioned (v. sup., p. 231) fact became unexpectedly appar- ent that the body, which, with a readiness that almost smacks of play, performs so many chemical transformations that put to shame the art of the chemist, is unable to bring about the simple substitution of the amine group of glucosamine by a hydroxyl radical; and that for this reason glucosamine cannot be classed among the typical sugar- and glycogen- formers. The logical conclusion was, therefore, that the sugar which in human diabetes, pancreatic diabetes, phlorid- zin-diabetes, etc., to all appearances originated from proteins is not a direct hydrolytic protein cleavage-product, but must be due to some more complicated chemical changes. Elimination of Sugar and Protein Decomposition. — For several decades a bitter controversy was waged upon the question of accepting the reality of sugar being formed from protein. Most prominent of all its opponents, Edward Pflüger, with an obstinacy here as inseparable from the char- acter of this great physiologist as was his earnestness in the search for the truth, never wearied of contesting by one new ingenious argument after another against the doctrine of sugar production from protein ; and yet in the end he was unable to prevent the theory, well founded as it now is, from taking a permanent place in our science. Today the details of this controversy have no more than historical interest; and there is no occasion for us to enter into the matter more fully at this time. It will probably be sufficient to briefly call to mind the steps by which we have come to know that sugar may be formed from protein. For this we are indebted primarily to a long series of metabolic researches upon human diabetes, and, too, upon pancreatic diabetes and phloridzin diabetes in animals, with which are prominently connected the names of Claude Bernard, Külz, Wolfberg, Naunyn, v. Mering, Minkowski, DECOMPOSITION OF PROTEIN 235 Cantani, Bendix, Prausnitz, Cremer, Lüthje, 0. Löwi, Mag- nus-Levy, Graham Lusk, Friedrich Kraus, Mohr, Falta, Gigon, and others. 38 It was proved time and time again that the glycogen supply of the body cannot possibly account for the large quantities of sugar excreted in the urine ; and the many observations upon the relation D | N (Dextrose: Nitrogen) indicate, in a manner which, to the writer's mind, must be convincing to any unprejudiced person, that there exists a relation between sugar formation and protein de- composition. Positive statements were sure to be given after findings like those of Lüthje, 39 who fed a dog whose pancreas had been removed upon carbohydrate-free diet for a protracted period and in the end found that four times more sugar had been excreted by the animal than could have possibly been deposited in the form of reserve carbohydrate (following Pflüger 's maximal figures for the quantity of glycogen in the tissues). Finally even the indefatigable sceptic of Bonn was forced to acknowledge that the sugar produced by a diabetic individual could not possibly come from the glycogen constituent of the body, and that it must arise from some other source which he was unwilling to accept as protein but was inclined to put down as fat. Later on the pros and cons of sugar formation from fat will be taken up ; here it is proper to say only that Pflüger did not continue in this interpretation. He found in dogs, whose supply of glycogen had been much reduced by starvation and phloridzin, and which were then fed upon codfish meat, which is very poor in carbohydrates, such a marked ac- cumulation of glycogen in the liver that he frankly acknowl- edged it as due to the formation of sugar from protein. 40 38 Literature upon Formation of Sugar from Protein : M. Cremer, Ergebn. d. Physiol., 1, 872-887, 1902; L. Langstein, ibid., 1, 62-109, 1902; 3, 453-496, 1904; R. Tigerstedt, Nagel's Handb. d. Physiol., 1, 502-508, 1905; E. Weinland, ibid., 2, 440^142, 1907 ; A. Magnus-Levy, Handb. d. Bioehem., 4', 340-345, 346- 356, 1909; J. Wohlgemuth, ibid., 3', 165-166, 1910. 89 H. Lüthje, Deutsch. Arch. f. klin. Med., 79, 498, 1904. " E. Pflüger and P. Junkersdorf, Pflüger's Arch., 131, 201, 1910. 236 FORMATION OF SUGAR FROM PROTEIN This ended the first act of this eventful scientific drama. All the more honor to the investigator to whom had fallen the role of the "spirit that would not down," that at the close of his arduous life he, as it were, went over to the enemies' camp ; this he did as soon as he was persuaded that there was where the truth lay. May the coming generations forget the acerbities and the many unfriendly words, but not forget that even this controversy was not entirely without value and that Eduard Pflüger is to be honored for what he stood in science, a true seeker after the truth. Respiration Experiments. — The formation of sugar from protein has also been determined, aside from the method of feeding experiments, in another way, by respiration experi- ments. It has been proved that if abundant protein be fed after a previous period of starvation practically all of the nitrogen of the protein employed will appear in the excreta, but a portion of the carbon will remain in the body. ' ' Here the only alternative," says Max Cremer, "unless one is will- ing to take refuge in unknown and hitherto unproven modes of retention, is to think of this carbon as contributing to form either glycogen or fat. To anyone who like myself holds the formation of fat in a strictly synthetic production only as succeeding a prior stage of glucose these experi- ments are thoroughly conclusive of the new formation of glucose. ' ' New Formation of Carbohydrate in Gly cog en-free Tis- sues. — Even the man who is dissatisfied with such observa- tions cannot gainsay the fact of the new formation of glyco- gen in the starving animal. Rabbits can be made entirely devoid of glycogen by being starved for a number of days and then subjected to strychnine convulsions. If such glycogen- free animals are allowed to starve further and are killed at the first sign of the premortal increase of nitrogen elimina- tion (indicative of the complete consumption of the supply of reserve material and that thereafter the protein constituents of the tissues are of necessity being drawn upon), they will SUGAR FORMATION IN GLYCOGEN-FREE TISSUES 237 again be found to contain glycogen, according to Roily. 41 Apparently a portion of the mobilized tissue proteins has been changed into sugar. In the same way new formation of carboh} T drate occurs in glycogen-free animals if an in- crease in protein decomposition be induced by an infectious fever (produced by inoculation with bacterium coli). 42 Pflüger himself proved that mere starvation, as a rule, is incapable of ridding the body of glycogen, because glycogen will be newly formed from substances which are not carbo- hydrates. 43 G. Embden 44 has proved that a marked access of sugar occurs when the living, excised, glycogen-free liver of a dog is perfused, referable to the sugar antecedents in the blood or in the liver ; and M. Löwit 45 has been able to show that the glycogen-free livers of cold-blooded and warm- blooded animals under proper circumstances can form sugar even postmortem (or in course of life after excision), although postmortem glyconeogenesis could not be proved for the blood or other tissues. It is safe to say that here, too, we can tentatively think of a formation of sugar from proteid substances, particularly as we have no ground for assuming the existence of any sort of unknown high mole- cular carbohydrates in the liver different from glycogen. There may be some justification in placing in this same cate- gory certain discoveries referable to the formation of sugar in autolysis, as that of Seegen in which sugar was said to have been formed from peptone in portions of liver, and that of Weinland, who assumed that sugar was produced from protein, supposed to have taken place in a mush of fly-larva? when oxygen was introduced. It should, however, be ex- plicitly stated that the former of these findings has not been "Roily, Deutsch. Arch. f. klin. Med., 83, 107, 1905. 42 C. Hirsch and Roily (Med. Clinic, Leipzig), Deutsch. Arch. f. klin. Med.. 78, 380, 1903. - 3 E. Pflüger, Pflüger's Arch., 119, 117, 1907. ** G. Embden, Hofmeister's Beitr., 6, 44, 1903 ; Biochem. Zeitschr., 6, 66, 1904. "M. Löwit (Innsbruck), Pflüger's Arch., 136, 572, 1910. 238 FORMATION OF SUGAR FROM PROTEIN confirmed, 46 and that Weinland 's work, 47 to the author's mind, should be repeated by a number of different methods before final credence can be given it. 48 Formation of Sugar from Various Proteins. — Consider- able work has been expended upon the question of the rela- tion of various proteins to the amount of sugar which they are capable of producing in metabolism, but with little other result, as far as the writer can observe, than the fact that the quantity of carbohydrate preformed in the proteid sub- stances (glucosamine) is by no means striking. Quite re- cently experiments have been repeated in M. Cramer's laboratory showing in comparative feeding tests on a phlor- idzin dog with meat and casein (casein is a carbohydrate- free protein) a difference in favor of casein. 49 The amounts of sugar which can be produced from protein (based on calculation of the D|N relation) have been given in highly varying proportion ; incidentally it was calculated that after feeding meat half of the energy of the protein can take on the form of sugar and can be stored as glycogen for future use. Such statements cannot, however, as yet be regarded as final. 50 Origin of Sugar from Aminoacids. — Having clearly be- fore us the facts of sugar formation from protein, we may proceed to the question of the chemistry of this process. Explanation of the mechanism of sugar production from protein has been sought by administering the individual "building stones" of protein, the aminoacids, to dogs with pancreatic diabetes and phloridzin diabetes, and determin- ing their influence upon sugar elimination and glycogen formation. Investigations with this in view (particularly itt Cf. L. Langstein, Ergebn. d. Physiol., 1', 108, 1902. 47 E. Weinland, Zeitschr. f. Biol., W, 421-466, 1907. 48 0. Krummacher and E. Weinland, Zeitschr. f . Biol., 52, 273, 1909. 49 P. Rohmer (M. Cremer's Lab.), Zeitschr. f. Biol., 54, 455, 1911. 50 Investigations by Külz, Lusk, Halsey, Lüthje, Bendix, Berger, Lehmann, Schumann-Leclerc, Mohr, Falta, Therman, G. Müller and others. Literature: P. Rohmer, 1. c. ORIGIN OF SUGAR FROM AMINOACIDS 239 those of Röhmann, R. Colin, Nebelthau, Neuberg and Lang- stein, Knopf, Halsey, F. Kraus, Enibden and Salomon, Glässner and E. Pick and Graham Lusk) 51 have led to the conclusion that sugar production from aminoacids, at least from glycocoll, alanin, asparaginic acid and glutaminic acid, cannot well be doubted. The conceptions framed to explain the process are entirely hypothetical. The synthesis of sugar may possibly take place through the formation of sub- stances containing one, two or three carbon atoms, as formaldehyde, glycolaldehyde and lactic acid, the fitness of which for taking part in direct construction of sugar is more or less to be expected. The author personally is more sympathetically attracted to such a conception as that which supposes leucin to be decomposed into two triple-carbon compounds 52 to be synthesized into sugar, than to any idea of a "stretching" of the branched LEUCIN LACTIC ACID GLUCOSE CH3 .CH3 \/ C H CH, CH 2 = > 2CH.OH — — > CeH^O« CH.NH 2 COOH COOH six-carbon chain of leucin to bring about its transformation into sugar. Ringer and Graham Lusk 53 by the employment of an excellently adapted method succeeded in producing in dogs a very uniform glycosuria ; and, after the D | N ratio in the urine had become constant, fed aminoacids to the animals. By measuring the resultant increases of sugar excretion they concluded that glycocoll and alanin can be all utilized in formation of sugar, but of the four carbon atoms of 51 Literature : J. Wohlgemuth, Handb. d. Biochem., 3', 165-166, 1910. 63 Cf. L. Langstein, Ergebn. d. Physiol., 3', 473, 1904. 53 A. J. Ringer and Graham Lusk (Cornell Univ.. New York), Zeitschr. f. physiol. Chem., 66, 106, 1910; Jour. Amer. Chem. Soc, 32, 671, 1910; cited in Centralis, f. d. ges. Biol., 10, No. 2348. 240 FORMATION OF SUGAR FROM FAT asparaginic acid and of the five of glutaminic acid presum- ably only three are available as material for sugar. The following schema of these authors may perhaps prove useful as guides in further investigations : GLYCOCOLL GLYCOLALDEHYDE GLUCOSE 3 CH 2 .NH 2 ^ 3 CH 2 .OH C6H 12 6 COOH COH — >■ ALANIN LACTIC ACID GLUCOSE CH 3 1 CH 3 O r^TT «~»TT 2 CH.NH 2 >■ '»w CeHi 2 Oe Z UH.Uxl COOH p COOH ASPARAGINIC ACID /3-LACTIC ACID GLUCOSE COOH 2 CH 2 l )- COOH 2 CH 2 C6Hi 2 8 CH.NH 2 COOH CH.OH >■ GLUTAMINIC ACID GLYCERINIC ACID GLUCOSE 2 COOH CH 2 CH 2 CH 2 .OH 1 ^>. 1 2 CH OH CaHi 2 Oe CH.NH 2 COOH ^ COOH FORMATION OF SUGAR FROM FAT Having adjusted our ideas as best we can in the matter of the production of sugar from protein, our attention should next be given the difficult problem of the formation of sugar from fat. It should primarily be understood that we are dealing here not with a single problem but with two. Fat is made up of two components (glycerine and higher fatty acids), and it is essential to clearly distinguish the production of sugar from glycerine from that involving the fatty acids. Formation of Sugar from Glycerine. — As far as the first of these items is concerned, we can be very brief. Experi- ments have been frequently made in which glycerine has been SEEGEN'S EXPERIMENTS 241 administered to diabetic human beings and animals, which have proved beyond doubt that this substance is a true sugar- and glycogen-producer. 54 . This was to have been expected when one recalls that Emil Fischer by condensation of the glyceroses, resulting by simple bromoxidation from glycerine, directly obtained sugar (i-fructose) : 55 CH 2 .OH CHj.OH CH.OH + CO COH . CH 2 .OH CH 2 .OH CH.OH CH.OH CH.OH CH,.OH. However, the fact that glycerine constitutes but a small part (about one-tenth) of the molecule of fat, indicates that the difficult part of the general problem is not in this, but in the question of the production of sugar from the higher fatty acids. 56 Let us therefore at once consider the fundamental facts which have been evolved for this latter. Seegen's Experiments. — Seegen's conception of a large volume of sugar, originating from protein and fat decom- position, passing from the liver by the hepatic vein and permeating the body, has not withstood criticism any more than his statements (supported by Weiss) of the existence of a process of an autolytic new-formation of sugar in bits of liver tissue digested with fat and soaps. 57 These matters are now all relegated to the past. 54 Van Deen, Lucksinger, Weiss, Salomon, Külz, Frerichs, Cremer, Lüthje. Literature upon the Formation of Sugar from Glycerine: M. Cremer, Ergebn. d. Physiol., 1' 888-890, 1902; J. Wohlgemuth, ibid., 3', 167, 1910. 66 Arranged without reference to the stereochemical configuration. 58 Literature upon the Formation of Sugar from the Higher Fatty Acids : M. Cremer, Ergebn. d. Physiol., 1, 888-895, 1902; A. Magnus-Levy, Noorden's Handb., 1, 178-181, 1906; Handb. d. Biochem., 4' 345-346, 1909. 57 A. Montuori, Ber. d. Acad. Neapel, 1895, cited in Handb. d. Biochem., 3', 167, 1910; M. Jacoby (Salkowski's Lab.), Virchow's Arch., 157, 255, 1897; E. Abderhalden and P. Rona, Zeitschr. f. physiol. Chem., l t l, 303, 1904. 16 242 FORMATION OF SUGAR FROM FAT Respiratory Quotient in Diabetes. — Attempts have also been made to prove that sugar is produced from fat from a study of the ratio of the respiratory quotient in diabetes. It is well known that the respiratory quotient, the ratio between the output of C0 2 and the intake of oxygen, wheu combustion of carbohydrates alone is in process, is ?&=if because the different sugars contain H and in the proportions of water and necessarily no oxygen is needed to oxidize the hydrogen into water. But as this is involved to a marked degree when fat (very poor in oxygen) is burned, the respiratory quotient falls to 0.7 in the actual consumption of fat. The portion of oxygen intake which is employed in the oxidation of H into water of course does not appear as C0 2 . In the combustion of protein the quotient is about 0.8. What may be expected, however, if in a diabetic there takes place transformation of higher fatty acids into sugar and this be excreted as such? As there must of necessity be a number of CH 2 complexes reformed into CH.OH., a considerable amount of oxygen will be re- quired ; and, as the new-formed sugar is not consumed but is excreted, the intake of oxygen cannot at all coincide with the C0 2 output. It is evident, therefore, that a marked lowering of the respiratory quotient must manifest itself. Magnus-Levy has calculated that the respiratory quotient will of necessity fall to 0.6 or lower under such conditions ; but even in diabetes of severe type he found an actually higher proportion, while Pflüger, taking the same observa- tions as his basis but other methods of calculation, actually obtained in the case of a severe diabetic a quotient of almost exactly 0.6, and believed that this proved beyond doubt that fat is a source of sugar. 58 Unfortunately it must be acknowledged that the elements, which have been basically assumed for calculation, are not as yet established with the 68 A. Magnus-Levy, Verhandl. d. physiol. Ges. Berlin, March 1, 1904; Centralbl. f. Physiol., 18, 373, 1904; Zeitschr. f. klin. Med., 56, 83, 1905; consult therein the Literature. E. PMger, Pfliiger's Arch., 108, 473, 1905. HIGHER FATTY ACIDS 243 certainty which is essential for precision in a physical ex- periment of this sort. Sugar Nitrogen Quotient. — Further evidence of the for- mation of sugar from fat has been recognized in observations upon the sugar-nitrogen quotient, D | N. In many cases of severe diabetes in man and the lower animals such large amounts of sugar, in comparison with the nitrogen output, have been met that protein decomposition does not seem sufficient to explain the sugar production ; and it has seemed impossible not to refer it in part at least to formation from fat. 59 The possible correctness of this mode of interpreta- tion cannot be well denied; but such observations cannot readily be accepted as complete proof, 60 particularly because fundamentally in these conclusions we have to take into consideration the tacit assumption that the nitrogen output at a given time is invariably regarded as a correct indicator of the protein decomposition going on in the body. O. Löwi 61 has, however, called attention to the important point that this assumption does not always obtain in full. Nitrogen retention in the body may exist at times, and it is possible that the protein decomposition may be actually greater than the coincident nitrogen elimination would indicate. Fat Impoverishment in Phloridzin Animals. — Kolisch's observation that white mice in chronic phloridzin intoxica- tion (in comparison with control animals) show extensive impoverishment of fat, is distinctly interesting, but not at all conclusive from this standpoint. 613 Influence of the Introduction of the Higher Fatty Acids Upon Sugar Elimination. — It might be supposed that the 69 Observations by Rumpf, Lüthje, Hartogh and Schumm, Rosenquist, Mohr, Hesse. Junkersdorf, Pfltiger's Arch., 137, 269, 1910, and of v. Noorden's school. 80 Cf. criticism of above by F. v. Müller, Landergren and Magnus- Levy, Literature: A. Magnus-Levy, v. Noorden's Handb., 2d ed., 1, 178, 1906, and Handb. d. Biochem., k ', 345, 1909. 61 O. Löwi (Lab. of H. H. Meyer, Marburg), Arch. f. exper. Pathol., 47, 68, 1902. OTa Kolisch, Wiener, klin. Wochenschr., 19, 559, 1906. 244 FORMATION OF SUGAR FROM FAT most simple and direct means of settling the question of the production of sugar from the higher fatty acids would be by determining whether on introduction of a large quantity of such substances into a diabetic the excretion of sugar would be increased. In the great majority of observations with this point in view, not only has such increase been missed as a matter of fact, but in addition in a number of cases there has been a noted lowering of the sugar excretion after ad- ministration of fatty acids (interpreted on the supposition that the combustion of the latter serves to protect the protein from undergoing destruction and in this way reduces the formation of sugar from the protein). 62 It would, however, be another mistake to regard such negative results as a satis- factory proof that formation of sugar from the higher fatty acids is impossible. Reference may be made here to the interpretation of A. Magnus-Levy, 63 whose perspicaciously conducted investigations have distinctly advanced the physiology of metabolism in so many ways, upon these points: ''In contrast to the conditions prevailing with varying introduction of protein, where as a matter of fact every addition of protein occasions a corresponding increase in protein exchange, the most marked addition of fat in- creases but little the fat exchange. Fat does not in any sense displace other food from metabolism. In the starving dog it replaces the previously consumed body fat; is con- sumed in its place. An excess is deposited almost in its total amount without essentially increasing metabolism. . . . We can also subscribe to the statement here outlined in this, that fat as the most passive of all the foodstuffs always takes part in the combustion process after all the other foods, after protein, the carbohydrates and alcohol, and takes part only as far as an existing demand is not 62 L. Mohr (F. Kraus's Clinic, Berlin), Zeitschr. f. exper. Pathol., 2, 463, 481, 1906; E. Pflüger, Pfliiger's Arch., 108, 115, 1905; S. Bondi and E. Rud- inger, Wiener klin. Wochenschr., 19, 1029, 1906; F. Maignpn, Jour, de Physiol., 10, 866, 1908; cf. therein the older literature. 63 A. Magnus-Levy, Handb. d. Biochem., k' 343-344, 1909. CONCLUSIONS AS TO ORIGIN FROM FAT 245 satisfied by some other material. Other things being equal it is always easy to force the other three foods into metab- olism by increasing their assimilation ; but this is impossible in the case of fat. Increased combustion of fat occurs in the diabetic animal, which is kept on protein and fat alone, only when protein is withdrawn. As a given degree of lowering of protein exchange is followed by a decrease in sugar, so some degree of increase of sugar elimination would be entirely covered after increased fat combustion." In this connec- tion it may be stated that (as in a case of very severe diabetes in v. Noorden's clinic 64 ) after administration of large amounts of fat an enormous sugar elimination has been ob- served, the sugar-nitrogen ratio (D|N) reaching the extra- ordinary height of 10. Falta, in his valuable efforts to es- tablish the rules of sugar elimination in diabetes mellitus, came to the conclusion that many facts of the pathology of metabolism are entirely inexplicable without the assumption that sugar may be formed from fat, and that the evidence in favor of this has been distinctly enlarged by his own and his associates ' investigations. 65 The question of sugar formation from fat today is in about the following status : that it has not been absolutely proved, but has on the other hand not been disproved or even become improbable. Magnus-Levy 66 very properly suggests that the body, as shown by all experiments in spon- taneous and experimental diabetes, has a relentless need for carbohydrates which it tries to satisfy under all circum- stances. For this end the carbohydrate supply in the body first comes in question; thereafter the formation of sugar from protein ; and only in the third place the formation of sugar from fat. If, however, the position be taken (and 84 S. Bernstein, C. Bolaffio, v. Westenrijk (v. Noorden's Clinic, Vienna), Zeitschr. f. klin. Med., 66, Heft. 5/6, 1908; cf. also W. Falta and A. Gigon, Zeitschr. f. klin. Med., 65, 326, 1908. 65 W. Falta (v. Noorden's Clinic), Zeitschr. f. klin. Med., 66; separate, p. 7, 1908. M A. Magnus-Levy, Noorden's Handb., 2d ed., 1, 179, 1906. 246 FORMATION OF SUGAR FROM FAT doubtless there may be much said for it) that the body can execute its manifestations of energy (in a certain sense, render its cash payments) only on a carbohydrate stand- ard, the assumption of sugar formation from fat is still asserted even if not in so many words ; for it can scarcely be questioned that in the starving animal the performance of work must draw upon its supplies of fat, or that a human being in a long continued fever, or a hibernating marmot, undoubtedly sustains the output of energy for the most part at expense of its body fat. If in such examples the direct source of mechanical and thermic energy were to be refer- able to the combustion of sugar alone, the conclusion must be that a great part of this sugar must come from fat. The problem of sugar formation from fat therefore falls in a certain sense in the same class with that of the sources of energy of the living body, and any one who adheres to Zuntz's belief (v. Vol. I of this series, p. 159, Chemistry of the Tissues) in the dynamic equivalence of the three main classes of foods, will necessarily refuse to acquiesce in the last mentioned argument. There is no doubt that in the plant in the germination of its oily seed (where the reserve substances in the cotyledons and in the endosperm furnish the material for the growth of the embryonal plant) there takes place a transformation of fat into carbohydrate to a very great degree. The fat that is disappearing from the reservoirs of reserve substance actually forms the material from which the cell-walls of the young plants are built up. But it is perhaps not apposite to apply facts which have been discovered in plant physiology to animal metabolism. 67 "Literature upon the Behavior of the Fat in Germination of Oil-bearing Seeds: O. v. Fürth, Hofmeister's Beitr., 4, 430, 1903. CHAPTER XI PANCREATIC DIABETES— HUMAN DIABETES PANCREATIC DIABETES The preceding lectures have introduced the fundamental facts of carbohydrate metabolism well enough to permit us to venture into one of the most interesting but at the same time one of the most difficult problems in the study of metab- olism, that of pancreatic diabetes. In the current of the sciences, just as in the life of man, there are periods when every good intention and the most honest effort are insufficient to make any decisive and pro- ductive progress possible; evil days when ability is com- pelled to employ a good part of its innate energy to keep from sinking into dejected inefficiency. Then all of a sud- den some new event takes place, changes the situation of affairs and brushes aside the impediments which have op- posed the free development of the long accumulated latent energy. And at once a period begins of heightened, feverish activity that endeavors to make up for all that was missed in the dull times of stagnation. Discovery of Pancreatic Diabetes. — One of these for- tunate events occurred in the development of metabolism study when in 1889 Oskar Minkowski and Josef v. Mering discovered pancreatic diabetes in Naunyn's laboratory in Strassburg. 1 At the same time, and independently of the authors just named, N. de Dominici, in Naples, also discovered the ex- istence of pancreatic diabetes (an example of the strange law of the doubling of events which often has appeared even in physiology, probably because a discovery cannot be made 1 Literature upon Pancreatic Diabetes: O. Minkowski, Ergebn. d. Pathol., 1, 69, 1896; C. v. Noorden, Handb. d. Pathol, d. Stoffwechs., 2d ed., 2, 38-43, 1907; A. Biedl, Innere Sekretion, pp. 375-399, 1910. 247 248 PANCREATIC DIABETES before the times are ripe for it, and in a sense it is in the air). Although it must be reluctantly confessed (for that mat- ter it would be recognized all too soon even without such a confession) that to-day, after almost a quarter of a century has elapsed since this discovery, we are not in position to offer a satisfactory explanation of the real nature of pan- creatic diabetes, yet it is impossible to mistake the fruitful influence which even this single possible means of artificially producing a metabolic disturbance analogous to human dia- betes has had on the whole field of physiology and pathology of metabolism. Interrupted Extirpation of the Pancreas. — It is well known that extirpation of the whole of the pancreas is requi- site to produce a typical pancreatic diabetes, and that the preservation of a small portion of the gland will suffice to prevent this metabolic fault. If the bulk of the gland is re- moved and the rest located subcutaneously diabetes does not ensue, but will promptly follow with all its symptoms upon subsequent removal of the transplanted portion. The fact that it is possible to cause a diabetes of the severest form by a trivial interference requiring but a few minutes for its com- pletion, in which the peritoneal cavity is not opened at all and in which there can be no question of irritation of the system of peritoneal nerves, eliminates all objections, as Minkow- ski 2 properly suggests, to the belief in a direct relation between diabetes and loss of pancreatic function. An inter- rupted process, recently suggested by Hedon, 3 makes the ex- tirpation of the pancreas in the dog, which is by no means easy of technic, decidedly less difficult. It is best in carrying out the procedure to first remove only the gastrosplenic portion of the gland and to transplant the lower part of the tail of the organ with its nervo-vascular pedicle under the 2 0. Minkowski, Pflüger's Arch., Ill, 13, 1906. 3 E. Hedon (Montpellier), Arch, intern, de Physiol., 10, 350, 1911; cf. therein Literature. FUNCTION OF THE ISLANDS OF LANGERHANS 249 skin, with the intention of taking it away some time later, after recovery is assured. It seems of much importance for the success of the operation that the separation of the head of the pancreas from the wall of the duodenum be properly performed. Hedon strongly advises that the gland be torn away from the bowel wall and the latter curetted ; a tedious stoppage of hemorrhage by ligature is avoided, a more thorough extirpation is accomplished, and necrosis of the intestine prevented, which is the principal point. A very severe diabetes is invariably thus produced, set- ting in after completion of extirpation and lasting until the death of the experiment animal (two to four weeks later). A diabetes thus produced proceeds in typical fashion with symptoms of hyperglycemia, polyphagia, polydypsia, poly- uria, wasting and acidosis. After partial extirpation a dia- betes of less marked severity may be induced, with a course of perhaps many months' duration, which if total extirpa- tion be completed later on will at once change to a severe type. By transplanting pancreatic tissue into the spleen 4 the duration of life of a dog with pancreatic diabetes has been successfully lengthened to a very distinct degree. The statement of Chauveau and Kaufmann that the re- sult of pancreatic extirpation may fail to appear if the cervical cord be divided has not been confirmed. Even if this be done and in addition the vagus and sympathetic nerves be cut, thus completely excluding influences from the cerebral centres, the glycosuria, according to Hedon, appears. 5 Function of the Islands of Langerhans. — In the literature upon pancreatic diabetes a very large part is devoted to experiments which have as their purpose determination whether the function of the organ concerned with carbo- hydrate metabolism is connected with the secreting paren- 4 Martina, cited by Jacoby, Einführung in die experimentelle Therapie, p. 136, 1910. B E. Hedon (Montpellier), Arch, intern, de Physiol., 11, 195, 1911. 250 PANCREATIC DIABETES chyma of the pancreas or with the ' ' islands of Langerhans. ' ' B. A. Kohn classes the islands of Langerhans along with the parathyroids, the epithelial part of the hypophysis and the cortex of the suprarenal glands, among glands without duct or glandular lumen. Here it may be sufficient to state as a matter of reference that U. Lombroso 6 after sifting the general material (col- lected experiments with ligature of the ducts, artificially induced atrophy of the parenchyma, pathological observa- tions and studies in comparative anatomy), came to the conclusion in a monograph upon the subject that both forms of epithelial tissue of the pancreas, acini and islands, take part in internal secretion. Swale Vincent, 7 in a recent critical review, says: " Whatever may be found out in the future in regard to the true function of the islands of Langerhans, their essential anatomical relation with the zymogenous tubules, the many transition forms met in all vertebrate genera, and the transformation of acini into islands and vice versa, apparently prove conclusively that the islands are not sui generis, but that they are integral constituents of the ordinary pancreatic tissue. Whether the temporary transformation into insular tissue means a particular specialization of function, our present evidence does not indicate. ' ' In the author's personal opinion, it can no longer honestly be doubted, in view of the recent investi- gations of Weichselbaum (vide infra), that the anatomical cause of diabetes is to be sought in lesions of the islets. Duodenal Diabetes. — Some years ago Pflüger took occa- sion to use the observation that section of the intramesen- teric nervous connections between the pancreas and duodenum in the frog may under certain circumstances give rise to a glycosuria, as a basis for calling into question the existence of a pancreatic diabetes, and for substituting for the latter a "duodenal diabetes." The whole discussion 6 U. Lombroso, Ergebn. d. Pbysiol., 9, 1-89, 1910. J Swale Vincent, Ergebn. d. Physiol., 9, 489-495, 1910. DUODENAL DIABETES 251 growing therefrom has proved practically fruitless, and was basically altogether superfluous as the above stated observa- tions of Minkowski and his numberless followers 8 permitted but one interpretation. But it served to show again that if a single bit of pancreas completely detached from its nerv- ous comunications be left it will suffice to prevent the appear- ance of glycosuria. The whole duodenum can be excised from a dog, as Minkowski had already shown, without pro- ducing diabetes, provided a portion of the pancreas has been transplanted under the skin ; but if the latter at a later time be removed from the animal the diabetes promptly sets in. Strictly speaking nothing at all has been gained in this whole campaign fought with an array of heavy artillery of au- thority and between strategists of note, except the reestab- lishment of a fact that has been known long before, that of the influence of nervous irritations of all sorts (corrosion of the duodenum or ileum, injection of nicotine into the stomach, chilling) to cause the liver to discharge its supply of carbo- hydrate and sometimes give rise to glycosuria. 9 We know conclusively that pancreatic diabetes is possible not only in many mammalia, but also in birds, turtles, frogs and fishes, and that in those instances in which a true glycosuria has not been found (as in many birds and in sharks) there may be noted at least a distinct hyper- glycemia. 10 We may apparently, therefore, regard the s Lepine, H6don, Gley, Thiroloix, Caparelli, Harley, Schabad, Cavazzani, S'andmeyer, Selig, Rumbolt and others; ef. Literature in A. Biedl, 1. c. ; also E. Hedon, Jour, de Physiol., l-' t , 907, 1912. 9 E. Pflüger, Pflüger's Arch., 106, 181, 1905; 118, 265, 267, 1907; 119, 227, 297, 1907; 122, 267, 1908; 123, 323, 1908; 124, 1, 529, 632, 1908; 128, 125, 1909; R. Gaultier, C. R. Soc. de Biol., 64, 826, 1908; A. Herlitzka, Pflüger's Arch., 123, 331, 1908; M. Löwit, Arch. f. exper. Pathol., 62, 47, 1908; U. Lom- broso, Arch, di Farmac, 9, 146, cited in Jahresber. f. Tierchem., 40, 811, 1910; O. Minkowski, Arch. f. exper. Pathol., 58, 271, 1908; S. Rosenberg (N. Zuntz's Lab.), Biochem. Zeitsehr., 18, 956, 1909; Pflüger's Arch., 12, 358, 1909; H. Eichler and H. Silbergleit, Berliner klin. Woohenschr., 1908, 1172; W. Tscherniachowski, Zeitsehr. f. Biol., 53, 1, 1909; A. Visintini (Pavia), Med. Kiin., 1908, 1613 ; E. Zack, Wiener klin. Wochenschr., 1908, 82. 252 PANCREATIC DIABETES existence of pancreatic diabetes as established beyond peradventure. Does the Internal Secretion of the Pancreas Pass with the Lymph Through the Thoracic Duct into the Blood? — We may, however, proceed farther and take up the question whether the presence of an internal secretion influencing the carbohydrate metabolism can in any manner be directly recognized in the blood. At the outset an interesting fact observed by Biedl n may be mentioned, namely, that in dogs in which the thoracic duct is tied or opened through a fistula, glycosuria will usually occur. This fact has been verified in Gottlieb's laboratory, 12 and has been supplemented, too, by clinical observations of the occasional coincidence of chyluria and glycosuria. 13 Naturally one feels a strong temptation to interpret these observations on the hypothesis that the lymph duct is carrying to the blood, a secretion originat- ing in the pancreas the cessation of which occasions the glycosuria; but it cannot be settled offhand whether the glycosuria may not have been induced in some very differ- ent way in such cases (as by nervous irritation, etc.). Blood Transfusion and Parabiosis. — Besides there is at least a chance that this "internal secretion" of the pancreas does not gain entrance into the general circulation indirectly by way of the lymph passages, but directly into the blood ves- sels. It is true the blood of the pancreatic vein has shown no influence upon sugar elimination in a dog with the pan- creas removed. 14 But when Hedon united two dogs by anastomosing their carotid arteries, one deprived of its 10 W. Kausch (Naunyn's Clinic), Arch. f. exper. Pathol., 37, 274, 1896; M. Löwit (Innsbruck), ibid., 62, 47, 1909; V. Diamara (Siena), Arch. Ital. de Biol., 55, 94, 1911; cf. therein the Literature. U A. Biedl, Centralbl. f. Physiol., 1898, 624; Biedl and Th. Offer (R. Pal- tauf's Lab.), Wiener klin. Wochenschr., 1901, 1530. 12 J. L. Tuckett, Jour, of Physiol., 41, 88, 1910. 13 A. Magnus-Levy, Zeitschr. f. klin. Med., 66, 4S2, 1908; 67, 524, 1909. 11 A. Alexander and R. Ehrmann (Berlin Pathol. Instit., Dept. Exp. Biol.), Zeitschr. f. exper. Pathol., 5, 367, 1909. HEPATIC FUNCTION IN PANCREATIC DIABETES 253 pancreas and diabetic and the other a normal animal, and procured thorough commingling of the blood of the two by "crossed carotid transfusion," there was occasioned in the normal animal only a transitory glycosuria, while in the dog deprived of its pancreas (the experiment being con- tinued a sufficient length of time) the glycosuria disap- peared sometimes but returned after the carotid anasto- mosis had been separated. 15 It is true these findings are neither constant nor entirely convincing, because a low- ering of the renal secretion takes place in connection with them. Forschbach, in Minkowski's Clinic, was more for- tunate in bringing about a blood mixture by an ingenious expedient by which he joined surgically a normal dog and one with pancreatic diabetes into one parabiotic double animal. In this experiment there not only occurred a low- ering and actual disappearance of the glycosuria in case of the animal with pancreatic diabetes, "but the condition of the animals scarcely differed from that of health, and the disappearance of cachexia suggests the idea that the diabetic metabolic fault was attacked at the root." 16 Basically con- sidered one can readily appreciate that in such a pair of animals the pancreas of the normal individual with its in- ternal secretory function is at the disposition of the partner without a pancreas, with the effect that the resultant symp- toms are not necessarily manifested. According to Carlson the internal secretion can apparently pass in pregnancy from the foetus into the blood of the mother. 17 Formation of Glycogen, Diastasic Power and Formation of Sugar from Carbohydrate-free Material in the Liver in Pancreatic Diabetes. — After complete extirpation of the pan- 15 E. Hedon (Montpellier) , C. R. Soc. de Biol., 66, 699, 1909; 67, 792, 1909; 68, 341, 1910; 72, 584, 1912; Rev. de MeU, 30, 617, 1910. "J. Forschbach (Minkowski's Clinic, Greifswald), Deutsch, med. Wochenschr., 1908, 910; Arch. f. exper. Pathol., 60, 131, 1909; cf. also E. Pflüger, Pflüger's Arch., 12k, 633, 1908. 17 A. J. Carlson and F. M. Drennan (Chicago), Amer. Jour, of Physiol., 28, 391, 1912. 254 PANCREATIC DIABETES creas there is always seen after a time a marked impoverish- ment of glycogen in the canine liver. The muscles, especially the heart muscle, always retain their glycogen supply more obstinately than the liver. In contrast to this loss of glycogen from the large depots a striking glycogenic infil- tration, according to Ehrlich, may be noted in the leucocytes ; which is to be interpreted as a carbohydrate engorgement of these cells caused by the enrichment of the blood serum with sugar. When we consider the dominant position of the liver in carbohydrate metabolism we cannot be far wrong in relating the real nature of pancreatic diabetes with a disturbance of the glycogen function of the liver. Actually what does the inability of the liver to fix glycogen in pancreatic diabetes (Naunyn has coined the term dyszooamylia for the condition) mean? Primarily it should be noted that the dyszooamylia applies only to dextrose, not to hevulose, Minkowski having shown that formation of glycogen from the latter substance may con- tinue without disturbance even in the severest pancreatic diabetes. According to the investigations of J. de Meyer 18 it would appear that the surviving liver of a dog with pancreatic diabetes may be found capable of storing glyco- gen provided pancreatic extract is added to the perfusing fluid. However, we can by no means assert an absolute in- ability of the liver to form glycogen in individuals with pancreatic diabetes, as Nishi 19 (in a research under the direction of 0. Löwi) has been able to show that in turtles with pancreatic diabetes the formation of glycogen is the same as in normal turtles when the liver is perfused with Ringer's fluid containing glucose. Here, too, matters are by no means simple. It is possible that an interpretation of the following kind 18 J. de Meyer (Instit. Solvay, Brussels), Arch, intern, de Physiol., 9, 1, 1910. 18 M. Nishi (Pharmacol. Instit., Vienna), Arch. f. exper. Pathol., 62, 170, 1910. GLYCOLYSIS 255 might be proposed: that the liver itself is not actually in- capable of building up glycogen in pancreatic diabetes, but that an increased activity of the hepatic diastasic ferment (normally kept in due bounds by the internal secretion of the pancreas) makes it impossible that the glycogen be re- tained. But apparently after extirpation of the pancreas the amount of diastase in the blood undergoes reduction. 20 Statements indicating that the diastasic power of the liver is increased in diabetes 21 are contradicted by other com- pletely negative results. 22 So there is no outlet apparent in this direction. Pflüger noticed that in dogs with pancreatic diabetes, with marked emaciation and their fat largely lost, the liver in contrast with other organs had distinctly increased in weight. This suggests that the hepatic function here is not depressed, but that the organ really is working under abnormal stress to form out of carbohydrate-free material the large amounts of sugar which appear in the urine. Yet in this connection it may be noted that in Embden's laboratory in the artificially perfused liver of the depancreatized dog, practically free of glycogen, the new formation of sugar proved to be no greater than in an organ freed of its glycogen by work or by strychnia convulsions. 23 For the present, therefore, we possess no satisfactory explanation for the mystery of pancreatic diabetes either in the formation of glycogen in the liver, its diastasic power, or in its ability to produce sugar from carbohydrate-free material. Glycolysis. — The hypothesis framed by Lepine seems much more attractive. The pancreas is supposed by him 20 W. Schlesinger (Vienna), Deutsch, med. Wochenschr., 1008, 593; L. K. Goult and A. J. Carlson (Chicago), Amer. Jour, of Physiol., 29. 165, 1911. 21 A. Hinselmann (Med. Clinic, Heidelberg), Zeitschr. f. physiol. Chem., 61, 265, 1909. 22 1. Bang, Hofmeister's Beitr., 10, 320, 1907 ; F. A. Bainbridge and A. P. Beddard, Biochem. Jour., 2, 89, 1907; cf. also O. J. Wynhausen (Amsterdam), Berlin, klin. Wochenschr., 1910, 2107. 23 L. Lattes, Biochem. Zeitschr., 20, 215, 1909. 256 PANCREATIC DIABETES normally to produce a glycolytic ferment, which is missing in the depancreatized animal, for which reason the animal is unable to bring the sugar to its usual catabolism ; the sugar, therefore, accumulates in the blood and thence passes into the urine. The original idea in this theory which would seek to place glycolysis normally in the blood, has been prac- tically altogether given up ; our present conception is that the sugar undergoes its destructive changes for the most part in the fixed tissues, not in the blood. Otto Cohnheim 24 believes he has evidence from studies upon the expressed juices of tissues that muscle contains an enzyme which is capable of inducing combustion of grape- sugar to carbonic acid ; which however does not exist in the muscle in active form for the most part, but requires for ac- tivation a material arising in the pancreas and passed thence into the blood stream, a "pancreatic activator." Occasion will be taken later, in summing up our conclusions upon gly- colysis, to discuss the objections which have been made to Cohnheim 's experiments. In view of the undoubtedly very great difficulty of posi- tively excluding interference from bacterial processes in experiments with expressed juices, it is a matter for special congratulation that certain recent comparative experiments upon the consumption of sugar by the normal and by the diabetic heart by one of the best of our living biological experimenters, E. H. Starling, 25 are available. By an in- genious experimental mechanism he has succeeded in arranging a heart and lung preparation 26 so as to keep a dog's heart beating for hours, working at normal arterial pressure and maintaining the normal amount of blood in 24 O. Cohnheim, Zeitschr. f. physiol. Chem., 39, 396, 1903; 42, 401, 1904; 41, 253, 1906. 25 F. P. Knowlton and E. H. Starling (Univ. College, London), Centralbl. f. Physiol., 26, 169, 1912; Jour, of Physiol., 45, 146, 1912. * Cf. E. Jerusalem and E. H. Starling, Jour, of Physiol., 40, 299, 1910. OXIDATION IN DIABETES 257 circulation. As shown by several investigators, 27 a normal heart in beating is able to take up and consume a not incon- siderable amount of sugar from the circulating fluid. Starling found that the sugar consumption by the heart of a pancreatic-diabetic dog (transmitting its own blood) is minimal or practically nil. If, however, a heart from an animal with pancreatic diabetes was supplied with normal blood, it was able to consume sugar. Moreover when a small amount of pancreatic extract was added to the diabetic blood there always followed a marked rise in the sugar con- sumption in the diabetic heart. Starling states that "meanwhile the conclusion seems justified that normally a hormone is produced by the pancreas, the presence of which in the blood is essential to the assimilation and utilization of the blood sugar. Our experiments are proof that pan- creatic diabetes is more likely to be caused by a diminished capacity of the tissue to make use of the sugar than by a primary exaggeration of its production. ' ' 28 The next question which presents itself is whether we are in position to frame some reasonable theory as to the kind of disturbance of function which interferes with the tissues consuming the sugar in a normal manner. Oxidation Processes in the Diabetic Economy. — That we are not dealing here with a diminution of the oxidizing power of the tissues in general has been long known. On the contrary, recent investigations upon animals with pancreatic diabetes show that an increased consumption of oxygen may be regarded as a characteristic symptom in severe diabe- tes. 29 It has also been shown that the first products of sugar oxidation, gluconic acid, glycuronic acid and saccharic acid, 27 Chauveau and Kaufmann, Locke and Rosenheim, and Rohde. 28 In reference to the hypotheses ( Chauveau, Hedon, Kaufmann and others ) which attempt to refer pancreatic diabetes to an increased sugar production dependent upon the effectiveness of the liver and of the central nervous system, see A. Biedl, 1. c, pp. 391-392. 28 C. v. Voit, Leo, Weintraud, Falta, Grote and Stähelin, Mohr; for Litera- ture consult R. Umber, Lehrb. d. Ernähr., p. 138, 1909. 17 258 PANCREATIC DIABETES undergo combustion readily also in the diabetic organism 30 GLUCOSE GLUCONIC ACID GLYCURONIC ACID SACCHARIC ACID CH 2 .OH CH 2 .OH COH COOH (CH.OH)< (CH.OH) 4 (CH.OH)« (CH.OH), COH COOH COOH COOH (as, too, it finds no difficulty in oxidizing a substance as close to dextrose as glucosamine, CH 2 .OH- (CH.OH) 3 - CH.NH 2 - COH). 31 It might possibly be thought that the diabetic has merely lost the ability to manage the first step in sugar combustion (the oxidation of glucose into gluconic acid). If that were the case, however, it would be difficult to interpret the fact that diabetics react to the introduction of chloral, camphor, etc., just as the normal individual does, by the excretion of combined glycuronic acids. 32 This shows clearly that the diabetic has the power to carry sugar oxidation as far as glycuronic acid. One might draw the conclusion from this that normal, physiological combustion of sugar does not follow the path through gluconic acid and glycuronic acid, that glycuronic acid is only produced excep- tionally, if occasion demands detoxification of some foreign substance or other. It might be conceived, too, that in diabetes the economy still retains the ability to oxidize the sugar into glycuronic acid, but loses its ability to effect the normal, physiological catabolism of the sugar (presumably disin- tegration of its molecule into compounds with two or three carbon atoms, like lactic acid). Metabolism in Pancreatic Diabetes. — As for the other metabolic features of pancreatic diabetes, the affection is characterized by a decided increase of protein and fat de- struction and by an increased elimination of the mineral 80 O. Baumgarten (Med. Clinic Halle), Zeitschr. f. exper. Pathol., 2, 53, 1905. 31 J. Forschbach (Minkowski's Med. Clinic, Cologne), Hofmeister's Beitr., 8, 313, 1906. 42 See H. G. Wells, Chemical Pathology, p. 530, 1907; O. Baumgarten, Zeitschr. f. exper. Pathol., 8, 206, 1910. METABOLISM IN PANCREATIC DIABETES 259 constituents. 83 Acidosis may be regarded as a symptom resulting from the destruction of fat (accumulation of ,/?-oxybutyric acid, diacetic acid and acetone), which is met in pancreatic diabetes as well as in severe human diabetes. Whether these features are entirely and satisfactorily ex- plained on the basis of a lowering of the consumption of sugar (by which the respiratory quotient is diminished) must for the present be left unanswered. 34 From the above it might be expected that if the con- sumption of sugar in a dog with pancreatic diabetes be raised by physical exertion or exposure to cold, there would result a lowering of the sugar elimination. This is, how- ever, by no means always the case, as shown by the studies of Embden and Lüthje. 35 According to Minkowski, in- creased sugar consumption from physical exercise occurs only if some functionable remnant of the pancreatic tissue is present in the subject. After complete exclusion of the pancreatic function it seems that sugar can practically no longer be utilized to defray an increased demand for energy, and at best the latter would only lead to a heightened mobilization of sugar at the expense of the noncarbohydrate stores and therefore (as the mobilized sugar is not oxidized but excreted unchanged) to an increased intensity of the dia- betes. Heat regulation in diabetic animals is correspond- ingly encroached upon apparently. J. de Meyer, from a study conducted in Heger 's labora- tory in Brussels, would have it that the impermeability of the normal kidney to sugar is related with the internal secretion of the pancreas. If Locke's solution containing about the same proportion of sugar as occurs in the blood be perfused through the fresh (taken from a dog) kidney, sugar passes into the " urine. " On adding a small amount 33 S. la Franca (Naples), Zeitschr. f. exper. Pathol., 6, 1, 1909. 34 Cf. F. Verzär (Tangl's Lab., Budapesth) , Biochem. Zeitschr., U, 201, 1912. 35 H. Lüthje, Verh. d. 22. Kongress f . innere Med., Wiesbaden, 1905, 268 ; G. Embden, H. Lüthje and E. Liefmann (Frankfurt, a. M.), Hofmeister's Beitr., 10, 265, 1907. 260 PANCREATIC DIABETES of pancreatic extract to the circulating fluid this "glyco- suria" diminishes appreciably. Other tissue extracts, how- ever, apparently have no specific influence upon the renal permeability. Although these experiments may well permit other interpretations, they seem to deserve further consideration from the standpoint in view. 36 Partial Extirpation of the Pancreas. — Attention should be directed to the recent reports upon partial pancreatic ex- tirpation issued by F. Beach 37 from Durig's laboratory. A long time since Sandmeyer noted an increased output of sugar in dogs after partial removal of the pancreas and administration of a mixed diet of raw horsemeat and raw pancreas; and explained it on the supposition of an aug- mentation in the utilization of the glycogen in the meat by the pancreatic ferment. Reach has shown that this explanation is not correct, but that the raw meat (compared with boiled meat) contains a poison, labile to boiling, which forces up the sugar-excretion in slightly diabetic dogs. Antipancreatin Serum. — One can readily believe that it has not been possible to entirely resist the temptation of applying the advances in immunology to the great puzzle of pancreatic diabetes. That the blood of animals after extirpation of the pancreas does not contain any "toxine" capable of rendering normal animals diabetic was shown a long time ago by Minkowski and Mering. Accord- ing to J. de Meyer, 38 after injection of extracts of dog pan- creas (heated previously to 70° C.) a supposedly specific antibody {"antipancreatin") appears in the blood serum of rabbits, which not only reduces in vitro the glycolytic power 36 J. de Meyer (Instit. Solvay, Brussels), Arch. Internat, de Physiol., 8, 121, 1909; Recherche sur la signification et la valeur de la secretion interne du Pancreas, Liege, Imprimerie H. Vaillant-Carmanne, 1910. " F. Reach (Durig's Lab.), Wiener klin. Wochenschr., 1910, No. 41; Biochem. Zeitschr., S3, 436, 1911; cf. also J. Thiroloix and Jacob, Bull, et Mem. de la Soc. des Höpit. de Paris, 1910, 492. 88 J. de Meyer, Arch, internat. de Physiol., 7, 317, 1909; 8, 121, 1909; 9, 1, 1910; 10, 239, 1910; 11, 131, 1911; Recherche sur la signification, etc., 1. c; Ann. Instit. Pasteur., 22, 778, 1908. ISOLATION OF THE "PANCREATIC HORMONE" 261 of dog's blood, but in the living animal induces a hyper- glycemia and a glycosuria of low grade. Objection has been raised to these findings, suggestion being made that the hyperglycemia is not due to "antipancreatin" but simply to withdrawal of the blood required for examination. The writer is not in position to decide whether this objection is well founded or not, but all these matters are so compli- cated and ambiguous that he is not disposed to promise any too much from them for the future of the problem. Isolation of the" Pancreatic Hormone." — There seems to be no more promise in the attempts to isolate the active principle of the pancreas which is concerned in carbohydrate metabolism, the so-called "pancreatic hormone." Realiza- tion of the pious wish to solve the enigma of pancreatic diabetes in the reagent glass seemed almost within grasp some few years ago when at the Congress of Internists of 1907 Zuelzer 39 excited attention by his announcement that by injection of pancreatic preparations it is possible to check the course of adrenin glycosuria, and by administering pancreatic substance to the experiment animal prior to in- jection of adrenin to prevent it. Based upon this observa- tion, which has been confirmed from many sources, 40 Zuelzer 41 later endeavored to apply the discovery to the treatment of human diabetes. The decided toxicity of the pancreas preparations (occasioned by the trypsin), how- ever, interfered. But Zuelzer believed, nevertheless, that by a method which he kept secret, he had successfully detoxified his "pancreatic hormone" sufficiently to permit him to venture to inject it intravenously into diabetic sub- jects. In a number of cases of diabetes he succeeded in 39 Zuelzer, Verb. d. Kongr. f. innere Med., 1907, 258; Berliner klin. Wochenschr., 1907, 474. 40 C. Frugoni, Makaroff, J. Gautrelet, J. Forschbach, K. Glässner and E. P. Pick; for Literature, cf. 0. v. Fürth, and C. Schwarz, Biochem. Zeitschr., SI, 114, 1911. "Zuelzer, Dohrn and Marxer, Deutsch, med. Wochenschr., 1908, 1380; Zuelzer, Zeitschr. f. exper. Pathol., 5, 307, 1909. 262 PANCREATIC DIABETES distinctly reducing the elimination of sugar and of acetone bodies. However, in these as well as in a test undertaken in Minkowski's Clinic, 42 the suggestive fact was manifested that injections of ''pancreatic hormone" were often fol- lowed by chills, fever of several days ' duration and malaise. Still later the writer's colleague, C. Schwarz, and the writer 43 were able to show, at least in case of intraperitoneal introduction of pancreatic substance, that this inhibition of adrenin glycosuria is not due to a mysterious antagonism between the "hormones" of the pancreas and of the adrenal glands, but probably is to be explained naturally and easily as depending upon a condition of peritoneal irritation (v. Vol. I of this series, p. 383, Chemistry of the Tissues). This may so affect the secretory ability of the kidneys that elimination of the dissolved constituents of the urine includ- ing sugar, is lowered without the quantity of fluid neces- sarily undergoing any coincident striking diminution. When this fact is considered along with the fact that a great variety of disturbances, perhaps not directly related with the peritoneum (as fever, suppression of urine, introduction of materials with lymphagogue action, etc.), 44 are capable of inhibiting adrenin diabetes, there is apparently not suffi- cient ground here for a pancreatic treatment of diabetes. If in addition the statements of numerous authors 45 are accepted, that injecting and feeding pancreatic substance not only does not arrest the glycosuria in animals with pancreatic diabetes, but in many instances actually intensi- 42 J. Forschbach (Minkowski's Clinic, Breslau), Deutsch, med. Wochenschr., 1909, No. 7. 4S 0. v. Fürth and C. Schwarz, Wiener klin. Wochenschr., 1911, No. 4, and Biochem. Zeitschr., 31, 113, 1911. " Cf . the report of Mikulicich upon the inhibition of adrenin diabetes by hirudin (O. Löwi's Lab., Gratz), Arch. f. exper. Pathol., 69, 128, 1912. 41 Cf. Literature in E. Leschke (Physiol. Instit., Bonn), Arch. f. Anat. u. Physiol., 1910, 401; Münchener med. Wochenschr., 1911, No. 26; cf. also N. Tiberti and A. Franchetti (Florence), Lo Sperimentale, 62, 81, 1908; E. L. Scott (Carlson's Lab., Chicago), Amer. Jour, of Physiol., 29, 306, 1912. HUMAN DIABETES 263 fies it, Schwarz and the writer 46 (as well as Leschke) can scarcely fail to be justified in characterizing the efforts to relieve human diabetes by intravenous injection of "pancre- atic hormone" as physiologically incorrect and (in view of the danger attending) entirely inadmissible. Nor can the attempts of Vahlen, 47 who believes he has isolated a sub- stance from the pancreas which, while not directly attacking the sugar, accelerates alcoholic fermentation of the sugar, and lowers the sugar excretion in phloridzin diabetes and adrenin diabetes, make any difference in their conclusion. The writer feels that it would be a good thing to consider carefully the above stated experiences in connection with the inhibition of glycosuria by administration of hydrazin, 48 opium, 48 atropine, 50 zyzigium jambolianum and other drugs. 51 HUMAN DIABETES Degeneration of the Pancreas in Human Diabetes. — We may now pass to the consideration of human diabetes. After the best informed experts in this affection, men like Naunyn, Minkowski and v. Noorden, as well as nu- merous pathological anatomists, had repeatedly pointed out a relation between diabetes and some functional fault of the pancreas, this in the author's opinion, seems to have been finally proved, whatever the contrary opinions, by the comprehensive investigations of the Viennese pathologist, Weichselbaum. He proved from a large amount of material at his disposal that degeneration of the islands of Langer- 4C O. v. Fürth and C. Schwarz, 1. c. 47 E. Vahlen (Halle), Zeitschr. f. physiol. Chem., 59, 194, 1909. 48 R. P. Underhill and M. Fine (Yale Univ., New Haven), Amer. Jour. Physiol., 10, 271, 1911. 40 A. Gigon (Basel), Verh. d. 26. Kongr. f. innere Med., Wiesbaden, 1909, p. 441. M Cf. Rudisch, Arch, f . Verdauungskr., 15, 469, 1909. 61 M. Mikulicich (O. Löwi's Lab., Gratz), Arch. f. exper. Pathol., 69, 133, 1912, holds a specific renal impermeability for sugar responsible for the inhi- bition of adrenin glycosuria by ergotoxin as well as for inhibition of the hyperglycemia. 264 HUMAN DIABETES hans is to be looked upon as the anatomical basis of diabetes, and that as a matter of fact the severity of the case stands in direct relation with the degree of involvement of the islands. Weichselbaum differentiates a hydropic degenera- tion of these structures, a peri- and an intra-insular sclerosis and a hyaline degeneration, the last characterized by swell- ing of the connective tissue about the vessels of the islands into a homogenous mass. The negative findings of other authors in this line may be satisfactorily explained by the fact that these changes may very readily be overlooked, if particularly attentive examination be not made and if im- perfect preservation of the material has obtained. 52 As far as the cause of the pancreatic disease is con- cerned many authors are disposed to believe that a relation- ship with previous acute infectious diseases, although cli- nically this can only rarely be proved, is more important than the influence of arteriosclerosis, alcoholism, syphilis, intes- tinal infections, etc. 53 That infectious diseases, even those of frankly mild type as influenza or angina, may make exist- ing diabetes permanently worse and that transient glycosu- rias often occur in the course of febrile affections has been long known. Doubtless, too, from the studies of Weichsel- baum, arteriosclerosis plays an important role in many forms of diabetes. The liver has frequently been examined for anatomical changes in diabetes ; but there can be little mistake in believ- ing that in the great bulk of cases in which diabetes has appeared as a sequel of hepatic disturbances, as cirrhosis or cholelithiasis, secondary lesions of the pancreas are the real point in fault. Thus we may appeal to the frequent connective tissue hyperplasia in the pancreas in connection with cirrhosis of the liver (chronic pancreatitis of Hanse- 52 A. Weichselbaum, Wiener klin. Wochenschr., 24, 153; Sitzungsber. d. Wiener Akad., 119, III, 73, 1910; consult therein, and also in U. Lombroso, Ergebn. d. Physiol., 9, 1-89, 1910, the Literature. M Cf . F. Hirschfeld, Deutsche med. Wochenschr., 1909, 137. GLYCOGEN CONTENT OF THE LIVER 265 mann) ; and in the occasional cases of diabetes in which previous examinations have found a cirrhosis of the liver along with an apparently normal pancreas, it is very ques- tionable whether there did not exist the above-mentioned hydropic changes of the islands of Langerhans, described by Weichselbaum, which are so difficult of recognition. 54 No doubt nervous lesions of very many types may give rise to glycosuria and be causative of true diabetes; and we may accept as a fact that nervous irritations are capable of inducing a liquidation of the glycogen stored in the liver, as seen in case of the experimental sugar puncture. This does not, however, at all affect our referring true human diabetes to the pancreas. We are, therefore, fully justified from the present status of our knowledge in regarding human diabetes as a special type of pancreatic diabetes. The writer can scarcely be expected to enter into any detailed description of this affection, the literature of which is enough to fill a whole library ; it will probably suffice if a few points are brought out which, considered from the standpoint of a biological chemist, seem for the time to be of special interest, and to refer for the rest to Naunyn's classical work upon diabetes 55 and to the recent excellent monographs by vonNoorden, 56 Magnus-Levy, 57 and Umber. 58 Glycogen Content of the Liver. — A point requiring at least brief consideration is the amount of glycogen found in the liver in human diabetes. As previously stated, Naunyn assigned to "dyszooamylia" an important position; and most writers are of the impression that, in severe diabetes at least, the power of the liver to store glycogen has been 64 Cf. F. Umber, Lehrb. der Ernähr, u. Stoffwechselkr., p. 161, 1909. 65 Naunyn, Diabetes Mellitus, 2d ed., Vienna, 1900. M C. v. Noorden, Die Zuckerkrankheit, 5th ed., 1910; Handb. d. Pathol, d. Stoffwechsels, 2, 1-113, 1907. 17 A. Magnus-Le'vy, Handb. d. Biochem., 4', 356, et seq., 1909. 68 F. Umber, 1. c, pp. 149-241. 266 HUMAN DIABETES more or less seriously disturbed. 59 Remembering the rapidity of postmortem disappearance of glycogen and the fact that the small amount of intake of nutrition before death may also influence the amount of glycogen in the liver, it is naturally somewhat difficult, to say the least, to come to any precise conclusions in a human diabetic as to his glycogen stock. The noted clinician, Frerichs, satisfied his curiosity in this respect (by a very direct method, but one scarcely to be commended for general use) by removing during life bits of liver tissue by puncture in two cases of diabetes ; one of the specimens contained glycogen. When we recall that diabetic patients are able to change laevulose into glycogen more readily than they can transform glucose, it is very suggestive that, as a study in the Vienna Phar- macological Institute 60 indicates, a rabbit's liver damaged by phosphorus poisoning makes a like differentiation be- tween dextrose and laevulose ; this particular power of dif- ferentiation is, therefore, by no means a characteristic of diabetes exclusively. It has been previously stated that quantitative examina- tion of the diabetic liver for its diastase has not led to definite conclusions. Hyperglycemia. — Hyperglycemia in diabetic cases is gen- erally accepted. While the amount of sugar in the blood of a normal human individual amounts to about 0.1 per cent., the proportions in the diabetic have been noted as high as one per cent, or even more. This in itself is sufficient reason for the presence of a considerable amount of sugar in the cerebrospinal fluid (obtained by lumbar puncture) ; notably, the highest figures (as high as three per cent.) having been met in comatose cases the urine of which contained only moderate amounts. 61 Methylene-Blue Reaction of the Blood. — This character- ■*Cf. Literature: A. Magnus-Levy, 1. c, pp. 357-358. 60 E. Neubauer (Pharmacol. Instit., Vienna), Arch. f. exper. Pathol., 61, 174, 1909. " N. B. Foster, Boston Med. and Surg. Jour., 158, 441, 1905. URINARY DEXTRINE 267 istic color reaction of the blood in diabetes is supposed to be due to the hyperglycemia. It has been noted that diabetic blood has the property of changing the color of methylene- blue to a yellowish red, apparently by some reduction process. Naturally it was supposed that the phenomenon is referable to the reducing power of the sugar in the blood. However, it was found that the red corpuscles of a diabetic, after they have been entirely freed from the sugar-contain- ing plasma by repeated centrif ugation with isotonic salt solu- tion, fail to give the normal appearance of erythrocytes (when stained with methylene-blue) . It is said, too, that this blood test may be met in cases long after the sugar has dis- appeared from the urine from withdrawal of carbohydrates. It may be, of course, that even in such cases the proportion of sugar in the erythrocytes is abnormally high. C. v. Noor- den properly calls attention to the need of making quantita- tive comparisons between the proportion of sugar in the blood and staining qualities in order to gain an explanation of this reaction. 62 Another question which is connected with the hyper- glycemia is whether the increased proportion of sugar in the blood in diabetes is necessarily due to an increased "impermeability of the kidneys for sugar." urinary Dextrine. — Very little attention has been given to the question whether besides the sugar it is possible that polymeric carbohydrates may also pass from the blood into the urine in diabetes. Alf tan 's 63 estimations of an aver- age excretion of 0.15 gram of "urinary dextrine" for a normal man and in severe diabetes of perhaps from 5 to 24 grams per diem apply here. The exact meaning of this "urinary dextrine," chemical study of which is as yet lacking, is another problem. 8a C. v. Noorden, Handb. d. Pathol, d. Stoffw., 2d ed., 2, 104-105, 1907; cf. therein Literature. 85 K. v. Alftan, Ueber dextrinartige Substanzen im diabetischen Harne, Helsingfors, 1904. 268 HUMAN DIABETES Protein Destruction.— In trying to come to some reason- ably clear appreciation of the general course of metabolism in diabetes we must take prominently in consideration what is known of protein destruction in this abnormal condition. While, as has been noted above, in the pancreatic diabetes of the dog the decomposition of tissue proteins is apparently markedly increased, in severe cases of diabetes mellitus (as shown by the metabolic studies of Falta and Gigon) 64 the destruction of protein does not proceed any more rapidly, and in special cases may be slower, than in normal persons who are examined under the same condition of nutrition. This is all the more remarkable when one considers that the diabetic patient undoubtedly fixes a distinctly smaller amount of material as reserve carbohydrate, and that the sugar excreted with the urine escapes combustion and can- not, therefore, serve to save the protein. The loss is apparently compensated for as far as possible by abundant intake of food protein. That the carbohydrate group in proteins is not of very great importance to the formation of sugar in the body has been shown to be true in case of human diabetes just as in other forms of glycosuria, 65 Falta 66 based his calculations upon the "excretion coef- ficient, ' ' that is, the ratio of sugar excretion, D, to the sugar value of the transformed material. He obtains this by the formula q = , in which N is the amount of urinary nitrogen and K the quantity of carbohydrate in the food ingested. This method of calculation is based upon Eub- ner's dictum that for each gram of transformed protein nitrogen a maximum of five grams of sugar are produced (that is to say, the sugar value of 100 grams of protein would be roughly 16 X 5 = 80 grams of dextrose). That a combustible material like alcohol may serve to 84 W. Falta and A. Gigon ( W. His's Clinic, Basel, and C. v. Noorden's Clinic, Vienna), Zeitschr. f. klin. Med., 65, 3-4, 1908. 95 Cf. E. Thermann (Helsingfors), Skandin. Arch. f. Physiol., 17. 1, 1905, 68 W. Falta, J. H. Whitney (v. Noorden's Clinic), Zeitschr. f. klin. Med., 65, 5-6, 1908. FATTY DIABETES 269 spare the proteins of the body and in this way lessen the sugar elimination at times in diabetics 6T is easily appreci- able; as, too, the fact that an influence tending to cause tissue break-down, as exposure to Röntgen rays, is likely to occasion an increase in the glycosuria in diabetes. 68 There seem to be times when aminoacids pass into the urine in diabetes in increased amount; but little certain knowledge is had of this phenomenon. 69 While to a certain extent toxic protein decomposition in human diabetes is, from what has been said, a matter of minor importance, the fat breakdown often dominates the general situation. This is really, as far as we can readily appreciate, what is actually meant when we are forced to say that the body must finally draw on some source to cover the necessary requirements for energy-production ; if the sugar is eliminated unconsumed, if the tissue protein is preserved from destruction, and if the food protein is insuf- ficient, there is nothing else left the body to draw upon except its stores of fat. Many writers are inclined to accept a possibility of sugar being formed from fat in severe diabetes. 70 Later, in connection with acidosis in relation to fat decomposition, this subject will be more fully reverted to. Brief reference should be made at this point to the rela- tionship of obesity to diabetes. Very frequently in litera- ture a "lipogenic diabetes" (diabete gras of the French) is spoken of. C. v. Noorden 71 has suggested a very plausible idea in this connection, that there exists a form of masked diabetes in which the sugar does not pass into the urine even though the capacity for sugar combustion is 67 H. Benedict and B. Török, Zeitschr. f. klin. Med., 60, 329, 1906. " P. Menetrier and A. Touraine, Arch. Maladies de Coeur, 8, 641, 1910. 69 P. Bergell and F. Blumenthal, Zeitschr. f. exper. Pathol., 2, 413, 1906; L. Mohr, ibid., 2, 665, 1906. 70 Cf. E. Gräfe and Ch. G. L. Wolf (Med. Clinic, Heidelberg), Deutsch. Arch. f. klin. Med., 101, 201, 1912. 71 C. v. Noorden, Handb. d. Pathol, d. Stoffwechs., 2d ed., 2, 25-26, 1907. 270 HUMAN DIABETES diminished; in such cases the excess of sugar is changed into fat and deposited as such. A ' ' diabetogenous obesity' T of this sort may, if glycosuria come to accompany it, be changed into the ordinary diabetes of the obese, and in the end into a severe diabetes, with development of a progressive loss of flesh. Diabetic Lipcsmia. — Finally among the disturbances of fat metabolism noted in diabetes, diabetic lipsemia 72 presents special interest. While the quantity of lipoid substances in the blood plasma is normally scarcely more than one per cent., in diabetes it may rise to many times this proportion. Cases have been known in whom the blood from the veins looks like chocolate and cream, 73 and in whom the vessels at autopsy appear as whitish cords. In one case more than one-fourth volume was obtained by ether extraction from the blood. 74 Sometimes the fat is present in practically normal amount, but the Cholesterin and lecithin are much increased. 75 Upon what the lipsemia essentially depends, it is impossible to say. The fact that it frequently parallels the acidosis, and becomes especially marked in the coma, strongly suggests a connection with tissue disintegration, especially some process which sets free the fat. At one time a reduction of the lipolytic power of the blood was sup- posed to be an important factor. In recent times, however, our views have changed essentially in that (as will be dis- cussed more fully hereafter) it has been recognized that the supposed destruction of fat in the blood is really a masking of the fat. We are likely, too, to view with a proper hesi- tancy statements to the effect that diabetic blood containing excessive fat shows a loss in the fat when mixed with normal blood. We did not in those earlier days clearly understand 12 Literature upon Diabetic Lipsemia: C. v. Noorden, Handb. d. Patbol. d. Stoffwechs., 2d ed., 2, 102-104, 135, 1907. 73 E. Neisser and E. Derlin, Zeitschr. f. klin. Med., 51. 428, 1904. '*C. Frugoni and G. Marcbetti (Florence), Berlin, klin. Wocbenschr., 1908? 1844. 76 G. Klemperer and H. Umber, Zeitschr. f. klin. Med., 61, 145, 1907. SUBSTITUTES FOR BREAD FOR DIABETICS 271 the importance of the great changes which physical-chemical adsorption is likely to induce in colloidal systems. Respiration Experiments in Diabetes. — Reference has al- ready been made to the fact that we cannot seriously attri- bute any general reduction of oxidation processes to the diabetic organism. Numerous respiration experiments, first by Pettenkofer and Voit and later by other investigators, 76 have shown in diabetics either normal ratios or in some instances a notable increase in the utilization of oxygen, 77 just as has been noted in case of animals with pancreatic diabetes and supposedly due to stimulations caused by the sugar and acetone bodies. 78 The hypothesis of Falta 79 that diabetes is due to some disturbance in the equilibrium between the internal secre- tory glands (pancreas, the chromaffine system, the thyroid and parathyroids) which are presumed to regulate the metabolism of carbohydrate material, will be reverted to later. Of course it is impossile here to take up fully the matter of dietetic treatment of diabetes, desirable and important as it is for the practitioner ; at best it must suffice to briefly refer to a few points of special biochemical interest. Substitutes for Bread for Diabetics. — The fact that lsevu- lose is invariably assimilated with more readiness by the diabetic economy than dextrose has led to the employment of its polysaccharide, inulin (which occurs in Jerusalem arti- choke meal, black salsify and artichokes) as an article of diet for diabetics. Experiments in ISTaunyn's clinic have, how- ever, indicated that while occasional use of this sugar or its polysaccharide is well tolerated, the toleration soon de- 76 Leo, Katzenstein, Weintraud and Laves, Magnus-Levy, Mohr. Litera- ture: A. Jaquet, Ergebn. d. Physiol., 2', 555-556, 1903; Umber, Lehrb. d. Ernähr., p. 171, 1909. 77 Cf. the experiments of W. Falta (with Benedict), conducted with the respiration calorimeter in Boston: Wiener klin. Wochenechr., 22, 565, 1909. 78 A. Leimdörfer (v. Noorden's Clinic), Biochem. Zeitschr., 40, 326, 1912. 78 W. Falta (v. Noorden's Clinic), Zeitschr. f. klin. Med., 66, 5-6, 1908. 272 HUMAN DIABETES creases in continued ingestion, in consequence of which little can be promised for these types of carbohydrate as a dia- betic food. 80 Nevertheless "inulin treatment" has recently been strongly recommended again. 81 The desirability of discovering some form of harmless carbohydrate for diabetics has led to many experiments in this line. 82 Thus it is said that a hemicellulose preparation made from agar-agar, which yields principally galactose on hydrolysis, has proved useful in continuous administration of diabetics, 83 although galactose occasions an increase of dextrose in the urine in diabetic animals. 84 From Iceland moss, which contains dextrine-like substances, hemicelluloses and pentosans, after modifying its bitter materials by mix- ing with proteins, a bread has been baked. 85 Aleuronat bread has been used as a carbohydrate-poor bread-substitute; it is prepared from a mixture of ordinary flour with aleuronat meal which is rich in protein but poor in carbohydrate. Oat-Treatments. — C. v. Noorden's observation as to the influence of "oat-meal teatment" in diabetes is interesting and of practical importance. The method presents the superficial paradox that many severe cases of diabetes pass less sugar in the urine when given considerable amounts of oat-meal than when kept on the strictest diet. 86 Most authors who have tried von Noorden's suggestion have been forced to acknowledge that the treatment in certain cases is followed by a diminution of the glycosuria and acidosis and by an increased tolerance for carbohydrates. 87 It is 60 Cf. F. Umber, Lehrb. der Ernähr., p. 201, 1909. 81 H. Strauss (Berlin), Berl. klin. Wochenschr., 1912, 1912. 82 Cf. A. Magnus-Levy, Berl. klin. Wochenschr., 1910, 233. 83 A. Schmidt and H. Lohrisch (Halle), Deutsch, med. Wochenschr., 1908, 2012. 84 W. Brasch, Zeitschr. f. Biol., 50, 113, 1908. 85 E. Poulsson, Festschr. f . O. Hammersten, Wiesbaden, 1906 ; cited in Centralbl. f. Physiol., 21, 196, 1907. 88 C. v. Noorden, Berlin klin. Wochenschr., 1903, 817. 87 Cf. Literature: C. v. Noorden, Handb. d. Pathol, d. S^offwechs., 2d ed., 2, 63, 1907, and F. Umber, Lehrb. d. Ernähr., pp. 208-210, 1909; Klotz (Strass- burg), Zeitschr. f. exper. Pathol., 8, 601, 1911. MEDICINAL TREATMENT 273 not known how to explain this remarkable influence. By some it is said that the oatmeal shows its full specific influ- ence only when used in whole form, the individual con- stituents failing of effect. 88 Others 89 hold that experiments with isolated oat-starch give practically the same favorable results as does ordinary oat-meal diet. Oat-starch, if this be true, is different from other starches in its behavior in the diabetic economy. Influence of Mineral Waters. — It is generally known that the use of certain mineral spring waters has long played a large role in the treatment of diabetes. ' ' The real factors in the undoubtedly favorable influence of residence in estab- lishments at Carlsbad, Neuenahr, etc., upon certain forms of diabetes," remarks F. Umber 90 in his excellent book, full of the spirit of the wholesome critic, "are certainly not due to the effects of the waters themselves, but to other condi- tions, as the rest, the well-regulated mode of living, change of surroundings and, by no means the last, to physicians especially experienced in this field of pathology. The dia- betic subjects who are most benefited on the spot from this sort of treatment are those belonging in the milder type of the disease, possibly afflicted by disturbances of the liver, the digestive organs, etc., upon which as experience shows the methods of treatment at the baths have an espe- cially good influence. These people are likely to be par- ticularly benefited at such cures if they are not in position, for special professional or other reasons, to live a properly regulated life at home. ' ' Medicinal Treatment of Diabetes. — Naunyn is disposed to pass a practically quashing judgment upon the medicinal therapy of diabetes, characterizing the whole numberless 88 O. Baumgarten and G. Grund (Halle, a. S.), Deutsch. Arch. f. klin. Med., 10k, 168, 1911. 88 A. Magnus-Levy, Verhandl. d. 28. Kongr. f. innere Med., Wiesbaden, p. 246, 1911; cf. also discussion ensuing. 90 F. Umber, Lehrb. d. Ernähr., usw., p. 236, 1909. 18 274 HUMAN DIABETES group of remedies which have been used and lauded, as untrustworthy or ineffective (with the exception of opium). It is not known which of the alkaloids of opium is responsible for the influence clearly manifested in many cases by the drug in lowering glycosuria. One can scarcely expect that as long as the real nature of diabetes is not thoroughly appreciated any particularly brilliant result will be attained by an essentially desultory prying around with physiologi- cally different or even indifferent substances. CHAPTER XII PHLORIDZIN DIABETES. L^EVULOSURIA. LACTOSURIA. PENTOSURIA. EXPERIMENTAL GLYCOSURIAS OF DIFFERENT KINDS PHLORIDZIN DIABETES Having familiarized ourselves in the last lecture with the general nature of pancreatic diabetes, our attention may be devoted to other physiologically important forms of glycosuria, of which phloridzin diabetes is the first to be taken up for consideration. As is well known we are indebted to J. von Mering for the discovery that a glucoside found in the roots of apple-, pear- and cherry-trees, known as phloridzin, when given to human beings or to animals, is capable of causing a notable excretion of sugar. On hydrolysis phloridzin splits into glucose and phloretin, the latter substance also manifesting diabetogenous properties. If the phloridzin be given regu- larly at intervals of a, few hours, a persistent " phloridzin diabetes" will result, as pointed out by M. Cremer and by Graham Lusk. 1 Absence of Hyperglycemia. — It was soon recognized that phloridzin diabetes differs essentially from pancreatic diabetes, and fails to show one of the principal character- istics of the latter, as well as of most of the other forms of glycosuria, namely, hyperglycemia. The amount of sugar in the blood, as proved by numerous investigators, is not only not increased in phloridzin diabetes, but on the con- trary is often diminished. 2 "While in pancreatic diabetes after removal of the kidneys or ligation of the ureters large literature upon Phloridzin Diabetes: M. Cremer, Ergebn. d. Physiol., 1', 883-886, 1902; K. Glässner, Centralbl. f. Stoffwechselkr., 1, 673-705, 1906; F. Umber, Lehrb. d. Ernährung, pp. 143-146, 1909; A. Magnus-Levy, Handb. d. Biochem., 4', 366-368, 1909; Graham Lusk, Ernährung u. Stoffwechsel (Ger- man translation by L. Hess), p. 247, et seq., 1910. 2 Cf. P. Junkersdorf (Bonn), Pfiuger's Arch., 136, 306, 1909; A. Erlandsen (Lund), Biochem. Zeitschr., 23, 329, 1910; 2k, 1, 1910. 275 276 PHLORTDZIN DIABETES amounts of sugar accumulate in the blood, here one cannot find evidence of any sugar stagnation. The explanation of this peculiarity is simply that the kidney is concerned here, not merely with the excretion of the sugar that is brought to it, but that in phloridzin diabetes the latter is, at least in large part, produced in the kidney itself. Here, therefore, we are dealing with a "renal diabetes." Role of the Kidney. — There can be no doubt as to the essential part played by the kidneys in phloridzin diabetes. This was impressed long ago by the experiment of N. Zuntz, who noted when he injected the glucoside into one of the renal arteries that the corresponding kidney eliminated sugar earlier and more freely than its fellow. 3 In the same line are to be classed the perfusion experiments upon ex- cised living kidneys conducted by Biedl and Kolisch 4 and by Pavy 5 and his collaborators. Exactly what is the renal activity in phloridzin diabetes? Wohlgemuth believes we are justified in concluding there is an increase of diastases in the kidneys, produced from the influence of the phloridzin, leading to an increased enzymic activity of the renal cells. 6 Otto Löwi 7 was able to show, contrary to opposite opinions, 8 that sugar elimination by a phloridzin kidney is not in- creased by setting up in addition a salt diuresis. There is, therefore, an increased activity of a special kind seen in the effect of phloridzin. Many writers were disposed to interpret phloridzin dia- betes on the assumption that normally the blood sugar is retained by the kidneys because it is not in a free state but in colloid combination ; that in phloridzin diabetes this com- 3 N. Zuntz, Verh. d. Berlin. Physiol. Ges., Arch. f. Anat. u. Physiol., 1895, 570. *A. Biedl and Kolisch, Verh. d. 18. Kongr. f. innere Med., p. 573, 1900. 8 F. W. Pavy, T. G. Brodie and R. L. Siau (London), Jour, of Physiol., 29, 467, 1903. 6 J. Wohlgemuth, and J. Benzur, Biochem. Zeitschr., 21, 460, 1909. 'Otto Löwi and E. Neuhauer (Pharm. Instit., Vienna), Arch. f. exper. Pathol., 59, 57, 1908. 8 S.Weber (Minkowski's Clinic) , Arch. f. exper. Pathol., 54, 1, 1905. FORMATION OF SUGAR IN THE KIDNEY 277 bination is broken in the kidneys and these organs are then enabled to niter the sugar out of the blood. Under the in- fluence of phloridzin very considerable quantities of sugar may appear in the urine, to be invariably made good at expense of the proteins after consumption of the carbohy- drate supply of the body. Possibly this may be explained by the idea that (very much as a Toricellian vacuum has a tendency to attract gases) the body does not permit the existence of a "sugar vacuum" in the blood but endeavors to repair the void with all the substances at its command. But in criticism of this attempt to explain the nature of phloridzin diabetes it must be recalled that, as has been previously discussed, we have no certain ground for predi- cating the existence of such a colloidal combination of the sugar in the blood. There is no reason for supposing this form of diabetes to be in any way related with the glycogen function of the liver. Frank and Isaak 9 were able to show that dogs in state of starvation continue to eliminate large amounts of sugar when jDhloridzin is administered even after inhibition of the hepatic function (that is, after severe changes have been produced) by phosphorus poisoning. Formation of Sugar in the Kidney. — These last authors have, in the writer's opinion, made a suggestion worth con- sidering, that the kidney under the influence of phloridzin is not only able to separate sugar from the blood, but be- comes capable of inducing synthesis of sugar and acquires the property of building up sugar from non-carbohydrate primary material. The observation that in animals with phloridzin diabetes there usually is to be found an increased quantity of sugar in the blood after some days of well marked hypoglycemia is explained on the supposition that the blood becomes charged with sugar from the kidneys. 9 E. Frank and S. Isaak, Verh. d. 17. Kongr. f. internal. Med., Wiesbaden, 1910, p. 586; Arch. f. exper. Pathol., 6If, 274, 293, 1911. 10 R. Lepine, C. R. Soc. de Biol., 68, 448, 1910. 278 PHLORIDZIN DIABETES Leprae's 10 discovery that in phloridzin poisoning the blood of the renal vein contains more sugar than that of the renal artery may be regarded as in the same line. Asher, 11 after stimulating the chorda tynrpani found such a marked in- crease of sugar in the blood passing from the submaxillary salivary gland that it must necessarily be assumed that under irritation sugar passes out of the glandular cells into the blood. It may be conceived that in an analogous manner the kidney under the stimulus of phloridzin becomes able to give off an excess of sugar to the blood. Frank and Isaak, how- ever, would explain the essential character of phloridzin diabetes as consisting of an acquired inability on the part of the kidneys to fix the glucose brought to them by the blood and utilize it in their own metabolism, and as a consequence becoming permeable to the sugar and excreting it. Just as the liver in pancreatic diabetes — and with the very same failure — the kidney attempts to make good the loss by con- tinuous reformation. The question whether the phloridzin kidney really acquires the ability to produce sugar de novo is one which apparently should receive further study. The hypothesis that the influence of phloridzin is pro- ductive of "a general glandular diabetes with predominant involvement of the kidneys" and of a disturbance of the vital sugar fixation 12 finds some support in the fact that, as proved upon a dog with a biliary fistula, after injection of phloridzin sugar is to be found not only in the urine but also in the bile. 13 No constant direct influence from phloridzin upon the formation of glycogen in the liver has been successfully proved by perfusion experiments. 14 It may, therefore, be conjectured that the sugar elimination in phloridzin diabetes results from activity of the glandular epithelium in much the same way as milk sugar is produced u L. Asher and Karaulow, Biochem. Zeitschr., 25, 36, 1910. u E. Frank and S. Isaak, Arch. f. exper. Pathol., 64, 326, 1910. U R. T. Woodyatt (Chicago), Jour, of Biol. Chem., 7, 133, 1910. 14 B. S. Schöndorff and F. S'uckrow (Bonn) , Pfliiger's Arch., 138, 538, 1911; opposed view, K. Grube, ibid., 128, 1909, and 139, 1911. FORMATION OF SUGAR IN KIDNEY 279 in the mammary glands. It is interesting to note, too, that phloridzin has been found by observations on milk cows to increase the amount of sugar in milk, 15 which may, how- ever, perhaps be due to a relative inspissation of the milk from the effect of the increased diuresis. 16 Significantly, Underhill has been able to show that hyperglycemia will result from administration of phloridzin after excluding the renal function. 17 Numerous investigations have been made upon the gen- eral metabolism in phloridzin diabetes. The same results have been obtained in the ratio between sugar elimination and nitrogen elimination in the dog with phloridzin diabetes as in the diabetic human being (D : N = 3.6 : 1) in the studies of Graham Lusk and his associates. 18 It is worthy of note that an injection of phosphorus, which in a normal dog is followed by a marked increase of nitrogen elimination, in the dog with phloridzin diabetes causes no greater heightening of toxic protein decomposition. 19 The increase of Creatinin excretion 20 and of elimination of aminoacids 21 seen in phloridzin diabetes may be regarded as evidence of this protein destruction. From the investigation of G. Rosenfeld, J. Baer and others it is evident that a disturbance of the nitrogen bal- ance, as that induced by starvation or carbohydrate-free diet, occurs in phloridzin diabetes with mobilization of fat and acidosis. The former of these features manifests itself as a fatty infiltration of the liver, the fat disappearing from other parts of the body and massing itself in the liver. 13 Cornevin, Compt. Rend., 116, 263, 1893. 16 C. Porcher, Compt. Rend., 1S8, 1475, 1908. 17 F. P. Underhill (Yale Univ., New Haven), Jour, of Biol. Chem., IS, 15, 1912. M G. Lusk, Ernährung und Stoffwechsel, p. 250, et seq., 1910; cf. also A. J. Ringer (Univ. of Penna.), Jour, of Biol. Chem., 12, 431, 1912. u W. E. Ray, T. S. McDermott and Graham Lusk, Amer. Jour, of Physiol., S, 139, 1899. 20 C. G. L. Wolf and E. Osterberg (Cornell Univ., New York), Amer. Jour. of Physiol., 28, 71, 1911. 21 J. Yoshikawa (Kyoto), Zeitschr. f. physiol. Chem., 78, 475, 1911. 280 PHLORIDZIN DIABETES Fate of Phloridzin in theBody. — In reference to this point it has been shown by a study emanating from the laboratory of M. Cremer 22 that a portion of the phloridzin is excreted in the form of a combined glycuronic acid. Another part apparently undergoes further change; after subcutaneous injection (according to investigation by K. Glässner and E. P. Pick) phloridzin can always be found for some time in the blood and tissues of the experiment animal. In nephrectomized animals, however, after small doses the glucoside disappears, which perhaps may be interpreted by supposing that this foreign substance is destroyed with in- creased readiness in the body if its normal elimination is prevented. 23 The real situation of affairs is, however, not clear. Little is known of the finer mechanism of this remark- able metabolic influence of phloridzin. Offhand it is diffi- cult to decide whether Biircker's 24 observation that phlorid- zin inhibits spontaneous oxidation of glucose in alkali solution, has anything to do with its effect in producing diabetes. It is worth noting, even if one cannot readily understand them, that experiments in Salkowski's labora- tory have shown that aliphatic alcohols with an odd number of carbohydrate atoms in the molecule (methyl alcohol, propyl alcohol, amyl alcohol, glycerol), but not those with an even number (as ethyl alcohol and butyl alcohol) give rise to an increase of sugar excretion in phoridzin animals. 25 Per- haps this may prove a loop hole through which we may approach a little nearer the secret of sugar synthesis in the phloridzin kidney. 22 J. Schiiller (Lab. of M. Cremer, Cologne), Zeitschr. f. Biol., 56, 274, 1911. 23 K. Glässner and E. P. Pick (R. Paltauf's Lab.), Hofmeister's Beitr., 10, 473, 1907; Pflüger's Arch., 133, 82, 1910; J. Schiiller, 1. c, p. 290; cf. also opposed view of E. Leschke, Arch. f. Anat. u. Physiol., 1910, 437; Pfliiger's Arch., 132, 319, 1910; 135, 171, 1910. ^Bürcker (Tübingen), Deutsch. Physiol. Kongr., München, 1911, Centralbl. f. Physiol., 25, 1091, 1911. 26 P. Höckendorf (Pathol. Instit., Chem. Div., Berlin), Biochem. Zeitschr., 23, 281, 1909. DETECTION OF LiEVULOSE 281 The capacity of glutaric acid to inhibit the glycosuria of phloridzin intoxication, discovered by E. Baer and Blum, must probably be regarded as in some way connected with an influence of the former upon the kidneys. 20 Having discussed the nature of the two most important, and as it were classical forms of experimental glycosuria, pancreatic diabetes and phloridzin diabetes, as far as is here possible and as far as the present status of our knowl- edge permits, our attention may next be given to several atypical forms of glycosuria, namely, Icevulosuria, pento- suria and lactosuria. LiEVULOSURIA The literature devoted to laevulosuria, as may be seen in the comprehensive articles upon the subject by Neuberg, 27 Magnus- Levy 28 and Umber 29 occupies a considerable space, — greater, probably, than the actual importance of the sub- ject requires. Many cases in the older literature, said to be instances of laevulosuria because of their Polarimetrie char- acteristics, should be. excluded for the reason that in their determination no care was taken to exclude the possibility that laevogyration might have been due to ß-oxybutyric acid or combined glycuronic acids. Detection of Lcevulose. — One should not be content in try- ingtyo determine the existence of a hevulosuria with the recog- nition of disproportion between Polarimetrie features, grade of reducing and fermentescibility of the specimen, but should employ more direct methods. Seliwanoff's test in its various modifications, producing a red color when the specimen is boiled with resorcin and hydrochloric acid, is the most prominent. The red color changes to a yellow 49 Cf. G. G. Wilenko, Ther. d. Gegenw., 1909, 227; E. Frank and S. Isaak, Verhandl. d. 27. Kongr. f. inner Med. (Senator's Clinic), Wiesbaden, 1910, p. 590. 21 C. Neuberg, Handb. d. Pathol, d. Stoffwechs., 2d ed., 2, 212-219, 1907. 28 A. Magnus-Levy, Handb. d. Biochem., //', 385-394, 1909. 29 F. Umber, Lehrb. d. Ernährung, pp. 249-253, 1909. 282 LiEVULOSURIA in acetic ether when the solution is alkalinized with soda. The interpretation of the result of this reaction, however, requires considerable judgment, as the presence of nitrites in the urine can be mistaken for the presence of laevulose. 30 Other color reactions are also employed for the detection of laevulose, among them the blue coloration resulting from boiling the urine with diphenylamine and hydrochloric acid. 31 According to Neuberg, the asymmetrical methyl- Phenylhydrazine, Nn— NH 2 , forms an osazone directly CeHs' only with laevulose of the sugars which are to be considered, but with the isomeric aldehyde sugars (glucose, galactose, mannose) it constantly forms only the hydrazone. Neu- berg 32 regards this as an available test (contrary to objec- tions which have been raised to the trustworthiness of the reaction). 33 Transformation of Glucose into Lcevulose. — In addition to the difficulty of analysis mentioned it is essential, in order to properly appreciate the statements about laevulosuria in literature, to keep prominently in mind the fact that glucose and laevulose under the influence of hydroxyl-ions may be very readily transformed one into the other both within and outside the animal body. Thus in an alkaline diabetic urine, especially after use of alkaline drinking waters a so-called "urogenous" laevulosuria can very read- ily appear. 34 Although formerly much was said and written about laevulosuria in diabetes under the name ''mixed melituria," most of the recent investigators agree that a true laevulosuria, if it ever occurs, certainly is of very great 30 H. Rosin, Salkowski Festschrift, cited in Jahresber. f. Tierchem., Slf, 917, 1904; L. Borchardt, Zeitschr. f. physiol. Chem., 55, 241, 1908; 60, 411, 1909; H. Malfatti (Innsbruck), ibid., 58, 544, 1909; O. Adler (Prague), Pflüger's Arch., 139, 93, 1910. 31 Cf. Literature: A. Jolles, Münchener med. Wochenschr., 51, 353, 1910. ™ C. Neuberg, Ber. d. deutsch, chem. Ges., 37, 4616, 1904. ^ R. Ofner (Prague), Ber. d. deutsch, chem. Ges., 87, 3362, 1904. 31 Cf . H. Königsfeld, Zeitschr. f . klin. Med., 69, 3-4, 1909. ALIMENTARY LtEVULOSURIA 283 rarity. 35 Contrary statements can be clearly explained as faults of observation. 36 Tttie Lcevulosuria. — A spontaneous true laevulosuria seems to be a very rare abnormality, seen beyond question in only a very few cases, and cannot be said to be related with diabetes. It may be influenced by ingestion of laevulose or cane-sugar, but not of grape-sugar, and disappears on a carbohydrate free diet. 37 The significance of presence of laevulose in the amniotic fluid and in the urine of new-born calves is unknown. 38 At- tention has been called with propriety to the possibility that the laevulose excreted by the calves in the first few days of their lives may have come from amniotic fluid which they swallowed. 39 Alimentary Lcevulosuria. — In the course of the last few years a number of observations upon alimentary laevulosuria have been collected, frequently occurring, as Strauss first pointed out, along with disturbances of the hepatic function. Thus a decrease in tolerance for laevulose has been met in cirrhosis of the liver, in marked biliary stagnation, phos- phorus poisoning, and analogous conditions. 40 The fact that an alimentary laevulosuria is a frequent complication of pregnancy (v. sup.) may also be suggestive of a hepatic functional disturbance. Such functional faults are in sharp contrast with true diabetes, as in this latter condition, as has been seen, the tolerance for laevulose tends to be less dimin- ished than that for dextrose. So much for laevulosuria. M L. Borchardt, 1. c. ; O. Adler, 1. c. ; H. Malfatti, 1. c. ; H. Chr. Geelumyden (Christiania), Zeitschr. f. klin. Med., 70, 287, 1910. 36 Cf. criticism by Borchardt upon the work of W. Voit, Zeitschr. f. physiol. Chem., 58, 182, 1909; 60, 411, 1909, and reply thereto, ibid., 61, 92, 1909. 37 Cf. Literature: F. Umber, 1. c, pp. 250-251, and Magnus-Levy, 1. c, p. 391. "L. Langstein and C. Neuberg, Biochem. Zeitschr., ! t , 212, 1907. **A. Magnus-Levy, 1. c, p. 390. 40 Cf. Literature in F. Umber, Salkowski Festschr., 1904, and in H. Hohl- weg (Giessen) , Arch. f. klin. Med., 97, 443, 1909. 284 LACTOSURIA LACTOSURIA Another anomaly of metabolism of some physiological interest is lactosuria. 41 The study of this condition began with a discovery by Hofmeister in 1877 of a reducing substance in the urine of lying-in women which he recognized as milk-sugar. Since then we have framed fairly clear conceptions of the conditions under which lactose passes into the urine. We know as the ability to hydrolyse the milk-sugar and to make full use of it is missing from the blood and the tissues generally, that lactose thus entering the circulation paren- terally is excreted in the urine, as above noted. It is pos- sible that the animal economy may in a measure acquire the ability to split milk-sugar parenterally and utilize it if re- peated injections of milk-sugar be given 42 ; but at any rate this capability does not exist in the normal body. Lactosuria of Puerperal Women. — There is nothing re- markable in the fact that if a woman in the Puerperium sud- denly has an interruption of suckling her infant and of thus eliminating large amounts of milk-sugar in her milk, the breasts do not at once stop producing lactose. This pent-up lactose will eventually be resorbed into the blood and thence naturally pass into the urine. Lactosuria, therefore, often appears, and has been frequently observed, as a reaction of the system to a sudden weaning ; and may continue for a long time, especially in good nurses. In milk-cows lacto- suria is a physiological phenomenon. 43 Again, lactosuria may occur shortly before delivery, as at this time the mammary glands are beginning to assume their duty and even the colostrum contains lactose. C. v. Noorden and Zuelzer have observed even in cases of abor- 41 Literature upon Lactosuria: C. Neuberg, Handb. d. Pathol, d. Stoflfw., 2, 238-241, 1909; A. Magnus-Levy, Handb. d. Biochem., 4', 378-384, 1909. 42 Cf. J. S. Leopold and A. v. Reuss (Pediatric Clinic, Univ. Vienna), Monatschr. f. Kinderheilk, 8, 1, 453, 1909. 43 Sieg, Arch. f. Tierheilkunde, 35, 114, 1909, cited in Jahresber. f. Tierchem., 39, 663, 1909. LACTOSURIA IN INFANTS 285 tion, in which there could be no occasion for colostrum pro- duction normally, that the system was distinctly disposed toward elimination of milk-suger in that, after administra- tion of large doses of dextrose, there did not occur a glycosu- ria but a lactosuria. Here the less readily assimilated carbo- hydrate was forced out of metabolism by the more easily consumed one. C. v. Noorden interpreted this as a provi- sion in the interest of the offspring by which the economy in preparation for lactation loses its power of destroying milk-sugar. 44 That the lactose appearing in the urine of nursing individuals has its origin from the mammary glands appears from observations of disappearance of the lactose from the urine of lactating guinea pigs if their mammary glands are amputated. On the other hand, however, ob- servations of P. Best and Porcher 45 are recorded showing that removal of the mammary glands gives rise to glyco- suria (not lactosuria) in lactating goats. This observation (not uncontradicted) 46 was supposed to indicate that the liver is thus interrupted in furnishing to the functionating mammary glands the large amounts of glucose which may be supposed to be converted in the latter into milk-sugar. If this glucose from the liver is transferred into the circula- tion without the chance of its proper use because of the absence of the mammary glands, of course its excretion into the urine takes place to prevent the impending hypergly- cemia. Lactosuria in Infants. — The lactosuria of infants at the breast is quite a different affair. It has been carefully studied by Langstein and Steinitz, 47 and is referred to a pathological insufficiency of enzymic milk-sugar cleavage in " Cf. C. Neuberg, 1. c, p. 240. "C. Porcher, Compt. Rend., 140, 1279, 1905; 141, 73, 1905; Arch. Internat, de Physiol., 8, 356, 1909; Biochem. Zeitachr., 23, 370, 1910. 46 C. Foä (Turin), Arch, di fisiol., 8, cited in Biochem. Centralbl., S, 1587, 1909; A. Magnus-Levy and L. Zuntz, Handb. d. Biochem., 4', 382, 1909; B. Moore and W. H. Parker ( Yale Med. School, New Haven ) , Amer. Jour, of Physiol., 4, 239, 1900. e L. Langstein and F. Steinitz, Hofmeister's Beitr., 7, 575, 190G. 286 LACTOSURIA the intestine. It can be readily appreciated that the lactose absorbed without preceding cleavage and in a sense a foreign substance to the tissues, cannot be utilized in the economy of the infant. It should be noted that C. v. Noor- den and Zuelzer 48 have also encountered appreciable amounts of lactose in the urine of children, not suffering from gastroenteric affections, in the first year of life, pro- vided about 30 grams of milk-sugar be added to the food. A further possibility is that of the lactose which is split in the intestine before resorption the easily assimilable glucose is completely burned, while the less assimilable galactose fraction passes undecomposed to the kidneys so that in the urine the lactose is found mixed with galactose. Luzatto 49 noted after free administration of milk-sugar to a dog under certain experimental conditions only galactose, but no lactose, in the urine. Alimentary Galactosuria in Disturbances of Hepatic Function. — This brings us to the much discussed question of alimentary galactosuria. There is no doubt that the economy possesses the ability to transform galactose into grape-sugar. This is indicated in the first place by the fact that galactose is a possible source of glycogen, and in the second place by its quantitative conversion into urinary sugar in severe diabetes. The actual availability of galactose in the economy is always very much lower than that of dextrose and laevulose. This is particularly notable in the Carnivora, in which even after small dosage of galactose this sugar may appear in the urine. 50 In the course of the past few years alimentary galacto- suria has frequently attracted the attention of clinicians, who have for a long time been seeking for means of chemical * s C. v. Noorden, Handb. d. Pathol, d. Stoffwechs., 2d ed., 2, 56, 1907. 49 R. Luzatto ( Scbmiedeberg's Lab., Strassburg), Arch. f. exper. Pathol., 52, 106, 1905. 00 Literature upon Alimentary Galactosuria: A. Mangus-Levy, Handb. d. Biochem., 4', 379-381, 1909. DISTURBANCES OF HEPATIC FUNCTION 287 diagnosis of the condition of the liver in the living patient. From the studies of E. Bauer, 51 of v. Neusser's Clinic, which have met with confirmation from numerous sources, 52 an alimentary galactosuria seems to always afford a limited possibility of testing the hepatic function. While a sound liver is able to handle almost all of the galactose, when from 30 to 40 grams of this sugar are administered, in certain disturbances of the hepatic function an appreciable por- tion of it tends to appear in the urine. Acording to Bauer the test is positive in the various cirrhoses of the liver, in catarrhal jaundice, in phosphorus poisoning, acute yel- low atrophy, and the fatty liver of tuberculosis; but in passive hyperemia of the liver, in cholelithiasis, cancers, tumors, echinococcus disease and abscesses and in peri- hepatic affections it is said to be negative, as well as in all other affections in which the liver does not take part. How- ever, there are exceptions to this rule. Thus alimentary galactosuria is found in occasional cases of Basedow's dis- ease, and was met in a case of paroxysmal tachycardia recently under observation in Ortner's Clinic, which pre- sented symptoms of vagotonia and sympathicotonia. 53 In cases of this sort the alimentary galactosuria is apparently associated with an alimentary glycosuria. Bierry succeeded in inducing experimentally an alimen- tary galactosuria by producing severe lesions of the liver in dogs by means of chloroform injections. Milk sugar in dosage of one or two grams, easily utilized by a normal ani- mal, caused in animals thus prepared an elimination of galactose in the urine. 54 51 R. Bauer, Wiener med. Wochenschr., 1906, 21, 2537; Deutsche med. Wochenschr., 1908, No. 35; Wiener klin. Wochenschr., 1912, 939-941; cf. Literature in the last. 62 V. Reuss. Bondi and König, Neugebauer, Jehn and Reiss and others. 83 H. Politzer (Ortner's Clinic, Vienna), Wiener klin. Wochenschr., 1912, 1303; cf. also E. Reis3 and W. Jehn, R. Roubitschek ( Schwenkenbecher's Clinic, Frankfurt a. M.), Deutsch. Arch. f. klin. Med., 108, 187, 225, 1912. M H. Bierry, C. R. Soc. de Biol., 61, 204, 1906. 288 PENTOSURIA PENTOSURIA Another abnormality of carbohydrate metabolism, of rare occurrence it is true, but of considerable physiological interest, is pentosuria, 55 which was discovered by Salkowski in 1892. X-xylose. — Attention has been called in a previous lec- ture (v. Vol. I of this series, p. 117, et seq., Chemistry of the Tissues) to the importance of the pentoses in the construc- tion of animal and vegetable tissue. Carl Neuberg's fine researches leave no doubt that the sugar which exists so widely in the nucleoproteins of animals is A-xylose. The discovery of Neuberg and Salkowski, who observed glycuronic acid pass over into A-xylose in putrefaction, COOH.[CH(OH)] 4 .COH - C0 2 = CH 2 .OH. [CH(OH)] 3 . COH, has cleared up the obscurity which previously involved the origin of tissue pentoses. One cannot make any serious mistake in supposing that glucose under certain physio- logical circumstances can be changed by oxidation into glycuronic acid and that the latter by having C0 2 split off can be transformed into tissue pentose, even though up to the present this series of changes has not been proved beyond peradventure. Another important connection which the pentoses have with the processes of animal metabolism is seen in the fact that pentosans, the primary substances of the pentoses, occur widely in the vegetable kingdom. What their part is in the nutrition of herbivora can be easily appreciated if one considers that many types of forage contain twenty-five per cent, or more of pentosans. Although there is but little probability that the pentoses are directly changed into glucose, that is to say, into glycogen (vide supra, p. 230), and that this is conceivable only when aided by complicated 60 Literature upon Pentosuria: C. Neuberg, Ergebn. d. Physiol., 3', 405-410, 1904, and v. Noorden's Handb. d. Pathol, d. Stoffwechs., 2d ed., 2, 219-224, 1907; A. Magnus-Levy, Handb. d. Biochem., J t ' 395-406, 1909; F. Umber, Lehrb. d. Ernähr., pp. 242-248, 1909. PENTOSURIA IN DIABETES 289 synthetical processes, it is also not very probable a priori that the pentoses are destroyed (as by fermentation in the intestine) to such an extent as to be entirely lost as sources of energy for the body. 56 While herbivora are able to con- sume considerable quantities of pentoses, the human assim- ilation limit for these sugars (although man always destroys in his economy at least a part of any large amounts which have been ingested) is strikingly low; so much so, in fact, that even after ingestion of a single gram or a fraction of a gram of xylose, arabinose, rhamnose, etc., the pentose reactions may become positive in the urine. Alimentary Pentosuria. — There is no wonder, therefore, that an alimentary pentosuria of low grade (as indicated by the studies of P. Blumenthal and others), particularly after indulgence in fruits (cherries, plums, apples) and other vegetable materials, is met with appreciable frequency. Pentosuria in Diabetes. — It should also be easy to under- stand why a small amount of a pentose may be found mixed with the glucose in the urine of severe cases of human diabetes and of the dog in pancreatic diabetes, as was proved by E. Kiilz and J. Vogel. The nature of this sugar with five carbon atoms is apparently not well known, and in fact its optical activity never seems to have been tested. 57 One would probably not be far wrong in supposing that a portion of the tissue pentoses, which are set free from the nucleo- proteins in the breaking down of the tissues, can make their appearance in the urine. This idea is not contradicted by the fact that it is not possible to recognize by analysis a pentose impoverishment of the tissues in connection with pancreatic diabetes and phloridzin diabetes in animals. 58 56 Literature upon Pentosans as Foods : A. Magnus-Levy, 1. c, pp. 396-398. OT C. Neuberg, Handb. d. Pathol, d. Stoffwechs., 2d ed., 2, 223, 1907; cf. also W. Voit ( Sandmeyer's Sanatorium), Zeitschr. f. physiol. u. diät. Ther., 12, 659, 1909. K S. Mancini (Siena), Arch, di Farmakol., 5, 309, 1906, cited in Centralbl. f. Physiol., 20, 642, 1906; cf. also L. Caminotti, Biochem. Zeitschr., 22, 106, 1909. 19 290 PENTOSURIA Chronic Pentosuria. — The idiopathic form of pentosuria is a complete puzzle to us. This is a comparatively uncommon anomaly of metab- olism. Some twenty cases were collected a few years ago from the general literature. Some of the cases were neuro- pathic individuals, cocaine and morphine habitues ; in some a family disposition was recognized. The abnormality does not give rise to any special clinical symptoms. Carl Neu- berg, to whom we owe much valuable progress in the chem- istry and physiology of the carbohydrates, found that the sugar eliminated by the case of chronic pentosuria which he studied was directly different from the tissue pentose, A-xylose, and was really an inactive arabinose. "It is a recognized fact," says Neuberg, 59 "that all sorts of living beings produce optically active forms exclusively, and acti- vate racemic types offered to them by consuming one of the components. The example of inactive urinary pentose is the first instance of the occurrence of a racemic body in the animal economy. By the construction of urinary pentose pentosuria is characterized as a phenomenon sui generis." While the inactive arabinose if introduced into the body of a healthy individual (as shown by Neuberg and Wohlge- muth) undergoes cleavage into its two components, one of which undergoes decomposition, the other, however, 8-arabinose, appearing in the urine, we see that this cleavage power may come to be lost by the economy of the pentosuric. O. af Klercker 60 comes to the conclusion from his own studies that in pentosuria the two complementally isomeric pentoses are excreted in very varying proportions. While they are sometimes, as in Neuberg 's case, in equal amounts, the A-component may in some instances be in excess. It is not known from what source the arabinose comes. Its excretion, according to Blumenthal and Bial, is independent M C. Neuberg, Handb. d. Patbol. d. Stoffwechs., 2d ed., 2, 221, 1907. 00 0. af Klercker (Instit. of Med. Chem., Lund), Deutsch. Arch. f. klin. Med., 108, 277, 1912. CHRONIC PENTOSURIA 291 of the quantity of pentoses in the ingested nucleoproteids. There is just as little reason to refer it to the pentoses in combination in the tissue proteins, as the whole supply of pentoses in the human body is estimated at only about ten grams, while the amount of urinary pentose which may be excreted in a single day may reach thirty grams or more. Ingested xylose in a pentosuric (just as in a healthy person) may at least in part pass into the urine. There is no more evidence of connection between pentosuria and the metab- olism of dextrose ; it is not influenced by total withdrawal of carbohydrates, or by administration of phloridzin; and glycuronic acid excretion takes place in normal measure, according to Blumenthal and Bial, after administration of chloral or menthol. There is nothing left except to conclude that the pentosuric individual synthesizes his urinary pen- tose; where, how, or why, we do not know. Neuberg has suggested a possible connection between galactose and arabinose on the basis of their stereochemical molecular configuration : «-GALACTOSE a-ARABINOSE C.OH COH H.C.OH H.C.OH ! I OH.C.H OH.C.H OH.C.H OH.C.H. I I H.C.OH CH 2 .OH I CH 2 .OH Finally, Luzzatto 61 has shown in a case of pentosuria that even the exhibition of large quantities of glucose, cane-sugar or starch is incapable of modifying it in any way. If, how- ever, galactose be given, it is in greater part decomposed, but a small portion would seem to be transformed into pentose and the amount of the five-carbon- atom sugar in- w R. Luzzatto (Camerino), Arch. f. exper. Pathol., Schniiedeberg Festschr., 1908, 366. 292 PENTOSURIA creased. Confirmation of this work with a refined technic which will permit the possibility of quantitative differentia- tion between pentoses and hexoses, is very much to be desired. Detection of Pentoses. — Most cases of pentosuria are for a time regarded as diabetics and treated as such ; and yet the diagnosis of pentosuria is not a difficult one. Even with Fehling's test an attentive observer will notice that the fluid at first remains clear and only after considerable boiling is suddenly and fitfully reduced. Neuberg explains this peculiarity upon the supposition that pentose may be in the form of a ureide in the urine : CH 2 .OH-(CH.OH) 3 -COH+^^)CO=CH 2 .OH-(CH.OH)3-CH= N I J l2 ^)CO +H 2 0. Eeduction can take place only in proportion as such a ureide is split by hydrolytic influences and the aldehyde group thus set free; for which reason it is probable that many statements as to the amount of pentose in urines are too low. With special attention it will be found in investigating the urine of a pentosuric individual that the reducing sub- stance of the urine is neither f ermentescible nor is it optically active. The Phenylhydrazine test yields beautiful yellow osazone-needles, the melting point of which is, however, dis- tinctly lower than that of glucosazone. Transformation of arabinose into a diphenylhydrazone is, according to Neuberg and Wohlgemuth, also applicable in testing quantitatively for arabinose in the presence of other carbohydrates. Finally, the Tollens tests in their different modifications, the greenish-blue color obtained by heating with orcin and hydrochloric acid and the analogous red color with phloro- glucin, and the spectroscopic behavior of the coloring matter obtained by shaking with amylalcohol, are very useful, even if they are not absolutely distinctive. Jolles recommends CAMMIDGE REACTION 293 distillation of the isolated pentosazone with hydrochloric acid and subjecting the distillate, containing furfurol, to the orcin test; by which means it is said confusion of pentose with a hexose or a combined glycuronic acid may be pre- vented. 62 Cammidge Reaction. — In concluding the consideration of pentosuria the reaction of Cammidge, which in recent years has attracted a great deal of attention, may be briefly spoken of. Cammidge 63 some years ago announced that he believed he had found a characteristic urine reaction for chronic dis- eases of the pancreas. The substance concerned in this reaction could be obtained by boiling the urine with hydro- chloric acid, neutralizing with lead carbonate, and precipitat- ing with alkaline lead. From the precipitate a solution can be obtained by the decomposing action of sulphuretted hydrogen, yielding the usual carbohydrate reactions and an osazone (apparently a pentosazone). Cammidge, who dis- covered this reaction in dogs in which a pancreatitis had been artificially produced, offered for it what was probably a correct interpretation, relating it with an excretion of sugars of five carbon atoms which were derived from the breaking down of nucleoproteins of the pancreas, which is rich in pen- tose. Many later investigators have much simplified the rather detailed process of Cammidge, at least to the end of satisfactorily demonstrating the presence of an osazone in an apparently sugar-free urine after hydrolysis with hydro- chloric acid. Cammidge 's contribution started up a perfect flood of publications, a detailed review of which is perhaps unneces- sary here. Whoever may desire to do so may easily find articles by the dozen in the last ten years of the journals devoting themselves to the publication of abstracts. The 02 A. Jolles, Biochem. Zeitschr., 2, 243, 1906; Münchener med. Wochenschr., 57, 353, 1910. 63 P. T. Cammidge, Proc. Roy. Soc, London, Series B, 81, 372, 1909. 294 PENTOSURIA author has often wondered what factors make particularly for the interest of the great medical public in a discovery in the field of his particular specialty. That such interest should be prominently shown for those points which may be expected to be of direct use in the practice of medicine is quite conceivable. But it is not so easy to understand why the greatest interest is so often not manifested for new facts that are clear and unambiguous and represent real advances, but by preference is turned to indefinite, vague and uncertain features in which there is no trace of really precise adapta- tion of chemical and physiological concepts. It is probably not wrong to believe that it is due to the fact that there are many people who at heart are glad to avoid the rather great inconveniences which a regulated course of chemical study brings with it, but who in their own interest and that of pos- terity are unwilling to forego having a new starlet blaze in the firmament of biochemical research. According to their experience these same people have an invincible and instinc- tive preference for those fields of investigation in which discovery is decidedly more easy than control of the dis- coveries made by others. But to return to Cammidge's reaction, opinions generally contradict its practical utility as an aid in functional diag- nosis of pancreatic affections. As to its real value it would seem from a few studies of critical character 64 to be fully shown to be neither distinctive nor especially reliable. It clearly does not deal with any one single substance; the presence of any poly- or disaccharide in the urine may by cleavage give rise to an osazone-forming atomic group. Even the small amounts of polymeric carbohydrates which exist normally in the urine, particularly after a diet rich in 44 O. Schumm and Hegler, Münchener med. Wochenschr., 56, 1878, 1909, Mitt. a. d. Hamburgischen Staatskrankenanstalten, 11, 1910, cited in Centralbl. f. d. ges. Biol., 10, No. 1933; L. Grimbert and R. Bernier, Jour, de Pharm, et de Chem., 30, 529, 1909, cited in Biochem. Centralbl., 9, No. 1643; J. E. Schmidt (Enderlen Clinic, Würzburg), Mitt. a. d. Grenzgebiete d. Med. u. Chir., 20, 426, 1909. VARIOUS EXPERIMENTAL GLYCOSURIAS 295 sugar, 65 may give a positive result by this reaction ; which may on the other hand, however, be due to excreted gly- curonic acids and pentoses. It cannot possibly be regarded, therefore, as specific for pancreatic break-down, the more so because any actual nuclear decomposition in the tissues may be followed, as has been seen, by passage of pentoses into the urine. This aside, of course the particularly large amount of pentoses in the pancreas (which is said to contain five times the quantity of pentose as the liver and other glan- dular organs and about twenty times more than do the muscles) 66 favors the passage of pentoses into the urine when this organ undergoes destructive changes. For this reason there is some value in examining the urine for pentoses as an aid to the diagnosis of pancreatic affections. EXPERIMENTAL GLYCOSURIAS OF VARIOUS KINDS The list of the recognized examples of this abnormality of metabolism is far from exhausted even after completing the above mentioned types of sugar excretion in the urine. We are acquainted also with a large number of possibilties of inducing experimental glycosurias by interferences of many kinds. A review of these may be considerably facilitated by dividing, as Pollak 67 does, the glycosurias due primarily to renal influence from the glycosurias following hyper- glycemia. Renal Glycosurias. — To the first of these groups, besides the phloridzin diabetes already considered, certain glyco- surias caused by renal poisons are to be referred. According to Ellinger and Seelig in rabbits in a certain stage of cantharides intoxication it is impossible to induce a glyco- suria by adrenin and the elimination of sugar by dogs with 65 Cf. R. Wilhelm, 8 internat. Physiol. Kongr., Vienna, 1910. (There would appear to be a synthesis therein of mono- to polysaccharides, which pass into the urine.) ,S G. Grund (Salkowski's Lab.), Zeitschr. f. physiol. Chem., 35, 111, 1902. 61 L. Pollak (Pharmakol. Instit., Vienna), Arch. f. exper. Pathol., 61, 366, 1909. 296 VARIOUS EXPERIMENTAL GLYCOSURIAS pancreatic diabetes may be lowered by inducing renal lesions. 68 In this stage of the poisoning, according to L. Pollak, 69 it is possible to call out a glycosuria by super- position of uranium poisoning, although the amount of sugar in the blood is raised but little or not at all above the normal by this measure. The explanation proposed is that while cantharidin restricts the permeability of the vessels of the kidneys for sugar, uranium makes them abnormally permeable; and it is not improbable that a comparable causative mechanism obtains in chromium and bichloride of mercury glycosuria. A renal glycosuria has also been observed occasionally in human beings with chronic nephritis. The long list of glycosurias which are accompanied by hyperglycemia includes, according to L. Pollak (exclusive of pancreatic diabetes which occupies a special position) a number of glycosurias induced by sympathetic nervous irri- tation. The factors producing the glycosuria are again to be grouped in two classes: first, those which (analogous to the sugar-puncture) occasion irritation of nervous centres (transmitted by sympathetic fibres, especially the splanch- nics, to the liver and cause the latter to throw off its glycogen deposit) ; and second, those which (analogous to adrenin glycosuria) cause an irritation of the peripheral endings of the sympathetics. Sugar Puncture. — We know enough about the essential nature of the sugar puncture of Claude Bernard to under- stand the parts the nerve impulses traverse which produce glycosuria after injury of the floor of the fourth ventricle of the brain. This nerve path extends from the "sugar centre" to the cervical cord, thence out through communi- cating branches to the lower cervical and upper thoracic 68 A. Ellinger and A. Seelig (Königsberg), Münchener med. Wochenschr... 1905, 345, 449, and Festschr. f. M. Jaffe, p. 349, 1901. 69 L. Pollak (Pharmakol. Instit., Vienna), Arch. f. exper. Pathol., 64, 415, 1911. SUGAR PUNCTURE 297 sympathetic ganglia, and thence along the splanchnics to the liver. We know, too, that stimulation of the central segment of the vagus and of numerous sensory (particularly visceral) nerves will induce activity of the sugar-centre, and that a great variety of anatomical lesions of the central nervous system (from traumatism, tumors, abscesses, hemorrhages, etc.) may act in the same way. Psychic influences may also induce glycosuria. For example, in cats that have been shut up in a cage and worried for half an hour by a dog enough to have had the latter very manifestly ' ' get on their nerves, ' ' a glycosuria may be observed. 70 We have no reason for doubting that the mechanism of the sugar puncture involves the liver and includes a process of discharging its glycogen supply; but it is not clearly understood in all points. Only recently two French investi- gators ( Wertheimer and Battez) have called attention to the effectiveness of the sugar puncture even in animals which have been placed under the influence of atropin sufficiently to induce a paralysis of all secretory nerves ; and they, there- fore, hold that the supposed connection of the sugar puncture with stimulation of glyco secretory nerves is decidedly ques- tionable. 71 The possibility of a connection between the su- gar puncture and the secretory activity of the adrenals will be taken up fully in the succeeding lecture, in which the re- lation between the ' ' internal secretory glands ' ' and carbohy- drate metabolism will be considered. Attention should also be called here to the occurrence of glycosuria, according to observations of Minkowski, Bedard, Pflüger and other authors, 72 after a great variety of opera- tive interferences ; it is especially likely to follow irritation of the bowel and peritoneum, according to Pflüger. Zak states that even the mechanical irritation of the intestine 70 W. B. Cannon, A. T. Strohl and W. S. Wright (Harvard Med. School), Amer. Jour, of Physiol., 29, 280, 1911. 71 E. Wertheimer and G. Battez, Arch, internat. de Physiol., 9, 140, 363, 1910; C. R. Soc. de Biol., 66, 1059, 1909. 72 Rose, Zak, Gaulthier, Eichler and Silbergleit, Visentini. 298 VARIOUS EXPERIMENTAL GLYCOSURIAS occasioned by introducing a drainage tube may produce this effect. Ulrich Rose invariably found a hyperglycemia, and sometimes, too, glycosuria, in rabbits after a simple laparotomy. And Kreidl and Winkler 73 have been able to prove that after opening the peritoneal cavity in dogs and cats, but less frequently in rabbits, it is almost a regular thing to find sugar in the urine. We would probably not be far out of the way in supposing that in all procedures of this sort, comparably with Bernard's puncture, we have to deal with an effect upon the sympathetic nervous apparatus and with a discharge of glycogen from its place of storage. Toxic Glycosurias. — L. Pollak is disposed to regard many forms of toxic glycosuria, as that in the course of caffein poisoning and strychnine poisoning, as due to a central nervous irritation analogous to that produced by the punc- ture. All sorts of explanations have been offered for the action of other harmful chemicals like ether, chloroform, morphine, nitrite of amyl or carbon monoxide, but thus far without any uniformity at all. 74 Salt Glycosuria. — "Salt glycosuria" is also supposed to be due to a direct irritation of the sugar centre. Injection of large amounts of a dilute (about one per cent.) solution of sodium chloride into the vascular system of an animal, is likely to occasion a glycosuria, according to M. H. Fischer, but fails to do so if the splanchnic nerves be cut. A glyco- suria of much more marked intensity can be induced if the salt solution be introduced directly into the vertebral artery of the experiment animal instead of into a vein, thus insuring a direct effect upon the nervous centres. Injections of sea water, diluted to the osmotic pressure of the blood, will also give rise to glycosuria. The glycosuria produced by sodium chloride can be inhibited to some degree by injection of 78 F. Winkler (under direction of A. Kreidl, Vienna), Centralbl. f. Physiol., 21t, No. 8, 1910; cf. therein Literature. "Literature upon Toxic Glycosurias: K. Glässner, Wiener klin. Wochenschr., 1909, No. 26. GLYCOSURIA FROM CHILL 299 potassium and calcium salts. 75 Injection of concentrated salt solutions produces pronounced hyperglycemia, but there is also induced an alteration in the renal efficiency (probably due to change of the osmotic pressure relations) and the glycosuria may fail to appear. 76 Glycosuria from Chill. — "Befrigeration glycosuria" should finally be briefly mentioned, observed first by Böhm and Hoffmann and later by Araki when they reduced the body temperature of warm blooded animals to a low level by cold baths, snow packs, etc. Grlässner 77 was in the same way able to recognize glycosuria in individuals who had at- tempted suicide by jumping into cold water. Whether the coincident occurrence of lactic acid, which may be regarded as due to the combined influence of oxygen impoverishment and increased muscular activity, has any direct connection with the glycosuria, may remain undecided. Even in frogs a glycosuria may be induced by exposure to intense cold, interpreted by M. Löwit 78 on the hypothesis that the oxida- tion processes are interfered with, and that as a result there may ensue disturbance in the consumption of sugar, altera- tion of the renal permeability and glycosuria. " M. H. Fischer, Pfltiger's Arch., 109, 1, 1905, and earlier contributions: O. H. Brown, Amer. Jour, of Physiol., 10, 378, 1904; T. C. Burnett (Univ. of California), Jour, of Biol. Chem., 4, 57; 5, 351, 1908. T6 G. Wilenko (Lemberg), Arch. f. exper. Pathol., 66, 143, 1911. " K. Glässner, Wiener klin. Wochenschr., 1906, p. 30. 78 M. Löwit, Arch. f. exper. Pathol., 60, 1908. CHAPTER XIII RELATIONS OF GLANDS WITH INTERNAL SECRE- TION TO CARBOHYDRATE METABOLISM. GLYCURONIC ACIDS RELATION OF THE ADRENALS TO CARBOHYDRATE METABOLISM In the course of a series of earlier lectures the writer attempted to present what is known of the role and the significance of "the glands with internal secretion," at least as far as the principal points are concerned. An important phase of this physiological problem has not, however, been systematically dealt with, the relations these puzzling organs bear to carbohydrate metabolism. In the present lecture it is proposed to fill in this hiatus and to consider the matter in an objective manner, so that it may be possible to come to some conclusion to what extent we are justified in the effort so often made in the course of the last few years to penetrate into the secrets of normal and pathological car- bohydrate metabolism and to interweave these with the no less secret and obscure mysteries of the "ductless glands" into one organic whole. Nature of Adrenal Diabetes. — The line of thought which is principally to occupy our attention in the present discussion, takes its inception from the discovery of adrenal diabetes. 1 In 1901 F. Blum, in Frankfurt, a. M., made the remark- able and important discovery that injection of the substance in the adrenals which induces increase of blood pressure (known in the present stage of science by the term Supra- renin or adrenin) is followed by an intense glycosuria of short duration in various experiment animals. The very great interest always manifested in the problem of diabetes 1 Literature upon Adrenal Diabetes: A. Biedl, Innere Secretion, pp. 202- 204, 1910; R. Hirsch, Handb. d. Biochem., 3, 322-325, 1910; G. Bayer, Ergebn. d. pathol. Anat., U h 101-105, 117-119, 1910. 300 NATURE OF ADRENAL DIABETES 301 from its clinical and physiological standpoints is sufficiently explanatory of the flood of publications persisting even now which had its beginning in this discovery. The glycosuric influence of Suprarenin is appreciable only when it is introduced subcutaneously, intravenously or intra- peritoneally. When given by the mouth even large doses are without effect upon the carbohydrate metabolism and the blood pressure. The glycosuria is directly related with the supply of glycogen in the liver and its liquidation. In- jection of adrenin directly into a mesenteric vein acts, ac- cording to Doyen, in an almost paroxysmal manner, although in dogs with Eck's fistulas it produces neither hyperglycemia nor glycosuria. 2 Between the amount of adrenin present in the blood and that of the sugar excreted in the urine there exists within certain limits a direct ratio, according to studies made in Straub 's laboratory. The occurence of a stage of latency is clearly due to the fact that a certain amount of time is necessary to put the mechanism of sugar mobilization in motion. Straub believes, as do others, that the parts influenced by the adrenin are the same sympa- thetic nerve fibres whose central irritation is responsible for the effectiveness of the sugar puncture. 3 As the Supra- renin acts on the endings of the sympathetic fibres, one can readily appreciate why splanchnicotomy does not prevent its glycosuric effect. 4 What actually takes place is that the liver under the influence of the poison passes its supply of glycogen into solution; if this supply is an abundant one (perhaps after preceding indulgence in carbohydrates) a stream of sugar will pass out from the liver into the blood. But if the store of glycogen has been used up through the 'Michaud (Kiel), Verh. d. Kongr. f. innere Med., Wiesbaden, 1911. 8 W. Straub, Münchener med. Wochenschr., 1909, 493 ; H. Ritzmann, Arch, f. exper. Pathol., 61, 231, 1909; cf. also F. P. Underhill and 0. E. Closson (Yale Univ., New Haven), Amer. Jour, of Physiol., 11, 421, 1906. 4 Cf. L. Pollak (Pharm. Instit., Vienna), Arch. f. exper. Pathol., 61, 1909; also the observations of K. Hirayama upon the interruption of sympathetic conduction by nicotine: Zeitschr. f. exper. Pathol., 8, 649, 1911. 302 ADRENALS IN CARBOHYDRATE METABOLISM influence of hunger, physical exertion, cold or earlier injec- tions of Suprarenin, phloridzin, etc., glycosuria may fail to appear. In a previous lecture occasion was taken to call attention to the fact that the economy is abundantly provided with means to maintain the sugar in the blood at constant level and that if the carbohydrate elements are used up it is in position to build sugar de novo from protein. An excel- lent example of this process is afforded in recent studies of L. Pollak, who has been able to show in the Pharmacological Institute in Vienna that in starving rabbits which have had their glycogen removed by strychnine convulsions, repeated administration of Suprarenin in increasing dosage may in- duce an accumulation of glycogen in the liver, of proportions elsewhere seen only in animals fed upon carbohydrates. It is of interest to note that the muscles, which usually (as the most important physiological sites of glycogen consump- tion) have a tendency to attract the carbohydrate stores from the liver for their own purposes, are under these circum- stances entirely or almost entirely free of glycogen. 5 Dependence of Adrenin Glycosuria Upon the Renal Function. — Suprarenin diabetes should undoubtedly be classed from its essential character among the glycosurias accompanied by hyperglycemia. Whether it is possible to actually find at a given moment after adrenin application that the level of sugar in the blood is distinctly raised, de- pends upon the precision and readiness with which the kid- ney affords passage of a surplus of sugar from the blood into the urine. Here, too, studies in the Vienna Pharmacologi- cal Institute have led to important conclusions. Thus it has been proved that in rabbits a proportion of 0.15 to 0.25 per cent, of sugar in the blood will give rise to glycosuria only in case a marked diuresis coexists (as after use of 5 L. Pollak (Pharmakol. Instit., Vienna), Arch. f. exper. Pathol., Gl, 166, 1909; cf. therein Literature: Doyen and Kareff, Gatin-Gruzewska, Agadschi- ananz, et al.; W. B. Drummond and D. Noel Paton, Jour, of Physiol., 31, 98, 1904. ADRENIN GLYCOSURIA 303 caffein) ; without the diuresis the glycosuria remains in abeyance. If the proportion of sugar in the blood exceeds 0.25 per cent, there is no longer necessity for the diuresis to put the glycosuria in operation. After repeated subcuta- neous injections of adrenin, however, the kidneys may acquire a sort of tolerance, making it possible that glyco- suria will not be manifested even when the proportion of sugar in the blood becomes very high. 6 While under certain conditions even with approximately normal proportions of sugar in the blood a glycosuria can be caused by certain nephrotoxines like chromium, uranium or corrosive sub- limate (" renal diabetes"), there are other renal lesions (as temporary ligation of the renal artery), according to Ellinger and Seelig, 7 which promptly inhibit the glycosuria caused by adrenalin. There is scarcely any doubt that some alteration of the renal circulation is directly concerned with the suppression of adrenin glycosuria, so often seen after injection of lymphagogues (extract of crab meat, 8 hirudin, 9 and Witte 's peptone 10 ), in infectious diseases, 11 and experi- mentally produced fever, 12 after extirpation of both adrenals, 13 and other severe operative interferences, after peritoneal irritations u (which may be produced by injec- tion of trypsin or pancreatic juice, vide supra, p. 262), after injections of calcium chloride, 15 etc. Occasion was taken in a previous lecture to point out that it is entirely reasonable to believe that passage of adrenin from the adrenal medulla into the circulating blood takes 6 L. Pollak, 1. c, p. 157. 7 A. Ellinger and Seelig, Münchener med. Wochensckr., 1905, No. 11. 8 A. Biedl and Offer, Wiener klin. Wochenschr., 1907, 1530. 8 Mikulicich, 1. c. 10 K. Glässner, and E. Pick, Zeitschr. f. exper. Pathol., 6, 1909. 11 G. Ghedini and G. Mascerpa, Folia Clinica, 2, H. 3, cited in Centralbl. f. d. ges. Biol., 11, No. 2508. 12 Aronsohn, Virchow's Arch., 174, 383, 1903. 13 J. Gautrelet and L. Thomas, C. R. Soc. de Biol., 66, 798, 1909. "O. v. Fürth and C. Schwarz, Biochem. Zeitschr., 31, 113, 1911. 15 F. Schrank, Zeitschr. f. klin. Med., 67, 230, 1908. 304 ADRENALS IN CARBOHYDRATE METABOLISM place as a physiological process. It is, therefore, not at all surprising that Herter should have succeeded in bringing on glycosuria by squeezing the normal adrenals in situ. Hypothesis of the Regulating Influence of Adrenin Upon Normal Carbohydrate Metabolism. — It is but a step from this point to the proposition that the passage of the adrenin from the adrenals into the blood stream has normally a regulative influence upon carbohydrate metabolism. Thence it is suggested that the adrenin having entered the circula- tion normally from the adrenals excites the sympathetic nerve terminations in the liver (in the same way as in elec- tric irritation of the sympathetic, in the sugar puncture, in stimulation of the central end of the vagus, in asphyxia, in the effect of carbon monoxide, caffein and many of the narcotics), 16 and that such a condition of stimulation can under proper conditions give rise to a discharge of the hepatic glycogen, to hyperglycemia and to glycosuria. Does the Sugar Puncture Act Through the Adrenals to Cause Glycosuria? — From this point of view a line of thought has been developed, that Claude Bernard's sugar puncture may possibly not have a direct effect upon the liver, but may perhaps act indirectly by way of the adrenals, inducing pri- marily a massive output of adrenin from the adrenals into the blood, with production in this way of a simple adrenin glycosuria, in complete analogy to the effect of an intra- venous injection of this very active substance into the cir- culation. Soon after F. Blum, 17 the discoverer of adrenal diabetes, published his belief that a close similarity exists between this condition and glycosuria from Bernard's puncture, the logical suggestion from which would be to investigate whether the latter does not have its influence on the liver 16 E. Starkenstein (J. Pohl's Lab., Prague), Zeitschr. f. exper. Pathol., 10, 1911. 17 F. Blum (Frankfurt, a. M.), Pfliiger's Arch., 90, 628, 1902. EXCLUSION OF ADRENALS 305 through the adrenals, the glycosuric puncture was studied from this view point by quite a number of researchers. Let us consider for a moment what objective facts we ought to demand in order to regard as proved such a thor- oughly complicated relation as the above mentioned hypothesis assumes. We should expect in the first place that if the adrenals were removed the glycosuric puncture would be without its customary effect. We might further very properly suppose that a massive discharge of this substance would be fol- lowed by a noticeable impoverishment in the adrenal medulla of its pressure-raising chromogen. And finally we ought to require evidence that the sugar puncture really is fol- lowed by an excessive presence of Suprarenin in the blood. Closer examination may now be made as to what results experiments have afforded which may be applied to each one of these postulates. Failure of Effect of Glycosuric Puncture After Exclusion of the Adrenals. — The first point to be made (determined coincidently by A. Meyers, E. Landau and R. H. Kahn) is that the puncture is no longer capable of producing glyco- suria when the adrenals have both been extirpated. This is all the more notable in case of operated animals which survive the operation for some length of time and are in good health. In such animals, too, no hyperglycemia is met from splanchnic irritation. 18 The plausible explanation that in animals deprived of their adrenals there is simply a reduction in the glycogen supplies may hold in case of rats and dogs, 19 but apparently not in case of rabbits, which may survive the bilateral removal of the adrenals for as much 18 J. J. R. Macleod and R. G. Pearce, Amer. Jour, of Physiol., 29, 419, 1912; cf. also the diuretin experiments of M. Nishi (Pharmacol. Instit., Vienna), Arch. f. exper. Pathol., 61, 401, 1909. 18 0. Schwarz (E. Pick's Lab., Vienna), Pflüger's Arch., 134, 259, 1910; O. Porges (v. Noorden's Clinic, Vienna), Zeitschr. f. klin. Med., 69, 3-4, 1909; 10, 1910. 20 306 ADRENALS IN CARBOHYDRATE METABOLISM as a year, take on weight, show a normal amount of glyco- gen, and differ from normal animals apparently only in the fact that the glycosuric puncture has no effect on them. 20 It should be recalled, too, that hypoglycemia and increased tolerance for sugar has been met in sequence to removal of the two adrenals in dogs and to Addison's disease in man. 21 Considerable importance on the other hand, should be attributed to the finding that even in animals deprived of the adrenals hyperglycemia may be caused by irritation of the central segment of the vagus nerve 22 ; this is a positive indication that the ' ' sugar centre ' ' can exert its power en- tirely independently of the adrenals. It seems possible, too, that the failure of puncture glycosuria and other re- lated glycosurias 23 after extirpation of the adrenals, in the course of which operation it is not unlikely that lesions of the network of nerves about the kidney may have been pro- duced (in line with what has already been said), may be ex- plained with much probability and quite fully on the assump- tion of some disturbance of the renal circulation. Chromium Affinity of the Adrenals. — An observation of Kahn is directly applicable to the second of our postulates. "If one adrenal be removed from a rabbit and glycosuric puncture be performed, and the second adrenal be extirpated some time after the appearance of glycosuria, it will be found on comparison of the two organs that the second one re- moved has undergone very decided alteration. The affinity for chromium is very greatly reduced, the cells are poor in granules and rich in vacuoles ; the more minute vessels are for the most part distended ; and its adrenin is very much 20 R. H. Kahn and E. Starkenstein (Prague), Pfliiger's Arch., 139, 181, 1911. 21 0. Porges, 1. c. 22 E. Starkenstein, 1. c. 23 Cf. J. J. R. Macleod and R. G. Pearce, Amer. Jour, of Physiol., 29, 419, 1912. ADRENIN IN BLOOD IN SUGAR PUNCTURE 307 reduced. Section of the corresponding splanchnic nerve protects the adrenal from these changes after glycosuria puncture. ' ' 24 Question of Increase of Adrenin After Sugar Puncture. — What of the third and most important of our postulates, on which in fact the whole hypothesis stands or falls, the detec- tion of an increased amount of Suprarenin in the blood after the sugar puncture? While Watermann and Smit 25 be- lieved they had found an increase of Suprarenin in the blood after sugar puncture by means of the frog's eye method, not only Kahn 26 but also E. Th. von Brücke 27 failed to recog- nize such an increase. It should be said that the latter used in determining the Suprarenin in the blood serum the very delicate Trendelenburg-Läwen test, which is based on the idea that the vasoconstrictor power of a solution under ex- amination may be tested by the variation in escape of fluid when perfused through the hind legs of a frog. Kahn 2S has objected to the force of this experiment because after sub- cutaneous administration of Suprarenin in small doses, but sufficient to cause glycosuria, it is impossible, by available methods, to recognize any accretion of this substance in the blood. The author confesses that he is not entirely convinced of the extent to which this objection is justified. For if the Suprarenin passes out from the adrenals into the circulation in sugar puncture, it does so not by a subcutaneous route but intravenously. As Kahn emphasized, adrenin-glycosuria will appear in half -grown rabbits from doses of 0.1 milli- gram upwards. A dose of this size given intravenously 24 R. H. Kahn (Prague) , Pflüger's Arch., 11(0, 254, 1911. 25 N. Watermann and H. J. Smit, Pflüger's Arch., 12k, 198, 1908. 16 R. H. Kahn (Prague), Pflüger's Arch., 128, 519, 1909. 27 E. Th. von Brücke (Leipzig), Münchener med. Wochenschr., 1911, No. 26; T. Negrin y Lopes (Physiol. Instit.. Leipzig) , Pflüger's Arch., 1^5, 311, 1912. 28 R. H. Kahn (Prague), Pflüger's Arch., 1U, 251, 1912. 308 ADRENALS IN CARBOHYDRATE METABOLISM would show a powerful vasoconstricting effect unless in injection it were spread out over a long space of time. Each one of us will necessarily value the above objection accord- ing to his own idea whether the adrenal medullary substance, from a stimulus passing along the nerve paths to it, dis- charges its store of Suprarenin suddenly, or whether it allows it to ooze out slowly. But this is the same old story: as long as an accumulation of adrenin in the blood after the glycosuric puncture is not actually proved, we will lack authority for assuming that the puncture glycosuria is caused by such means. Here is a place in physiology where we are forced, if we are not willing to lose the ground under our feet, to hold on to things we can see, even if we are very well aware that there is plenty which we do not see. It has been well said "that the negative finding in reference to the sugar puncture proves nothing more than the impossibility of demonstrating an adrenaemia from this disturbance but in no way contradicts the possibility of its existence. ' ' 29 We are here not after something that is possible (many things are possible), but something that is probable. The fact that a hypothesis starts up in an investigator's brain and that at the time we are not in position to fully prove its incorrect- ness does not force us by a great deal to accord reality to it. It does not lie at our door to bring proof that a hypo- thesis is incorrect; but whoever proposes a hypothesis has to bring the positive proof that it is correct. In this way and not in the reverse we hold in other exact sciences, and so, too, it should be held in physiology. Here is a rare little verse which perhaps has occurred to some in this connection : 1 l Oh, learned man, 'tis thus I know you, face to face ! What you don 't touch stands miles from you apace ; What you don't grasp, for you is gone for woe and weal; 29 R. H. Kahn, Pflüger's Arch., 1U, 271, 1912. DO ADRENALS HAVE REGULATING INFLUENCE ? 309 What you don't see, you're sure cannot be real; What you don't weigh, ne'er has a weight for you; What you don't coin, you think don't count a sou." The author will not evade the point. But for the present in biochemistry we make the most progress if we hold to the things we can see, and weigh and count. Do the Adrenals Have a Regulating Influence on the Normal Carbohydrate Metabolism? — Even were a positive proof obtained that the sugar-puncture acts essentially like an adrenin glycosuria, it would still be far from proving that under normal physiological conditions the internal secretion of the adrenals possesses a regulative effect on the metabolism of carbohydrates. Even if a sudden massive expulsion of Suprarenin from the adrenal medulla does cause a glycosuria this might very well be a toxic glycosuria, of which there are so many examples. Whether the minimal quantities of adrenin which in normal conditions enter the blood have anything to do with the liquidation of the glycogen stores and with the regula- tion of carbohydrate metabolism, remains, therefore, an open question, although many writers today regard a rela- tion of this sort as a settled fact. Thus Falta 30 thinks that every diabetic disturbance of metabolism may be looked upon as a predominance of sympathetic impulses over the autonomous ones. "If the cause of it is seen rather in some insufficiency of the pancreas it is proper to speak of a pan- creatogenous diabetes ; if it is rather in an excessive func- tion of the circulatory system, to say it is an adrenalogenous one. ' ' It should be added, however, that the recognition of a hypoglycemia and increased tolerance for carbohydrates in a few cases of Addison's disease 31 is in direct contrast to 30 W. Falta, Prager. med. Woehenschr., 1910, No. 7. 31 O. Porges, 1. c. ; H. Eppinger, W. Falta and C. Rudinger, Zeitschr. f . klin. Med., 66, 50, 1908. 310 ADRENALS IN CARBOHYDRATE METABOLISM observations in which in rabbits with adrenals removed the amount of sugar in the blood was, as a rule, found not reduced. 32 0. Schwartz, who found that the glycosuric effect of phloridzin was entirely preserved in rats deprived of their adrenals, concludes after a critical discussion of the above statements that ' ' the assumption of a sugar-mobiliz- ing function of adrenin, hitherto accepted without any direct experimental basis, can no longer be maintained. " 33 In view of the recognized antagonism between the sympathetic and autonymous nerve systems and the fact that almost always, where organs have a double innervation, this double supply is an antagonistic one, and stimulation of the sym- pathetic fibres produces characteristically the opposite action from that of the autonymous nerves, 34 it is quite well worth observing that the glycosuria produced by adrenin is not influenced by its antagonists, cholin and pilocarpin. This certainly does not speak in favor of the assumption that sugar metabolism is subject to a delicate regulation by Suprarenin through stimulation of the sympathetics. 35 An idea has also been suggested that the adrenals influ- ence sugar metabolism in some way through the pancreas. But for this view, also, there is no definite basis ; in geese and ducks, in which extirpation of the pancreas does not occasion sugar excretion, Suprarenin produces glycosuria even after removal of the pancreas. 36 The pancreas is, of course, not responsible for the production of this effect. So much for the relation of the adrenals to carbohydrate metabolism. 83 E. Frank and S. Isaak (Wiesbaden), Zeitschr. f. exper. Pathol., 7, 326, 1909. 83 O. Schwarz (E. Pick's Lab., Vienna), Pflüger's Arch., 13k, 257, 1910; cf. also J. J. R. Macleod and R. G. Pearce, Amer. Jour, of Physiol., 29, 419, 1912. 31 Cf. H. H. Meyer and R. Gottlieb, Exper. Pharmacol., p. 122, 1910, and A. Fröhlich (Pharmakol. Instit., Vienna), Med. Klinik, 1911, No. 8. 85 E. Frank and S. Isaak, 1. c. 38 Noel Paton, Jour, of Physiol., 32, 59, 1909. REMOVAL OF THYROID 311 RELATIONS OF THE THYROID GLAND TO CARBOHYDRATE METABOLISM We next turn to a no less knotty and troublesome sub- ject, the problem of the relation of the thyroid gland to the carbohydrate metabolism of the body. Removal of the Thyroid and of the Parathyroids. — In or- der to prevent confusion it is necessary to strictly differenti- ate between the thyroid gland and the parathyroid bodies. From studies of cases of myxcedema 37 and upon dogs after extirpation of their thyroids, a decided increase of sugar tolerance has been observed following loss of the thyroid function, and with this a marked general slowing of the transformation processes. 38 Thus a dog without its thyroid can tolerate very large quantities of ingested sugar without the appearance of an alimentary glycosuria. So, too, adrenin glycosuria may fail to appear in different animals under certain conditions (although by no means always) after extirpation of the thyroid. 39 Removal of the para- thyroids, however, has precisely the opposite effect (as indi- cated in the studies of R. Hirsch, Underbill and Saiki, and of Eppinger, Falta and Rudinger). Even after extirpation of three parathyroids in dogs there may be noted, in the course of a latent tetany (which is without spasmodic twitching and is only told by a typical change in electrical excitability) , an enormous lowering of the assimilation limit for grape-sugar. If there be combined with the extirpation of several of the parathyroids also extirpation of the pan- creas, a marked rise in the protein exchange in fasting and " J. Hirschl, W. Knopf elmacher (cf. Vol. I of this series, p. 439, Chemistry of the Tissues) and C. v. Noorden, Die Zuckerkrankheit, 5th ed., p. 47, 1910. 38 Pari, Falta and Gigon, H. Eppinger, W. Falta, C. Rudinger (v. Noor- den's Clinic), Zeitschr. f. klin. Med., 66, 1908; 67, 1909. 29 Eppinger, Falta and Rudinger, 1. c. ; E. P. Pick and F. Pineles, Biochem. Zeitschr., 12, 473, 1908; F. P. Underhill and W. H. Hilditch, Amer. Jour, of Physiol., 25, 66, 1909; F. P. Underhill. ibid., 21, 331, 1911; Ritzmann (Straub's Lab.). Arch. f. exper. Pathol., 61, 231, 1909; E. G. Grey and W. T. de Santelle (Johns Hopkins Univ., Baltimore), Jour, of Exper. Medicine, 11, 659, 1909; J. McCurdy, ibid., 798. 312 THYROID GLAND IN CARBOHYDRATE METABOLISM coincidently an increase in the D/N quotient to as much as 3.5 (as seen in the height of phloridzin diabetes). 40 Possibly with these facts in view we may assume that we are justified in looking upon the thyroid and parathyroids as antagonistic in their relations to carbohydrate metabolism. However, the whole matter seems by no means thoroughly established. Hyperthyroidism. — Hyperthyroidism lowers the assimi- lation limit for sugar. Transitory glycosurias have been met time after time after feeding thyroid tissue (cf. Vol. I of this series, p. 448, Chemistry of the Tissues). In special cases the occurrence of true diabetes has been noted, which may, however, be nothing more than the manifestation of a previously existing disposition toward the affection (as C. v. Noorden believes). In Basedow's disease, too, which we are for the present justified in regarding as a form of hyperthyroidism (cf. Vol. I of this series, p. 456, Chemistry of the Tissues), there may be frequently seen instances of alimentary glycosuria (first discovered by Ludwig and Kraus, and by Chvostek and confirmed by many later ob- servers). This is to be expected, according to the observa- tions of Eppinger in C. v. Noorden 's Clinic, in those cases of Basedow's disease which show symptoms of sympathetic irritation, and is usually absent from those cases in which symptoms of vagotonia are prominent. 41 Interaction of the Internal Secretory Glands. — Eppin- ger, Falta and Rudinger have come to very definite ideas as to the interaction of the internal secretory glands, to which they ascribe a regulative influence over normal carbohy- drate metabolism. They assume that exclusion of a "blood gland" will give rise to two distinct effects, primarily direct influences from the loss of the specific secretion and sec- ondarily indirect influences through disturbance of its inter- relation with other glands. 40 Eppinger, Falta and Rudinger, 1. c. ; also R. Hirsch (F. Kraus's Clinic, Berlin), Zeitschr. f. exper. Pathol., 3, 393, 1906; 5, 233, 1908; Handb. d. Biochem., 8', 295, 329, 1910. 41 C. v. Noorden's Die Zuckerkrankheit, 5th ed., p. 46, 1910. INTERACTION OF INTERNAL SECRETORY GLANDS 313 The following schema is intended to represent the mutual relations of the thyroid, adrenals and pancreas : PANCREAS CHROMAFFINE Inhibition 8YSTEM If we keep perfectly clearly before us the fact that this interrelation is of an absolutely hypothetical nature, the most of us will not be disposed to deny a heuristic value to such schematic presentation. It may well be that we can classify the ductless glands in two groups, one group with an accelerator, the other with a retarding functional effect upon the metabolism. The first group, whose "hormones" apparently excite sympa- thicotonic impulses, includes the thyroid, the chromaffine sys- tem and perhaps the hypophysis. To the second, seem- ingly antagonistic to the first group, belong the pancreas and the parathyroids. The first group could stimulate the basic protein destruction, carbohydrate mobilization, fat meta- bolism, output of water and salt, and the galvanic irritabil- ity of nerves; the second group, however, acts according to all appearance in an inhibitory manner. 42 The strongest criticism, unquestionably, comes when we discriminatively examine the mutual relations of these functions. The idea that a centre in the medulla regulates the provision of sugar to the various tissues, which, as Falta supposes, "continu- ously mobilizes, by way of the sympathetic nerves and the adrenals, sugar in the liver, but exerts inhibitory influences 42 W. Falta, Wiener klin. Wochenschr., 1909, p. 1059 ; Caro, Med. Klinik, 1910, 136; W. Falta, L. H. Newburgh and E. Nobel (v. Noorden's Clinic, Vienna), Zeitschr. f. klin. Med., 72, 97, 1911. 314 HYPOPHYSIS IN CARBOHYDRATE METABOLISM on this mobilization by the autonymous nerves and pancreas, and thus the level of sugar in the blood is kept at a constant level, ' ' is, it is true, very plausible. But actually such inter- dependence is far from proven (and this should be clearly understood) . RELATION OF THE HYPOPHYSIS TO CARBOHYDRATE METABOLISM Perhaps nothing can illustrate more clearly how ex- tremely difficult it is to come to any definite conclusions in this field of our study than a statement of the relations of the hypophysis to carbohydrate metabolism. As previously stated (Vol. I of this series, p. 484, Chem- istry of the Tissues), Borchhardt succeeded in inducing a glycosuria in rabbits, but not in dogs, by injections of ex- tract of the hypophysis. Later, after a careful review of the whole literature bearing upon the subject, he came to the conclusion that glycosuria is a more constantly associ- ated feature in cases of acromegaly (in which it may be assumed that the hypophysis is the part primarily involved) than in any other affection. 43 It is very interesting in this connection that B. Aschner, 44 from recent investigations of his own carried out in R. Paltauf 's Institute, holds that there exists a sugar centre in the vicinity of the hypophysis. After Kreidl and Karplus, colleagues of the writer, had succeeded in the Vienna Physiological Institute in showing a sympa- thetic nervous centre in immediate proximity to the tuber cinereum, Aschner found that it was possible to produce a glycosuria with absolute certainty by injuring the tuber cinereum. "If we grant," suggests Aschner, "that over- activity of the hypophysis itself, just as overactivity of the thyroid in Basedow's disease, can give rise at times to a glycosuria, it seems much more probable from the foregoing experiment (production of glycosuria from injury to the 43 L. Borchhardt ( Jaffe's Lab., Königsberg), Zeitschr. f. klin. Med., 66, 3-4, 1908. **B. Aschner, Pfliiger's Arch., 1^6, 114, 1912. INTERACTION OF INTERNAL SECRETORY GLANDS 315 tuber cinereum) that the glycosuria of hypophyseal affec- tions may be explained as depending upon an irritation of the base of the brain. This is the more likely because of the existence here of both vagus and sympathetic paths, and an irritation of the sympathetic is apparently directly capable of giving rise to glycosuria." In this connection it seems to be of great interest that it has been found possible in R. Gottlieb 's laboratory to pro- duce a sensitization of sympathetic nerve endings by consti- tuents of the hypophysis in the same way that this had been previously accomplished by components of the thyroid (Vol. I of this series, p. 457, Chemistry of the Tissues). Proof of sensitization of the points of attack of Suprarenin was ob- tained both by the Läwen-Trendelenburg frog preparations and by mydriasis of the enucleated frog's eye. Emphasis has been properly laid upon the point that as long as we know nothing of an internal secretion of the constituents of the hypophysis we are really not in position to insist upon the physiological significance of this phenomenon. We can- not help, however, considering such sensitizing of the points of attack of one internal secretion by another internal secre- tion in trying to explain the physiological balance of the system and its disturbances. 45 A rather impressive complement to the above-mentioned discoveries may be recognized in the observations of B. Aschner 46 according to which in young dogs the glyco- suria caused by adrenin may be markedly suppressed by extirpation of the hypophysis, possible not only in the first few days after the operation but for months. It is espe- cially noteworthy that the skin necroses, which so often occur in case of adrenin injections, are not met in such in- stances at all or only rarely. This, it might be suggested, may be due to the fact that the vaso-constricting influence of the adrenin has been weakened. "Kepinow (R. Gottlieb's Lab., Heidelberg), Arcb. f. exper. Patbol., 67, 247, 1912. «Aschner, 1. c, pp. 105-106. 316 HYPOPHYSIS IN CARBOHYDRATE METABOLISM Consistently with the failure of adrenin glycosuria in animals deprived of their hypophyses, in hypophyseal obesity (degeneratio adiposo-genitalis), which is believed to be connected with a lowered function of the hypophysis, a higher tolerance for carbohydrates has been observed, as in myxcedema. 47 As previously stated, the hypophysis may be regarded as associated with the thyroid and the chrom- affine system in a group of organs with sympathetic innerva- tion, having an accelerator influence upon metabolism, and under proper circumstances increasing the respiratory metabolism as well as the carbohydrate-, protein- and fat- exchange. Aschner 48 expresses this relation by the follow- ing modification of the previously presented Eppinger-Falta schema (including the ovary and the parathyroids) : THYROID HYPOPHYSIS PANCREAS PARATHYROIDS OVARY Inhibition CHROMAFFINE SYSTEM Without any desire to place any excessive provisional estimate upon the reality of all these matters, it will be well for us to follow with proper interest the further development of this modern scientific magic, in which internal secretory organs replace the planetary orbits of the ancient astrol- ogers, with their favoring and inhibitory influences upon every phase of life. With this, these little known subjects may be dismissed, and with them that of glycosuria, attention being directed to other matters connected with carbohydrate metabolism ; of which the first to demand our interest are the glycuronic acids. 47 Cf. C. v. Noorden, Die Zuckerkrankheit, 5th ed., 1910, p. 48. "'Aechner, 1. c, p. 110. GLYCURONIC ACID 317 GLYCURONIC ACID Constitution. — Glycuronic acid, which was discovered as a product of metabolism in 1878 by M. Jaffe and by 0. Schmiedeberg and H. H. Meyer, contemporaneously but in- dependently, and the constitution of which was verified by its synthetic production by Emil Fischer and Piloty, is without doubt a direct oxidation product of grape-sugar : GLUCOSE GLYCURONIC ACID COH COH H.C.OH H.C.OH I I OH.C.H OH.C.H I > I H.C.OH H.C.OH I I H.C.OH H.C.OH I I [CH 2 .OH COOH. Glycuronic acid in metabolism always appears in the form of conjugated glycuronic acids which are laevorotary, an optical dextrogyration being characteristic of the free glycuronic acid. The conjugates are in general of the nature of alcohols or phenols. According to the synthesis by Neuberg and Neimann and the discovery that glucoside-splitting fer- ments (like emulsin and kefirlactase) are also capable of splitting conjugated glycuronic acids, there is no room for doubt that in a general way these latter correspond to the glucoside type of Emil Fischer. Starting with the tauto- meric, accessory form of glucose CH.OH H.C.OH OH.C.H ni H.C.OH I CH 2 .OH, the reaction with an alcohol obviously takes place with separation of water and oxidation of the terminal CH 2 .0H 318 GLYCURONIC ACID group to a carboxyl, thus giving a phenol-glycuronic acid the constitution: CH.O.C 6 H 6 H.C.OH I OH.C.H I H.C I H.C.OH COOH. According to Emil Fischer it is very improbable that free glycuronic acid is first formed, as it cannot well be imagined why the CH 2 (OH) group should be oxidized while the much more labile aldehyde group is preserved. However, this objection is of less force if one supposes that the aldehyde group is protected by first fixing an alcohol or a phenol group, after which the terminal CH 2 OH group undergoes oxidation into COOH. Conjugation Conditions. — Very many foreign substances when introduced into the body are finally excreted with the urine in the form of conjugate glycuronic acids. However, the alcohols and phenols alone are capable of direct conjuga- tion. Aldehydes and acetones must first be reduced to alco- hols (as chloral, CCl 3 .COH, to CCL.CH 2 .OH; acetone, CH 3 . CO.CH 3 , to CH 3 .CH(OH).CH 3 ). Hydrocarbons of the aro- matic and hydroaromatic series are hydroxylized (as ben- zol into phenol) ; and heterocyclic compounds undergo analogous hydroxylation (as indol into indoxyl). These relations have been thoroughly proved by numerous studies by Jaffe, Neuberg, Fromm, Hildebrandt, Neubauer, Hämä- läinen, and others. 49 ** Cf. Literature: C. Neuberg, Handb. d. Pathol, d. Stoffw., 2d ed., 2, 225, 228, 1907, and Ergebn. d. Physiol., 3, 385-390, 1904; C. Neuberg and W. Nei- mann, Zeitschr. f. physiol. Chem., M, 114 1905; E. Salkowski and C. Neuberg, Biochem. Zeitschr., 2, 307, 1906; R. Hildebrandt (Halle), Hofmeister's Beitr., 7, 438, 1906; Hämäläinen (Helsingfors) , Skandin. Arch. f. Physiol., 27, 141, 1912; J. Schüller (M. Cremer's Lab., Cologne), Zeitschr. f. Biol., 56, 274, 1911; J. Saneyoshi (C. Neuberg's Lab.), Biochem. Zeitschr., 86, 22, 1911. ORIGIN OF GLYCURONIC ACID 319 In determining the amount of aromatic substances ex- creted in the urine it is necessary besides the conjugated sulphuric acids to also keep in mind the conjugated gly- curonic acids, with possible complex interrelations. Thus Neuberg isolated 50 from the urine of dogs to which cresol had been fed, the barium salt of a double combination of cresol-glycuronic acid and cresol-sulphuric acid: CH.O—C^.CH, H.C.OH OH.C.H. I H.C.OH COO— Ba— O— S0 2 — O— CeH^.CHs. Properly considered the conjugation of foreign substances with glycuronic acid is really a detoxifying process. Ap- parently the economy sometimes provides a larger amount of glycuronic acid than is absolutely needed. At least F, Blumenthal noticed in a case of lysol poisoning the form- ation of considerably more glycuronic acid than required for fixing the amount of cresol introduced. Origin of Glycuronic Acid from Oxidation of Sugar. — The origin of glycuronic acid from sugar by oxidation (which can be performed artificially in vitro by careful oxi- dation of dextrose by means of peroxide of hydrogen) 51 directly explains a positive dependence of the physiological formation of this substance upon the body supply of carbo- hydrates. One can readily appreciate that an animal, with its glycogen reduced from long continued starvation, would react to dosage with camphor with only a slight excretion of glycuronic acid, and that this would increase progressively with exhibition of sugar. And when it is recalled that sugar may be formed from protein, there is just as little occasion 60 C. Neuberg and E. Kretschmer, Bioehem. Zeitschr., 36, 15, 1911; cf. also F. Stern (Kiel), Zeitschr. f. physiol. Chem., 68, 52, 1910. 51 A. Jolles, Bioehem. Zeitschr., 3-',, 242, 1911. 320 GLYCURONIC ACID for surprise to find that a practically glycogen-free indivi- dual is still capable of producing some glycuronic acid. The problem of its production is to-day not so much as to its derivation from sugar (for this surely can no longer be a subject of doubt), but whether physiologically the normal catabolism of sugar proceeds through glycuronic acid. In the sense of Paul Meyer's much, discussed "theory of in- complete sugar oxidation" many instances of pathological glycuronic acid excretion, not satisfactorily explicable on the assumption of an increased introduction or formation of substances able to combine with it, may be looked on as due to a primary disturbance of the normal sugar catabolism, here becoming stationary in the intermediate stage of glycuronic acid. This might be thought of in many in- stances of glycuronic acid excretion in respiratory disturb- ances, in many intoxications, in very free glycogen supply, and therefore in diabetes. Paul Meyer believes, too, that a heightened excretion of glycuronic acid without evident cause and without coincident increase of elimination of aromatic conjugates in apparently healthy individuals may be looked upon as a precedent of diabetes and as a symptom of incomplete oxidation of sugar. Neuberg very properly suggests, however, that Paul Meyer has not presented exact proof of the correctness of his theory ; that it can only be looked on for the present as a simple expression of the facts observed by him and may perhaps eventually be ex- plained in a totally different way. 52 C. von Noorden, who does not favor the theory above outlined, thinks it incorrect to assign importance to increase of glycuronic acid in diabetes. 53 Oxaluria. — The circumstance that free supply of sugar and glycuronic acid may lead to an increased elimination of oxalic acid and to an increased accumulation of this acid in the liver, and the fact that oxaluria and glycuronic acid ex- 52 C. Neuberg, Handb. d. Pathol, d. Stoffwechs., 2d ed., 2, 233-235, 1907. 63 C. von Noorden, Handb. d. Pathol, d. Stoffwechs., 2d ed., 2, 60, 1907. CONVERSION OF GLYCURONIC ACID 321 cretion are at times found associated with diabetes, has been the basis for regarding oxalic acid as an end-product of sugar catabolism: Sugar > Glycuronic Acid >• Oxalic Acid. That oxalic acid may be an end-product of all sorts of sub- stances has probably been the uncomfortable experience of every chemist, who after having carried out some tiresome oxidation experiment has time after time been able to obtain, instead of the product he hoped for, only the inevitable and unwelcome oxalic acid. Of course, then, it cannot be denied that sometimes sugar may also be converted into oxalic acid in the body. When and under what circumstances this is true is, however, unknown ; and thus far all told there has very little of a positive nature been obtained from the many studies of the elimination of the relatively incom- bustible oxalic acid in physiological and pathological conditions. 54 Conversion of Glycuronic Acid into ^-xylose. — A very im- portant phase of the glycuronic acid question has been previously referred to, the conjectural relation of the acid with the tissue pentoses. From E. Salkowski's and C. Neu- berg's discovery it is at least quite probable that the tissue sugar with five carbon atoms, A-xylose, arises from glycuronic acid by cleavage of C0 2 , and that the latter may be thus converted into pentose by the agency of putrefactive microorganisms : GLYCURONIC ACID X- XYLOSE COH COH H.C.OH H.C.OH. OH.C.H OH.C.H. H.C.OH H.C.OH H.C.OH H.C.OH COOH H + C0 2 Literature upon Oxaluria: A. Magnus-Levy, Bd., 1, 155-159, 1906. 21 322 GLYCURONIC ACID Occurrence of Glycuronic Acid in the Body. — As for the occurence of compounds of glycuronic acid in the economy, this substance has been recognized by C. Neuberg and Paul Meyer and by Lepine and Boulud, as a normal constituent of the urine and of the blood, although apparently no notable amounts are apt to accumulate in the tissues generally. 55 According to the opinion of the last-named authors the increment in reducing power of blood extracts after hydro- lytic cleavage ("sucre virtue!") may at least partly be referred to glycuronic acid compounds supposed to be pres- ent especially in the formed elements of the blood. 56 Importance of Glycuronic Acid in Diagnosis of Intestinal Disturbances and Diseases of the Liver. — Excretion of gly- curonic acid in the urine apparently depends primarily upon digestive conditions and intestinal putrefaction, and thereafter upon the quantities of indol and phenol available for conjugation. According to E. Mayerhofer's studies a new reaction, originated by Guido Goldschmiedt {v. infra), producing a green color from the action of a-naphthol and concentrated sulphuric acid upon glycuronic acid, is in gen- eral the most delicate mode of detecting the presence of intestinal disturbances in infants. While the test is in- variably negative in well nourished breast-fed children, it is sufficiently delicate to give a positive result with even the slightest nutritive disturbances with an associated height- ening of intestinal putrefaction, the intensity of color in- creasing and decreasing with the grade of the affection. In artificially fed infants, who are practically never free, as is known, from nutritive disturbances, the absence of 55 Literature upon the Occurrence of Glycuronic Acid Compounds in the Body: C. Neuberg, Handb. d. Pathol, d. Stoffw., 2, 226, 1907. 68 R. Lupine and Boulud, Compt Rendu, 1^1, 453, 1905, and earlier con- tributions; Jour, de Physiol., 7, 775, 1905. DETECTION OF GLYCURONIC ACID 323 glycuronic acid excretion in the nrine is really an exception. 57 Apparently, however, urinary glycuronic acid presents a practical clinical interest in another direction. Studies by K. v. Stejskal and Grünwald 58 have indicated that, while the normal body tends to carry out the synthesis of conjugate glycuronic acid after administration of camphor with such precision that almost the whole quantity of camphor- giycuronic acid, as calculated theoretically, appears in the urine in the course of twenty-four hours, in a patient suf- fering from hepatic cirrhosis or severe catarrhal jaundice, the process is distinctly inhibited. The expectation of pos- sible employment of the synthetic production of this sub- stance as the basis for a method of functional hepatic diagnosis is apparently, therefore, not entirely without rea- son, although there is not the least reason for referring the processes of glycuronic acid formation exclusively to the liver. It has been shown, however, in studies carried on in Hofmeister 's laboratory, 59 that hepatic destruction from acid infusion may be without any influence upon the syn- thesis of glycuronic acid after administration of chloral; and J. Pohl 60 has found that severe hepatic changes due to intoxication with ethylendiamine inhibit, it is true, the formation of urochloralic acid, but do not inhibit that of phenol-glycuronic acid. Detection and Estimation of Glycuronic Acid. — In conclu- sion a few words may be devoted to discussion of the modes of detection and estimation of glycuronic acid. 57 B. von Fenyvessy (Pharmakol. Instit., Budapesth), Arch, internat. des Pharmacodyn, 12, 407, 1903; C. Tollens and F. Stern, Zeitschr. f. physiol. Chem., 64, 39, 1910; C. Tollens, ibid., 67, 138, 1910; E. Mayerhofer (Franz Josef's Hospital, Vienna), Zeitschr. f. Kinderheilk., 1, 226, 1910; Zeitschr. f. physiol. Chem., 10, 391, 1910. 58 K. v. Stejskal and H. Fr. Grünwald (Second Med. Clinic, Vienna), Wiener klin. Wochenschr., 1909, No. 30. 59 F. Pick ( Hofmeister's Lab., Prague), Arch. f. exper. Pathol., 33, 315, 1894. 00 J. Pohl (Prague), Arch. f. exper. Pathol., 41, 971, 1895. 324 • GLYCURONIC ACID In a general way we may suspect a given urine of con- taining conjugate glycuronic acid if it polarizes to the left without showing fermentescibility and reducing power, the lasvogyratory power changing to dextrogyratory and coin- cidently a marked reducing power developing after boiling for a long time with dilute acid. C. Neuberg recommends in the determination of glycur- onic acid to first concentrate it by lead precipitation, after hydrolytic cleavage of the urine. After decomposing the lead precipitate by means of sulphuretted hydrogen or sul- phuric acid one sometimes succeeds in obtaining the beau- tiful crystalline cinchonin salt of the acid. The p-brom- phenylhydrazine compound of glycuronic acid may also prove of important service, according to Neuberg ; it can be distinguished from the bromphenylosazones of sugar by its almost complete insolubility in warm alcohol, but is espe- cially characterized by the fact that its optical rotating power in a pyridinalcoholic solution is much more marked than that of any of the analogous sugar compounds. The brilliant colors produced in the orcin- and phloro- glucin-tests may also serve for the detection of glycuronic acid. According to Neuberg, these tests are not to be at- tributed to the separation of furfurol, and to speak of them as ' ' furfurol reactions " is a mistake. They are, however, not at all characteristic of glycuronic acid, but are well known reactions also of the pentoses. It seems, moreover, that they respond to any sugar with an uneven number of carbon atoms in its molecule. The naphtho -res orcin reaction of B. Tollens 61 offers dis- tinct advantages over these reactions. It depends on the fact that naphtho-resorcin [1.3 dioxynaphthalin, C 10 H G (OH) 2 ] changes the glycuronic acid, on boiling with hydro- 81 B. Tollens (Göttingen), Ber. d. deutsch, ehem. Ges., 41, 1783, 1908; Zeitschr. f. physiol. Chem., 56, 115, 1908; C. Tollens (Kiel), Münchener med. Wochenschr., 1909, 652; C. Neuberg and 0. Schewket, Biochem. Zeitschr., 44, 502, 1912. DETECTION OF GLYCURONIC ACID 325 chloric acid, into a coloring matter which gives a blue color in ether, and in solution gives a dark spectral band in the vicinity of the sodium lime. It is true that this reaction is also not specific for glycuronic acid ; it occurs also (as shown by A. Mandel and C. Neuberg) 62 with many aliphatic CO aldehyde- and ketonic-acids containing the group | COH beginning with glyoxylic acid, | , and seems to be due COOH to a certain combination between carboxyl and carbonyl groups. Indoxyl may also cause confusion. 63 The test is suited, however, for positive differentiation of glycuronic acid from the pentoses. If glycuronic acid in a mixture of different sugars is precipitated with these as an osazone, the glycuronic acid osazone alone will give the Tollens color reaction. 64 A very valuable recent reaction for glycuronic acid has been originated by Guido Goldschmiedt, as above stated ; he found that glycuronic acid with a-naphthol and concentrated sulphuric acid will give an emerald green color (passing into violet and blue when diluted with water). Neither hexoses nor pentoses give this reaction, which is directly applicable in human urine only when the diet is free from nitrates (as milk, white bread and meat) . The urine of dogs and rabbits is always free from nitrites, as proved by the diphenylamine reaction. 65 A method of quantitative determination of glycuronic acid has been recently proposed by Lefevre and Tollens. It depends on the fact that if urine is precipitated by acetate of lead and ammonia, the furfurol appearing in distillation 62 A. Mandel and C. Neuberg, Biochem. Zeitschr., 13, 148, 1908; C. Neuberg, ibid., 24, 436, 1910. "R. Bernier, Jour. d. Pharm, et de Chim., series 7, 2, 401, 1910; cited in Jahresber. f. Tierchem., 40, 301, 1910. 84 C. Neuberg, and S. Saneyoshi, Biochem. Zeitschr., 36, 56, 1911. 66 G. Goldschmiedt (Prague), Zeitschr. f. physiol. Chem., 65, 389, 1910; 67, 194, 1910; cf. also L. v. Udränsky, ibid., 68, 88, 1910. 326 GLYCURONIC ACID of this precipitate with acids has its actual source in glycu- ronic acid. A molecule of the latter breaks down into a molecule of furfurol and C0 2 . The former may be weighed after precipitation of the distillate with phloroglucin ; and the C0 2 taken up in a potash apparatus and estimated. As furfurol formation is also possible from the pentoses, but the accompanying separation of carbonic acid is entirely characteristic for glycuronic acid (from the carboxylic group of which it arises) the method serves to determine glycuronic acid in the presence of pentoses. 66 It is to be expected now that we possess this method, our knowledge of the role and significance of glycuronic acid in the economy will advance more rapidly than has hitherto been the case. 66 B. Tollens, Zeitschr. f. physiol. Chem., 61, 95, 1909; U. Lefevre and B. Tollens, Ber. d. deutsch, chem. Ges., J f 0, 4513, 1908. CHAPTER XIV SUGAR DESTRUCTION IN THE ECONOMY Glycolysis. — The author feels that it is desirable to con- clude the series of lectures upon the question of carbohydrate metabolism by a statement of the problem of glycolysis. Although the ground is by no means sure and we know perfectly well that whatever is to be said is far more of a negative than of positive import, the question cannot well be neglected from the list of important problems of physio- logical chemistry — how the economy proceeds in the cata- bolism of sugar. Efforts to discover the answer of this problem reach rather far back. It is, of course, quite natural that in part these took inception in an example of glycolysis in nature, a process which has achieved a tremendous economical im- portance (not exactly to the best interest of human welfare), that of alcoholic fermentation of sugar by yeast fungi. It is entirely apropos to inquire whether other animal and vegetable cells may not have in some degree a power similar to that of the yeasts to split sugar into alcohol and C0 2 . Distribution of Zymases in the Vegetable Kingdom. — Al- though Pasteur and Pfeffer had originally suggested that the first phase of the decomposition of sugar in plants con- sists of an anaerobic formation of alcohol which then under- goes combustion into C0 2 and water upon the access of oxy- gen, Stoklasa, of Prague, the biochemist, and his pupils de- serve the credit of first having experimentally furnished support for the idea. After E. Buchner in the latter part of the nineties made the important discovery that an enzyme elaborated by the vital activity of the yeast cells, zymase, is capable of setting up fermentation of sugar into alcohol and carbonic acid, the basis was prepared for the recognition of the zymases of higher types of living beings. By employing the methods used by Büchner for recovering the zymase of 327 328 SUGAR DESTRUCTION IN THE ECONOMY yeast Stoklasa and his collaborators succeeded (by precipi- tating expressed juices by alcoholic ether and rapidly drying the precipitate) in obtaining zymases from beets, potatoes, peas and green types of plants, i.e., enzymes capable of in- ducing an active fermentation of sugar into alcohol and car- bonic acid in complete absence of microorganisms. 1 The studies of Palladin and Kostytschew mark a further step, showing that the anaerobic respiration of plants is appar- ently at least in part an alcohol fermentation which inde- pendently of the life of the cells takes place from the influ- ence of enzymes even after the cells have been killed by freezing. Besides the zymases, according to Stoklasa there are held to be also " lactolases' ' concerned in the anaerobic plant respiration, which give rise to formation of lactic acid. The related older view, that a molecule of sugar is first separated into two molecules of lactic acid and these later fermented into alcohol and carbonic acid, can no longer be regarded as correct from the modern viewpoint of the fer- mentation theory. However, lactic acid can originate from labile intermediate products through transposition proc- esses. In the later stages of the fermentation process the alcohol fermentation (the zymase being injuied by the ad- vancing acidity) may lag behind the lactic acid fermenta- tion, and in still later phases the latter may in turn be limited by a butyric acid fermentation. Supposed Occurrence of Zymases in Animal Tissues. — There is thus no ground for question as to the occurrence of an alcohol fermentation in the anaerobic respiration of plants. Stoklasa, however, believed his findings applicable to the field of the animal economy, and thought that he could recover zymases from all sorts of tissue (muscle, liver, pan- creas, leucocytes, etc.). 2 These latter discoveries, which were subjected to control tests by many authors, have, how- 1 Literature upon the Zymases of Higher Plants : C. Oppenheimer, Die Fermente, 3d ed., 466-474, 193 0. 2 Cf. Literature: E. Abderhalden, Lehrb. d. physiol. Chem., 2d ed., 89, 1909. ZYMASES IN ANIMAL TISSUES 329 ever, been confirmed only here and there, 3 but have for the most part excited opposition. Thus Blumenthal and others found that sugar catabolism proceeds in the tissues with formation of C0 2 , it is true, but not with a corresponding production of alcohol. 4 Small amounts of alcohol have actually been found in animal tis- sues by various observers, 5 but Landsberg believes this can be fully explained as the result of absorption of alcohol from the gastroenteric canal, where from the influence of yeasts and bacteria upon the carbohydrates there is always oppor- tunity given for alcohol production. Then, too, it has been shown that only the putrefaction of tissues, not their auto- lysis, increases their alcohol content. Battelli maintained the circulation in freshly killed animals for two hours by compressing the exposed heart, and coincidently kept up artificial hydrogen respiration through a tracheal canula; under which circumstances it was shown that in the absence of oxygen the economy of the higher animals does not form carbonic acid, that, as a matter of fact, therefore, there apparently does not exist here an " anaerobic respiration." 6 It is certainly worth considering, too, that in the anoxybiosis of ascarides and earth worms, as shown by the studies of Weinland and Lesser, there is nothing in the way of an alcoholic fermentation of sugar (in these, however, as an important product of carbohydrate cleavage there is formed, besides carbonic, a volatile fatty acid, apparently valerianic acid). 7 The most weighty objection to Stoklasa's idea, however, is to be seen in the circumstance that many careful 3 R. Robert, Pflüger's Arch., 99, 176, 1909; F. Maignan, Compt. Rend., 140, 1063, 1124, 1905; F. Ransom (Cambridge), Jour, of Physiol., 40, 1, 1910. 4 F. Blumenthal, Deutsch, med. Wochenschr., 1903, No. 51; J. Feinschmidt (F. Blumenthal's Lab.), Hofmeister's Beitr., 4, 511, 1904; E. Bendix (F. Blumenthal's Lab.), Zeitschr. f. physik. und diät. Ther., 2, 218, 1899. 5 Ford ; Rajewski ; G. Landsberg, Zeitschr. f. physiol. Chem., 41, 505, 1904 ; Reach (A. Durig's Lab.), Biochem. Zeitschr., 3, 326, 1907. 8 F. Battelli, Arch, intern, de Physiol., 1, 47, 1904. T Literature upon Anoxybiosis: E. J. Lesser, Ergebn. d. Physiol., 8, 742- 796. 1909 ; Zeitschr. f. Biol., 52, 282, 1909. 330 SUGAR DESTRUCTION IN THE ECONOMY observers, 8 in their control tests of his statements (with provision of thorough asepsis), failed to obtain any fermen- tation. "The fact," states Vernon, 9 "that Stoklasa and his collaborators were unable to find any bacteria by their cultures does not necessarily prove that they were not pres- ent. As far as can be made out from their studies the cultures, as a rule, were placed in aerobic surroundings, al- though the fermentations were anaerobic. The cultural con- ditions were, therefore, not favorable for the growth of the bacteria which occasion the alcoholic fermentation." Harden and Maclean, 10 who have recently subjected the work of Stoklasa to severe criticism, found that if to solutions of glucose they add tissue juices, or sediments obtained therefrom by alcoholic ether, in the course of the first few hours a very trivial gas development takes place, the cause of which is purely physical, and which is noticeable both when there is no sugar present and when disinfectants are added. Later on, however, a gas production begins, continuously increasing in intensity, which goes on in pro- portion to the growth of bacteria. If toluol be added in quantities which are harmful to bacteria but which are known from experience to be without influence upon the zymases, the gas formation ceases. That the test for bac- teria in Stoklasa's experiments so frequently resulted nega- tively, may, in the opinion of these authors, have been due to the fact that it was made at the end of the fermentation, therefore at a time when the bacteria may have been al- ready destroyed by the concentration of their own metab- olic products (alcohol, lactic acid). However highly Stoklasa's discoveries as to the impor- tance of zymases in the anasrobic respiration of plants may be regarded, it must be said that analogous processes in the s Battelli, Maze, Portier, Colmheim, Embden, Arnheim and Rosenbaum, Harden and Maclean. 9 H. M. Vernon, Ergebn. d. Physiol., 9, 205, 1910. 10 A. Harden and H. Maclean (Lister Instit.), Jour, of Physiol., 42, 64, 1911. ZYMASES IN ANIMAL TISSUES 331 animal economy have not only not been proved but are not even probable. The author acknowledges that this is only his own sub- jective impression, and desires to add that others who have gone at length into this problem have arrived at a contrary opinion. Thus Carl Oppenheimer does not look on the bac- terial objection, even if it cannot be completely put aside, as the real nucleus of the question. "Stoklasa found a satis- factory reproduction of yeast fermentation in all its phases. Where are the bacteria which are carrying this on? We are aware, of course, that in bacterial fermentations alcohol occurs as a more or less insignificant by-product. Either Stoklasa's alcohol findings are false — when there is nothing further to be said to the contrary ; or they are correct — and in this case they cannot be referred to the action of bacteria. That true yeast cells are present in Stoklasa's ether-precipi- tated extracts of tissues no one has, to the best of my knowl- edge, as yet asserted. A priori it is much more probable that the investigators following Stoklasa have not had the same fortunate hand as he. . . . Other sources of error are in all probability to be apprehended in the antiseptics employed; which perhaps may have so thoroughly elimi- nated the bacteria so often called into question that the easily affected zymase has also been destroyed by them. . . . We have, in fact, here one of the most curious of spectacles, one which only an exact research can afford — on one side a series of experiments published in fullest extent, with all details, which always give the same result; on the other, a list of reinvestigators who are unable to find anything and who introduce into the field unsupported contradiction of the experiments. As long as no one appears to show . . . where the alcohol and lactic acid come from in Stoklasa's experiments his experiments are going to stand like a rocher de bronze.'''' Even as objective an observer as Hammersten 11 con- 11 O. Hammersten, Lehrb. d. physiol. Chem., 7th ed, 382, 1910. 332 SUGAR DESTRUCTION IN THE ECONOMY eludes that the statements of Stoklasa and his collaborators have not been controverted, and believes that we cannot deny the possibility that an alcoholic fermentation can exist in animal tissues as well as in plant tissues in anaerobic respiration. Here again is an example of how differently a given situation may strike the minds of different individuals. The same impression will be aroused when we attempt to discuss what is thought to be known of the glycolytic power of ground-up tissues and the expressed juices of tissues. Cohnheim's Pancreas-Muscle Experiment. — In view of the importance to be attributed to the pancreas in carbohy- drate metabolism since the discovery of pancreatic diabetes, it was desirable to determine whether a special extracorpor- eal glycolytic power is inherent to the pancreas. Otto Cohn- heim believes that this may be answered affirmatively, in the sense that the muscles contain a glycolytic ferment which owes its activation to the pancreas. The " pancreatic acti- vator" is believed to be a thermostable substance, soluble in dilute alcohol, the effect of which in increasing amounts is peculiar, first increasing and then diminishing. Cohn- heim compares this last with the phenomena of complement deviation and overfixation in the combined action of amboceptor and complement. In extraction of the glyco- lytic enzyme of the muscle, which is very easily affected by salt solutions, an ice-cold solution of sodium oxalate is re- commended. The quantity present in muscles is believed to be subject to marked variation even in physiological condi- tions ; thus it is supposed that cat muscle is strongly active in glycolysis if the cats have been fed upon milk and sugar, but of little glycolytic power if the animals are fed on fat or fatigued from physical exercise. 12 13 O. Cohnheim (Heidelberg), Zeitschr. f. physiol. Chem., 39, 396, 1903; 1,2, 402, 1904; J,3, 547, 1905; 47, 253, 1906. OBJECTIONS OF CLAUS AND EMBDEN 333 Objections of Claus and Embden. — B. Claus and G. Emb- den 13 after subjecting Coknheim's experiments to careful control investigation, came to believe that they are dealing with an action due to contaminations, probably of bacterial nature. The apparent increase of glycolysis on addition of pancreatic material might possibly be due to nothing more than that corpuscular elements may withdraw some of the toluol from the fluid saturated with this substance, and in this way really favor the development of bacteria. The writers named also express the opinion that toluol, as well as other antiseptics, if not added in large amounts affords no very great protection against bacteria, and that the com- mon method of studying the action of glycolytic ferments in tissue pulp with antiseptics added is not a serviceable one and should be abandoned. There is no doubt of the very great difficulty of entirely excluding confusion of results because of the influence of microorganisms in experiments of this sort, and this has been confirmed from another point of view. 14 The convic- tion is borne in upon us that even if we do not employ a tissue pulp, but work with acetone-precipitates from the expressed juices, and whether toluol or chloroform be used as an antiseptic, we are by no means sure of not finding scattered bacteria in the sediment. 15 However impressed the writer may be that the greatest scepticism for all the discoveries in this field is apropos, that moreover many of them are merely without objection, and that especially all estimates of the quantity of glycolytic enzyme in the tissues are apparently extremely problem- atical, he would not willingly be persuaded that everything which has been observed and noted here is actually nothing but the effect of bacteria. U R. Claus and G. Embden (Frankfurt a. M.), Hofmeister's Beitr., 6, 214, 343, 1905. 14 G. C. E. Simpson (Liverpool), Biochem. Jour., 5, 126, 1910. 15 L. Rapoport (First Med. Clinic, Berlin), Zeitschr. f. klin. Med., 57, 208, 1905. 334 SUGAR DESTRUCTION IN THE ECONOMY No great importance can be attributed by the author to experiments with tissue pulp, 10 as the technical difficulties of antiseptics with such material seem at the present, at least, not overcome. The author, however, acknowledges an im- pression that it may be a mistake to set aside without further consideration as ' ' the effects of bacteria ' ' the results of all experiments upon the glycolytic action of expressed juices provided they are conducted with necessary antiseptic pre- cautions. Possible Existence of Glycolytic Tissue Ferments. — One should remember, for instance, that, according to Fein- schmidt, 17 in the expressed juices from the pancreas, liver and muscles, even in the presence of 0.9 per cent, of sodium fluoride, appreciable glycolysis, with development of C0 2 and organic acids, but with only traces of alcohol, is said to take place. It is without doubt of importance, too, that Cohnheim 's work should have been confirmed by the Amer- ican, Hall, 18 in a very carefully conducted and critical con- trol experiment. Hall expressed the juice from muscle and pancreas; and with these and with alcoholic extracts (by boiling) from the latter, mixed or separate, acted upon a dilute sugar solution at incubator temperature and in pres- ence of toluol, the length of the experiment not exceeding three days. That he should have found as an average from his experiments that pancreatic juice alone destroyed only 0.3 per cent, of the amount of glucose employed, muscle juice alone only 1.6 per cent., the combination of muscle juice and pancreas 4.4 per cent., but the combination of the alcoholic extract of pancreas and muscle-juice as much as 18.3 per cent., is very remarkable. The objection that such results are entirely due to bacterial influences seems decidedly im- 10 Literature: (Blumenthal, Ssobolew, Herzog, Feinschmidt, Arnheim and Rosenbaum, Sehrt, Rapoport, Braunstein, Simacek, R. Hirsch) in Rosenberg, Handb. d. Biochem., 3', 253, 1910. 17 Feinschmidt, Hofmeister's Beitr., 4, 511, 1904. 18 C. W. Hall (Harvard Medical School), Amer. Jour, of Physiol., 18, 283, 1907. GLYCOLYTIC TISSUE FERMENTS 335 probable. While we are coming to recognize from a grow- ing and more and more convincing experience that even with the presence of toluol it is not at all easy to protect a tissue pulp or especially suspensions of such a material from bacterial invasion with certainty, we have no reason, as far as the writer is aware, to doubt the efficiency of toluol in a fluid medium. Hall, in addition, controlled the sterility of his tests by both aarobic and anaerobic culture methods. More- over the fact that the glycolytic power of pancreatic and muscle extracts failed to manifest itself upon solutions of fructose and lactose, and appeared only in connection with glucose, specifically contradicts any bacterial influence, bac- teria being in no wise selective enough to make such a difference. 19 Recently P. A. Levene, who has at his disposal the great technical facilities of the Rockefeller Institute, of New York, has investigated the glycolytic influence of various animal tissues with and without addition of "activators." The tissues as such proved in practically all cases inactive. Addition of pancreatic extract to muscle was without effect in case of canine tissues, in rabbit tissues resulted in show- ing a lowered reducing power of the sugar solutions (but, in the dog, for example, splenic extract in combination with other tissues seemed to be feebly active). Levene 's experi- ments have shown, however, that we are not justified in regarding a loss in reducing power of the glucose solutions employed as due only to "glycolysis." It seems that con- densation processes, converting simple sugar to higher molecular products, may be involved. The reducing power of a sugar solution which has been lowered by the combined 19 Stoklasa, Zeitschr. f. physiol. Chem., 62, 35, 1909, found precisely the opposite, viz., that preparations made from pancreas juice by alcoholic ether would break up disaccharides in the absence of antiseptics, with formation of lactic acid, alcohol and carbonic acid. However, hexoses apparently ferment only when produced by fermentation hydrolysis. All these effects are decidedly interfered with by the presence of antiseptics. Here, too, may be applied the remark that bacteria do not tend to be very selective which if correct to one is fair to the other side. 336 SUGAR DESTRUCTION IN THE ECONOMY influences of muscle plasma and pancreatic extract, can be restored by boiling with, weak hydrochloric acid. Levene also succeeded in recovering from such a solution the osazone of a disaccharide. 20 What now, in conclusion, is the exact meaning of this long disquisition ? No one doubts in the least that glycolysis takes place in the tissues and that the living tissues are able to catabolize sugar. The point in question is, however, whether we are in position to reproduce the process with dead fragments of tissue and tissue extracts, whether in such case we are justified in referring the glycolysis to the influ- ence of ferments which can operate independently of the liv- ing cells. As far as the writer sees, the question cannot at present be either affirmed or denied with certainty; and it seems not improbable that under proper conditions ferments can be obtained from tissues which in some way chemically change the sugar, either to induce cleavage or synthesis. But that is probably all that can be said. For the assump- tion that this chemical cleavage is to be regarded as analogous to the vital combustion of sugar, not to say equivalent, there is in the writer's opinion not the least basis. And, too, it certainly has not been shown that the activity of Cohnheim's pancreatic activator has anything to do with the puzzling influence which the living pancreas exercises upon the metabolism of sugar. If Hall {vide sup.) found that alcoholic extract of pancreas is more efficient than the pancreas itself, and if Levene found the pancreas inactive in many cases, but did find an extract of spleen to be active, it must seem that an unprejudiced person would conclude that they are perhaps dealing with a non- specific effect. In the course of the last few years we have heard so much about the activating influences of lipoids, and especially tissue lipoids, upon the processes of fermentation and intoxication of the most varied types, that one may well 20 P. A. Levene and G. M. Meyer, Jour, of Biol. Chem., 9, 97, 1911 ; 11, 347, 353, 1912. EXPERIMENTS UPON SURVIVING TISSUES 337 consider the possibility of a relation with such substances of the rather inconstant effects of the activators which Cohn- heim and his followers have noted. All in all the writer believes that the expectation of nearer approach to the secret of sugar catabolism in the living body by experiments upon tissue pulp and expressed juices is at the present unfortunately decidedly depressed. It was both logical and essential in forcing the path along this way to make and perseveringly repeat the experiment. But it is quite as logical after being quite convinced of the impracticability of this route of approach to look about for other paths which lead further. Experiments upon Surviving Tissues. — A more promis- ing way is apparently to be found in perfusion of living organs removed from the body. Occasion has been taken in a previous lecture to state in detail the fact that in R. Gottlieb's laboratory and elsewhere it has been found possible to measure continuously the amount of sugar used by the beating mammalian heart artificially perfused. Ref- erence has already been made, too, to the expectations we are justified in cherishing for the pancreatic problem by ap- plying the fine perfusion methods of E. H. Starling. Oppor- tunity may be taken here, moreover, to refer to the important experiments of the American, McGuigan, who perfused or- gans with blood diluted with Ringer-Locke solution and mixed with different kinds of sugar, and then calculated the amount of sugar consumed (with reference to the amount of glycogen shown before and after perfusion) . His experi- ments show that living muscles are able to induce a rapid combustion of dextrose, laevulose and galactose, but not maltose, and that this is true to a greater degree when the muscle is in activity than when at rest. However, in perfu- sion experiments on dead muscles, which, of course, should always offer more natural and more favorable experiment- conditions than a ground-up muscle or an expressed juice, 22 338 SUGAR DESTRUCTION IN THE ECONOMY scarcely any loss of sugar could be determined. 21 In the last count there will be nothing left to do but to accept the same verdict in the sugar problem as we long ago arrived at in the case of the protein question ; that the secret of the process is hidden within the living cells and cannot be ex- tracted by any solvent. The hope of preparing some fer- ment solution and by some clever manipulation bring about the combustion of protein at 40° C. into carbonic acid, ammonia and water has long since been abandoned. The same thing will have to be accepted in the case of the sugar problem. It is the same in science as in life; when we stop striving for the unttainable our efforts to reach the attain- able are pressed forward with just so much the fresher courage. Glycolysis in Blood. — Interesting results in relation to glycolysis may be expected from the study of sugar catabol- ism in blood. This is the more noteworthy because for a long time there was nothing at all promising in view from this point of investigation. Claude Bernard observed long ago that the sugar content diminished in blood on standing for a time, and expressed the idea that this was perhaps due to a destr action of sugar with formation of lactic acid. Later blood glycolysis was made the subject of careful investigation, especially by Lepine, who framed a new, much discussed and soon aban- doned theory of diabetes upon the fact that he found the phenomenon decreased in diabetics and in animals deprived of the pancreas. Lepine, and with him Arthus, referred the glycolytic process in the blood to the leucocytes ; the latter was disposed to recognize it as a postmortem phenomenon related with the disintegration of the blood corpuscles. 22 21 McGuigan ( Washington Univ., St. Louis, Mo. ) , Amer. Jour, of Physiol., 21, 334, 1908; H. McGuigan and C. L. von Hess (Northwestern Univ. Med. School), ibid., 30, 341, 1912. ö Cf. the Older Literature upon Blood-glycolysis : C. Oppenheimer, Die Fermente, 3d ed., 478, 1910. IMPORTANCE OF LEUCOCYTES IN GLYCOLYSIS 339 In recent times a series of carefully conducted investiga- tions have afforded important results in this connection, for which we are indebted to A. Slosse, of Brussels, and his collaborators, J. de Meyer and E. Vandeput. Primarily these show that aseptic glycolysis in the blood is not an alcoholic fermentation (even the most delicate tests indicate not the slightest trace of either alcohol- or carbonic acid- formation). The catabolism takes place in a much more interesting manner, one molecule of glucose separating into two of lactic acid, and the latter in turn into acetic acid and formic acid. From the formic acid very small quantities of CO can be produced. Sugar catabolism, therefore, accord- ing to Slosse, characteristically follows this schema: Glucose Lactic Acid Lactic Acid Acetic Acid Formic Acid Carbon Monoxide This, as will be referred to later, presents striking analogies to the mode of decomposition of sugar under the influence of alkalies. 23 Importance of Leucocytes in Blood-glycolysis. — J. de Meyer 24 suggests that in the formation of the glycolytic blood ferment this is secreted as a proferment by the leucocytes and that this is then activated by a substance produced by the islands of Langerhans in the pancreas (the latter, then, as a "substance sensibilatrice" or amboceptor). E. Vandeput finds, in consonance with the older statements of Lepine, that the glycolytic power of the blood of dogs is distinctly reduced after removal of the pancreas, and that a A. Slosse (Instit. Solvay, Brussels), Arch, internat. de Physiol., 11, 153, 1911. 24 J. de Meyer (Brussels), Ann. de l'lnstit. Pasteur, 22, 778, 1908; Arch, intern, de Physiol., 7, 317, 1909; 8, 204, 1909; Centralbl. f. Physiol., 23, No. 26, 1910; cf. therein Literature. 340 SUGAR DESTRUCTION IN THE ECONOMY the addition of pancreatic extract restores it. 25 If with these findings we take into account the previously described observations of E. H. Starling (v. sup., p. 256), who ob- served a distinct increase of the amount of sugar used up by artificially perfused hearts of depancreatized animals after addition of pancreatic extract to the perfused blood, we cannot help feeling that the active manifestation of glycolytic power by the corpuscular elements of the blood is no more than an isolated example of what is really a gen- eral rule, that perhaps every living cell is capable of decom- posing sugar and requires in carrying out this function the combined influence of an activator secreted into the circulation by the pancreas. However, that the formed elements of the blood, espe- cially the leucocytes, are directly connected with the glyco- lytic processes in the blood cannot well be doubted from the uniform confirmation of numerous investigators. Thus Lepine and Boulud refer glycolysis in the blood (in their estimation not only the " immediate" but the "virtuelle" hsemic sugar as well should be included) to the colorless blood cells which in this point are of decidedly more impor- tance than the red cells. 26 The observations of Nadina Sieber upon a glycolytic ferment in the blood fibrin may perhaps be related with the white corpuscles in the fibrin. 27 P. Bona and A. Döblin look on the observation that even a sparing haemolysis occasioned by addition of water will stop glycolysis (while dilution with Ringer's solution or with physiological salt solution has no influence upon it) as favor- ing the idea that sugar catabolism in the blood is surely not a simple oxidation process, being seen even in complete absence of oxygen, and is connected with the integrity of 28 E. Vandeput (Slosse's Lab., Brussels), Arch, intern, de Physiol., 9, 293, 1910; cf. also J. Edelmann (Odessa), Biochem. Zeitschr., 1,0, 314, 1912. 20 R. Lepine and Boulud, Jour, de Physiol., 13, 353, 1911; C. R. Soc. de Biol., 60, 901, 1906. 27 N. Sieber (Petersburg), Zeitschr. f. physiol. Chem., 1,1,, 560, 1905. SUGAR CATABOLISM FROM ALKALI 341 the formed elements. 28 In serum they could recognize no loss of sugar or only a very slight diminution. The recent observations of Levene, of the Rockefeller Institute, of New York, are also of special importance in this relation. The latter noted that solutions of glucose under the influence of leucocytes lose a part of their reducing power (and this is not restored by boiling with a mineral acid, and is, therefore, surely not the result of a simple molecular condensation). The fact that a neutral reaction was maintained in this ex- periment by means of Henderson's phosphate mixture is of special significance. If water was employed instead the effect was lost ; addition of tuluol also proved deleterious. Levene, just as Slosse, found that lactic acid appears in the catabolism of sugar. 29 Sugar Catabolism from the Influence of Alkali. — In re- viewing the foregoing material which, in spite of the extent of literature devoted to it, is decidedly inadequate, and in trying to obtain a picture of the process of sugar decomposi- tion in the living body, in the author's opinion the above mentioned observations of Slosse upon the catabolism of sugar in the blood stand out prominently. Slosse very properly referred to the analogies which his observations suggested to the behavior of sugar under mild influence of caustic alkalies. Kiliani, about the beginning of the eighties made the development of lactic acid from sugar the subject of careful investigation ; Framm 30 later on observed the appearance of aldehyde and of formic acid when air is passed through alkaline solutions of sugar. Still later Buchner, Meisen- heimer and Schade 31 noted that if they allowed a solution of sugar in dilute caustic soda to stand for some weeks or 28 P. Rona and A. Döblin (Krankenhaus am Urban, Berlin), Biochem. Zeitschr., 82, 489, 1911. 28 Levene and Meyer, Jour, of Biol. Chem., 11, 301, 1912; 12, 265, 1912. 30 F. Framm (0. Nasse's Lab., Rostock), Pfliiger's Arch., 64, 587, 1896. 81 Buchner, Meisenheimer, Schade, Ber. d. deutsch, chem. Ges., 88, 623, 1905; 39, 4217, 1906; 41, 1009, 1908; Schade, Zeitschr. f. physikal. Chem., 57, 1, 1906; cited in Biochem. Centralbl., 5, No. 2311. 342 SUGAR DESTRUCTION IN THE ECONOMY months, with the air excluded, half or more would be trans- formed into inactive lactic acid, the remainder for the most part into polyoxyacids (dioxybutyric acid, etc.), with only small amounts of formic acid, carbonic acid and alcohol appearing along with the latter. According to J. de Meyer 32 glucose breaks up in soda lye in the presence of platinum sponge with formation of lactic acid, formic acid and oxalic acid, without the appearance of alcohol and carbonic acid. However, Jolles 33 saw, when he allowed the alkaline cleavage to proceed in the presence of oxidizing agents, as peroxide of hydrogen and silver oxide, at the body temperature, only formic acid develop in any important quantity. That sugar can undergo extensive catabolism from the influence of dilute alkalies even at the temperature of the body cannot be doubted. There is a question, however, whether the degree of alkalinity of the blood and of the animal juices is high enough to permit one to seriously consider whether this mode of break-down can take place physiologically. In view of a result obtained by Michaelis and Bona 34 in a fluid containing the same or somewhat higher amount of hydroxyl ions as the blood, in which prac- tically no sugar catabolism occurred in the course of twenty-four hours at body temperature one might feel dis- posed to deny off-hand any importance to this factor. Importance of Catalyzers in the Catalysis of Sugar. — As a matter of fact, however, conditions are not as simple as this would presuppose; the presence of catalyzing agents is certainly a matter which may make a complete difference. Thus Walter Lob has proved that in salt-free solutions of sugar of the same degree of alkalinity as the blood, with low concentration of hydroxyl ions (differing very slightly from 32 J. de Meyer, Rev. Med. Memoirs Lepine, 1911, 517, cited in Centralbl. f. d. Ges. Biol., 12, No. 2887. 33 A. Jolles, Biochem. Zeitschr., 29, 152, 1910; Centralbl. f. innere Med., 1911, No. 1. 84 L. Michaelis and P. Rona, Biochem. Zeitschr., 23, 364, 1910. ELECTROLYTIC CLEAVAGE OF SUGAR 343 that of pure water), glycolysis is insignificant; but if phos- phates be added a very appreciable increase takes place. 35 The same author also makes the very instructive statement that the iron-containing coloring matter of the blood, in spite of its unequivocal peroxidase character, is not able to affect glucose even in the presence of peroxide of hydrogen ; while substances which may be obtained from an alcoholic extract of pancreas when treated with salts of iron, effect a marked cleavage of sugar in which (along with small quanti- ties of formic acid, polyoxyacids and carbonic acid) the chief products are pentose and formaldehyde. 36 Manganese and potassium are also capable of accelerating alkali-hydrolysis of sugar, according to Slosse. 37 It is without question not an easy matter to properly judge the physiological value of such observations ; but we cannot go far astray in assuming the association of some kind of catalyzing agencies in the processes of sugar cleavage in the economy. A wide and gratifying field of research in physical chemistry is opening in this connection. The closely related question, how the economy, if it be capable of catabolizing sugar with such readiness, should come to have the special power of storing up large reserves of carbohydrate, may, perhaps, in the opinion of Jolles, be answered by supposing that glycogen, because it does not possess free aldehyde groups, is acted upon with much more difficulty than sugar ; and that in this manner the economy protects the dextrose from the catabolizing influence of the alkali by storing it as glycogen in the liver and muscles. 38 Electrolytic Cleavage of Sugar. — Walter Lob 39 and, inde- pendently, Carl Neuberg, 40 undoubtedly followed a valuable 85 W. Lob (Virchow Krankenhaus, Berlin), Biochem. Zeitschr., 32, 43, 1911. 35 W. Lob and C. Pulvermacher, Biochem. Zeitschr., 29, 316, 1910. 37 A. Slosse, Bull, de la Soc. Roy. des Sciences m€d., Brussels, May 5, 1911. S8 A. Jolles, Wiener med. Wochenschr., 1911, No. 45. "W. Lob, Biochem. Zeitschr., 17, 132, 1909. 40 C. Neuberg, in association with L. Scott and S. Lachmann, Biochem. Zeitschr., 17, 270, 1909; 2> h 152, 1910. 344 SUGAR DESTRUCTION IN THE ECONOMY line of thought in endeavoring, by studying the electrolysis of different sugars, to bring out analogies to the physio- logical destruction of sugar in the living body. Apparently formalydehyde and pentose may appear as stages of sugar cleavage and of sugar synthesis, both in the electrolytic reduction of grape-sugar and in the action of oxygen on glucose. We are probably here dealing with a change of equilibrium : GLUCOSE PENTOSE FORMALDEHYDE OH.,0» - ^ OH.nOs + CH 2 0. W. Lob, who was able to detect in the electrolysis of grape-sugar in dilute sulphuric acid (using lead electrodes) gluconic acid, saccharic acid, arabinose, arabonic acid, tri- oxyglutaric acid, formaldehyde and formic acid, 41 suggests the following schematic plan for the process : GLUCOSE >■ ARABINOSE -f- FORMALDEHYDE C6H 12 6 C 6 H 10 O 6 CH 2 j^ ^ j^ ^ / \ CH 2 .OH COOH CH 2 .OH COOH H CO, II II I (CH.OH), (CH.OH), (CH.OH) 3 (CH.OH) 3 COOH CO COOH COOH COOH COOH Gluconic Acid Sacchario Acid Arabonic Acid Trioxyglutaric Acid Formic Acid. The influence of ultraviolet light rays is also followed by cleavage of sugar with formation of aldehydes and volatile acids, and if continued may lead to the appearance of formaldehyde and carbonic acid. 42 Speaking in general terms it can be readily recognized that we have not progressed in the study of the catabolism of sugar in the economy much beyond the stage of hypothesis. As it is well known that everything that is printed is not for that reason true, there are a great many 41 Concerning formic acid as an intermediate product in sugar cleavage in the body, cf. O. Steppuhn, and H. S'chellenbach, Zeitschr. f. physiol. Chem., 80, 274, 1912. 44 P. Meyer (Neuberg's Lab.), Biochem. Zeitschr., 32, 1, 1911; A. Jolles, ibid., 33, 252, 1911; H. Bierry, V. Henri and Ranc, Compt. rend., 152, 1629, 1911, and earlier contributions. ALCOHOLIC FERMENTATION OF SUGAR 345 publications on the subject which the writer gladly forbears to inflict. It may be sufficient to refer here to one of these only, 43 that of Wohl: 44 Methylglyoxal Lactic Acid Alcohol COH COH COH COH COOH C0 2 CH.OH C.OH CO CO CH.OH — > CH 2 (OH) CH.OH CH CH 2 CH, CH 3 CH 3 — >• — >■ — >■ Glycerine Aldehyde — >- Methylglyoxal Lactic Acid CH.OH CH.OH CH.OH COH COH COOH III I I I CH.OH CH.OH CH.OH CH.OH CO > CH.OH III I J. 1 CH 2 OH CH 2 .OH CH 2 OH CH 2 .OH CH 3 CH 3 . Mention may also be made here of pyroracemic acid (CHg.CO.COOH), which, according to Paul Meyer, 45 may be placed among the sugar-formers, and which, according to Neuberg, 46 can be fermented by yeast to aldehyde and carbonic acid (CH 3 .CO.COOH = CH 3 .COH + C0 2 ), as open to consideration as a possible intermediate stage of physi- ological sugar catabolism. Mechanism of Alcoholic Fermentation of Sugar. — It was originally thought that by thorough study of alcoholic fer- mentation of sugar we might attain definite information as to the catabolism of sugar in the animal economy. The fact, already stated, that we have no sound support for the as- sumption that alcohol formation is of any physiological importance in the animal body, makes the value of studies in this line of questionable worth. Then, too, it is obvious that it is impossible, in what must be merely a brief sketch of the subject of fermentation, to properly deal with a branch which in the last few decades has grown into an independent science. The only thing which can be appropriately done here is to briefly indicate the intermediate stages, as we 43 A. Wohl (Danzig), Biochem. Zeitschr., 5, 54, 1907. 44 Without reference to the stereochemical configuration. "P. Mayer (C. Neuberg's Lab.), Biochem. Zeitschr., 40, 441, 1912. 40 C. Neuberg, Berlin, physiol. Ges., Nov. 1, 1912. 346 SUGAR DESTRUCTION IN THE ECONOMY know them at present, which are of the most importance in the transformation of sugar into alcohol and carbonic acid. At first many thought that the molecule of sugar was primarily separated into two molecules of lactic acid in fermentation, and that these were thereafter broken up into alcohol and carbonic acid: C 6 H 12 6 = 2C 3 H 6 3 ; C 3 H 6 3 = C 2 H 5 .OH + C0 2 . We know that lactic acid may very readily be broken down to form acetaldehyde and formic acid: CH 3 .CHOH. COOH == CH 3 .COH + H.COOH. Euler, of Stockholm, noted the appearance of alcohol and carbonic acid after sub- jecting lactic acid (or, instead, a mixture of equal amounts of acetaldehyde and formic acid) to the influence of the ultraviolet rays of a uviol lamp. 47 However, the idea that lactic acid is an intermediary product of alcoholic fermenta- tion was in time abandoned. Were it correct, it would be necessary to suppose the lactic acid to be as easily fermented by yeast as sugar, or even more easily. This, however, is not the case; in fact, when lactic acid is added it remains unaffected by the fermentation. 48 Later on Eduard Buchner and Jacob Meisenheimer, 49 two investigators who have rendered distinguished service in the elucidation of the fermentation process, were disposed CH 2 .OH to regard dioxyacetone, isomeric to lactic acid, CO , as CH 2 .OH an intermediate product of alcoholic fermentation, because they found that this substance uniformly undergoes fer- mentation, not only from living yeast cells but from ex- pressed juice of yeast as well, if juice extracted by boiling ( containing the ' ' coenzyme " ) be added. Eecently A. Slator 47 H. Euler (Stockholm), Zeitschr. f. physiol. Chem., 11, 311, 1911. 48 A. Slator, Ber. d. deutsch, ehem. Gesellsch., J,0, 123, 1907. ** E. Büchner and J. Meisenheimer, Ber. d. deutsch, chem. Ges., J^S, 1773, 1910. BUTYRIC ACID FERMENTATION 347 has rejected, it is true, the view that dioxy acetone is an intermediate product of alcoholic fermentation, from the fact that while, for example, a decigram of dextrose in an experiment was completely fermented by yeast in twenty minutes, a decigram of dioxyacetone in parallel experiment underwent practically no change. 50 The justice of such criticism is, however, not accepted by the first-named in- vestigators. 51 Recent studies of Lebedew, Harden and Young, and of Euler seem to indicate that the phosphoric acid contained in the fermentation mixtures plays an important role in the fermentation, uniting with hexose to form an ester which much more readily lends itself to fermentation cleavage; perhaps, too, the possibility of an intermediate formation of a triose or triose-ester in the course of the fermentation ought to be thought of. 52 At best the whole matter is at present vague and uncertain. Butyric Acid Fermentation. — In the author's opinion there may be some interest in the observations which have been made upon the mechanism of butyric acid fermentation in their bearing upon the study of the processes of physio- logical catabolism of sugar. Butyric acid may be formed from hexoses as well as from lactic acid from fermentation. Büchner and Meisenheimer 53 conceive of the process in this fashion : that the sugar first is split into lactic acid, and this in turn into acetaldehyde 54 and formic acid, two molecules 50 A. Slator, Ber. d. deutsch, ehem. Ges., 45, 43, 1912. 51 E. Buchner and Meisenheimer, Ber. d. deutsch, ehem. Ges., 45, 1632, 1912; cf. also Harden and Young, ibid., 40, 458, 1912; Chick (Lister Instit., London), Biochem. Zeitschr., 40, 479, 1912. 62 A. v. Lebedew (Instit. Pasteur), Ber. d. deutsch, ehem. Ges., 44> 2932, 1911; Biochem. Zeitschr., 36, 248, 1911; Ann. Instit. Pasteur, 25, 847, 1911; H. Euler (Stockholm), with Kullberg, OhlsSn, Fodor, Zeitschr. f. physiol. Chem., 14, 15, 1911; 76, 468, 1911; Biochem. Zeitschr., 86, 401, 1911. 83 E. Büchner and J. Meisenheimer, Ber. d. deutsch, chem. Ges., 41> 1910, 1908. 54 Cf. statements of Kostytschew (St. Petersburg), Zeitschr. f. physiol. Chem., 19, 130, 1912, upon the formation of acetaldehyde in alcoholic fermenta- tion of sugar. 348 SUGAR DESTRUCTION IN THE ECONOMY of the aldehyde then condensing into aldol and this finally becoming converted into its isomer, butyric acid : ALDOL BUTYRIC ACID -Formic Acid CH 3 CH 3 ^ I I lactic acid — >■ Acetaldehyde — > CH.OH CH 2 SUGAR > I = I lactic acid — > Acetaldehyde — > CH 2 CH 2 ^Formic Acid COH COOH. The equation for butyric acid fermentation is usually written: C 6 H 12 6 = C 4 H 8 2 + 2C0 2 + 2H 2 . Citric Acid Fermentation. — In conclusion, observations upon citric acid fermentation seem to be not without some interest in connection with the question of sugar catabolism in the body, particularly because citric acid is present in the animal body as a constituent of milk. In culture of certain forms of citromycetes on appropriate media which are poor in nitrogen and contain an addiment of carbonate of lime to fix the citric acid as the relatively insoluble citrate of calcium and thus prevent its further decomposition, the fermentation of the sugar may be governed so that actually only citric acid and carbonic acid, but neither alcohol, acetic, lactic or succinic acid, will appear. 55 Obviously the branched chain of citric acid can be explained only on the supposition of a synthesis. It is thought that it can be explained, for example, by the combination of three molecules of glycolic CH 2 .OH ^OOH citric acid !ÖH.;CH 2 .COOH CH 2 COOH '' [H[ OH.C— COOH — — > OH.C.COOH JH'j "j" CH 2 .COOH- iÖHJCH 2 .COOH But it must be said that there is as little proof that the synthesis of citric acid actually takes place in this way, as 65 E. Büchner and H. Wüstenfeld, Zeitschr. f. Biochem., 11, 395, 1909. acid, I ' with separation of water: CC~ CITRIC ACID FERMENTATION 349 there is for the statement that the hypothetical intermediate product, glycolic acid, actually is produced from sugar. All in all there is no wonder that the real nature of sugar catabolism has remained a secret thus far, locked in the depths of the economy, when we have never been able to satisfactorily explain processes like alcoholic fermentation and the various types of acid fermentation, which have been known so long, which are completed in the greatest quanti- ties in vitro, and which are so favorable to objective study. CHAPTER XV DIGESTION AND RESORPTION OF FATS Heeewith we turn our steps to a new field, that of the biochemistry of fats. It will undoubtedly be found best to continue very much in the same way as in the discussion of protein and carbohydrate metabolism, beginning with the introduction of the fats into the digestive tract, then dealing with their digestion and resorption, thereafter following them on their way through the chyle- and lymph-paths and through the tissues, up to where they and their cleavage products begin to sink into the depths of the intermediate metabolism and pass out of sight. The Lipase of the Stomach. — Beginning then with consid- eration of the process of fat digestion, the first phase to be considered is the gastric digestion of fats. 1 It is true there has been much written upon this subject, but really there is very little that can be said about it. Doubtless, according to the investigations of Volhard and others, the stomach con- tains a lipolytic ferment and physiologically the splitting of fat begins at this point. Thus London 2 found in a dog with gastric fistula that about one-third of the fat introduced in emulsified form was split in the stomach into glycerol and fatty acids. There is probably no doubt, too, that at times some fat is resorbed in the stomach. Otto Weiss 3 observed a resorption process of this sort in the stomach of ringed snakes and in the stomachs of newborn dogs and cats; in adult human beings, however, it is unquestionably insigni- ficant. Apparently, too, the lipolytic power of pure gastric literature upon Gastric Lipase: C. Oppenheimer, Die Fermente, 3d ed., 11, pp. 15-16, 1909. 2 E. S. London and M. A. Wersilowa, Zeitschr. f. physiol. Chem., 56, 545, 1908. 3 0. Weiss (Physiol. Instit., Königsberg) , Pflüger's Arch., lJ f / f , 540, 1912; cf. also W. Lamb. Jour, of Physiol., J,0, Proc. Physiol. Soc, xxiii, 1910 (Fat Resorption in the Stomach of Kittens). 350 PROBLEMS OF FAT RESORPTION 351 juice is distinctly less than we were formerly disposed to believe. 4 From the previously mentioned investigation of Pawlow and Boldyreff we have learned that immediately after taking fatty food it is very easy to have a reflux of the duodenal contents into the stomach. The first portion of fat on entering the duodenum may in the same manner as acid give rise to a pyloric reflex ; although this does not (as in case of acid stimulation) lead to closure of the pylorus, but to an inhibition of the normal antral peristalsis. In this manner the pancreatic steapsin may gain entrance into the stomach and take part here in the cleavage of fats. 5 A number of authors used to be disposed to deny for the pure gastric juice any active lipolytic function. This apparently, how- ever, is not the case ; fat cleavage in the stomach being due in part, at least, to the influence of an enzyme secreted by the gastric mucous membrane, 6 the secretion of which goes on about parallel with that of the pepsin. 7 Then, too, the state- ment of Laqueur that the gastric lipase is not activated by bile does not bespeak an identity with the pancreatic steap- sin. 8 Nevertheless, no particular physiological importance is to be attached to the lipolytic processes in the stomach in the author's opinion. PROBLEMS OF FAT RESORPTION Of very different significance is the question as to the manner in which fat resorption takes place in the intestine. It is well known that, after the fat has become mixed in the small intestine with the bile and pancreatic secretion, the greater portion of it is changed into a fine emulsion. For thirty years there has been an open question whether the *E. S. London, Zeitschr. f. physiol. Chem., 50, 125, 1906. 6 Cf. S. J. Levites. (St. Petersburg), Biochem. Zeitschr., 20, 220, 1909; Zeitschr. f. physiol. Chem., 49, 273, 1906. 6 Cf. S. v. Pesthy (F. Tangl's Lab., Budapesth), Biochem. Zeitschr., 84, 147, 1911. 7 Heinsheimer, Deutsch, med. Wochenschr., 1906, 1194. 8 E. Laqueur (P. Gottlieb's Lab., Heidelberg), Hofmeister's Beitr., 8, 281, 1906. 352 DIGESTION AND RESORPTION OF FATS emulsified fat is taken up as such by the intestinal epi- thelium, or whether the fat is first broken up in the intestine into glycerol and fatty acids and these components absorbed in a dissolved state, to reunite into neutral fat after the absorption is accomplished. The writer would prefer not to enter into the detailed phases of this conflict of opinion here. 9 It will perhaps be sufficient to state categorically that the view maintained by W. Kühne, J. Muni?:, M. Nencki, E. Pflüger, 0. Frank and many others, that a process of solution must precede the resorption of the fat (at least of the bulk), has been upheld time and again. "That the great part of the fat is split before being absorbed and thereby changed into a water- soluble form," 0. Cohnheim 10 believes, "is certain, and there is no single ground and not a single observation to prove that this is not also true of all the fat, even though the negative proof that no known portion of the fat passes the epithelium in emulsied form has not yet been established and in fact would be difficult of establishment except by con- siderations of a general nature." Probably it would be well to indicate the most important reasons which support this view. Fat Cleavage and Solution of the Products of Cleavage. — It should be clearly understood that the fat in the intestine, in by far its greatest proportion, is present, not as neutral fat, but in state of cleavage, so that, especially in the lower portions of the intestine, the neutral fat occurs in far smaller amounts than the soaps and the free fatty acids. Moreover, one should keep in mind the important fact (known today, but not known in the early days) that, be- cause of the presence of salts of the biliary acids, of lecithin, Literature upon Cleavage, Besorption and Synthesis of Fat in the Intes- tine: J. Munk, Ergebn. d. Physiol., 1, 317-323, 1912; O. Cohnheim, Nagel's Handb. d. Physiol., 2, 555, 618-621, 1907; Physiol, d. Verd. u. Ernähr., 165- 169, 1908; E. H. Starling, Handb. d. Biochem., 3", 226-233, 1909. 10 0. Cohnheim, Biochem. Centralbl., 1, 174, 1903. FAT IN THE INTESTINAL WALL 353 and of Cholesterine from the bile, physical-chemical condi- tions in the contents of the small intestine are determined in which the cleavage products of the fat are kept in solution no matter whether an acid or an alkaline reaction may prevail. This is true not only of the free fatty acids, but also of their relatively insoluble calcium and magnesium salts. Synthesis of Fat in the Intestinal Wall. — The possibility of fat synthesis in the wall of the bowel is very clearly indi- cated from the experiments of J. Munk, and those of 0. Frank; it has been shown that in the chyle of the thoracic duct neutral fat is present, not only after the administration of neutral fat, but also when the fatty acids, their alkaline salts and esters are ingested. Where the glycerol comes from which is necessary in such a synthetic formation is not known; it must suffice for the present to know that it can be produced apparently in unlimited amount by the intes- tinal wall. If this were not true it would be impossible to understand how, after the ingestion of large quantities of soaps or free fatty acids, we invariably find only the tri- glycerides in the lymph of the thoracic duct. The 'discovery that soaps are poisonous when introduced directly into the circulation and that for this reason it would be injudicious to have them enter the circulation unchanged, is, of course, no explanation. We are here brought face to face with another unsolved puzzle. If, however, the wall of the bowel manages to carry out the synthetic formation of fat from fatty acids and glycerol even when no glycerol is provided for it, why should there be any doubt of its ability to com- plete the synthesis if coincidently with the fatty acids equivalent amounts of glycerol are resorbed from the lumen of the bowel? From recent experiments of 0. Frank " it appears that ingested monoglycerides do not enter the chyle as such but "A. Argyris and 0. Frank (Munich), Zeitsch. f. Biol., 59, 143, 1912. 23 354 DIGESTION AND RESORPTION OF FATS are changed into triglycerides. The fatty acids essential for this synthesis requisite to completely transform the monoglyceride into triglyceride must first, however, be ob- tained from cleavage of the former. The inference that any extensive cleavage of ingested fat must occur is not to be made, however, from this point. Behavior of Non-saponifiable Emulsions. — The clearest indication that the intestine does not take up the fat ex- clusively in the form of an emulsion, but at least partly in a dissolved state, may be seen, to the author's mind, in the fact that even the finest emulsion of lanolin, 12 petroleum or paraffin (material which from its chemical peculiarities cannot be converted into a dissolved form in the intestine) is not open to absorption. If neutral fat be incorporated with soft paraffin by melting them together and the mixture introduced into the intestine in finely emulsified form, the bowel wall takes up only the fat, but rejects the paraflfin. 13 The writer is disposed to regard this as a thoroughly crucial experiment. The statement of Hofbauer and S. Exner, 14 subsequently confirmed by Lafayette Mendel, that fat colored with suitable staining reagents can pass into the chyle ducts as stained fat has no further reference to the method of resorption in the opinion of Pflüger, and, too, of L. B. Mendel, than to indicate that these staining substances are soluble in free fatty acids or in a biliary solution of fatty acids and soaps, the latter being quite capable of acting as a vehicle to aid the passage of the stain- ing substances through the wall of the intestine into the chyle ducts. 15 12 W. Connstein, Arch. f. Anat. u. Physiol., 1899, 30; A. v. Fekete (Physiol. Instit., Budapesth), Pflüger's Arch., 139, 211, 1911. 13 V. Henriques and C. Hansen, Centralbl. f. Physiol., Ik, 313, 1900. "L. Hofbauer (S. Exner's Lab., Vienna), Pflüger's Arch., 81, 263, 1900; 8k, 619, 1901; Zeitschr. f. klin. Med., kl, 475, 1902; S. Exner, Pflüger's Arch., 84, 628, 1901 ; L. B. Mendel (Yale Univ.) , Amer. Jour, of Physiol., 24, 493, 1909. "E. Pflüger, Pflüger's Arch., 81, 375, 1900; 85, 1, 1901; L. B. Mendel, 1. c; cf. also L. B. Mendel and A. L. Daniels (Yale Univ.), Jour, of Biol. Chem., 13, 71, 1912. OBSERVATIONS UPON FAT RESORPTION 355 Finally, as 0. Cohnheim believes, the very existence in the intestine of fat splitting ferments seemingly would be inexplicable and superfluous, if it were true that the resorp- tion is exclusively that of fat in emulsion. Histological Observations Bearing Upon Fat Resorption. — To Pfluger's objections, however, Sigmund Exner 16 sug- gests that histological examination always has made it look as though a part of the fat is resorbed without being split. "I have always regarded it as probable," says Exner, "that fat is absorbed in part unsaponified, having followed since the sixties a number of investigations conducted in the Vienna Physiological Institute bearing upon the path of resorption, 17 and having invariably found features which upheld this idea. ... As the hypothesis that all fat is changed into a soluble form before absorption and is re- formed after absorption gained more and more adherence I was forced to say to myself : If in the first place we see the fat droplets (possibly, too, droplets of fatty acids) in the contents of the intestine, in the rodded border of the cells, in the protoplasm of the epithelial cells, and finally in the chyle vessels, is it not more likely that all these droplets are of essentially identical origin and that we are catching them first before absorption and then step by step in their progress, than that the droplets which we find within the intestinal lumen are as yet to undergo solution, and that those right alongside of them in the rodded cell border or in the cell protoplasm have already reseparated from such a solution, that is to say, have been regenerated? . . . Solutions go through all forms of epithelium; fat in any important amount especially through that of the small in- testine; is it purely a matter of chance that no other epithelium but that lining the small intestine is provided with that peculiar border which in its construction from parallel rods arranged in palisade manner cannot help 16 S. Exner, Pfluger's Arch., 8-$, 628, 1901. 17 E. Brücke, S. v. Basch, F. v. Winiwarter. 356 DIGESTION AND RESORPTION OF FATS suggesting a sieve-like purpose? ... I am well aware that this is all based on probabilities, and would not care to bring it forward if there were clear proof evident on the other side. But there, too, I recognize nothing but argu- ments based upon probability. For even if anyone had proved that in this or that particular instance all the fat had undergone cleavage or saponification before being absorbed, it would be far from proving that invariably this is the case or even usually true. ' ' Many histological studies have been made to come nearer the solution of the secrets of fat resorption. The synthetic production of fat from soaps and glycerol formerly main- tained by many has as a fact not been confirmed by experi- ments upon excised living intestinal mucosa ; 18 studies from Fano 's laboratory in Florence 19 have, on the contrary, shown that the epithelial cells of the intestinal mucosa when brought in contact with oleic acid or a solution of sodium oleate become loaded with fatty acid which can be demon- strated by staining with osmic acid ; neutral fat, however, is not taken up. We are in this case undoubtedly dealing with a true physical solution phenomenon, one not mani- fested by the epithelium of the oesophagus and stomach, but which can be demonstrated with intestinal mucosa even when hardened in f ormol, and which is probably due to some solvent which the cells contain for oleic acid. Formerly a great deal of importance was ascribed to tracing the fat droplets in their passage through the epi- thelial cells of the intestine. For example, observations like those of Cuenot upon the intestine of Crustacea, in which the fat globules are apparent only in the portion of intestinal epithelium exposed to the lumen, were regarded as particu- larly valuable. Since we have come to realize that fats may be masked in the presence of proteins so that they are no 18 O. Frank and A. Ritter (Physiol. Instit., Munich), Zeitschr. f. Biol., 47, 251, 1906; Moore, Proc. Roy. S'oc, 72, 134, 1903. 18 G. Rossi, Arch, di Fisiol., 5, 381, 1908. RESORPTION OF SOAPS 357 longer demonstrable by means of osmic acid, 20 the interest in these and similar observations has perceptibly diminished. According to the studies of Noll and those of many of the older observers, a portion of the fat taken up by the intestinal epithelium leaves the latter immediately ; another portion first runs together into larger droplets, these then gradually passing out into the chyle vessels. 21 Step by step the process first shows the fat granules in the lymph clefts of the villus ; passing thence with the lymph which transudes from the capillaries into the central chyle vessel, and finally along the mesenteric lymph passages into the thoracic duct, the contraction of the musculature of the villi acting as the propelling force. When fat resorption is in active process the exposed intestine shows the chyle passages filled with milky fluid. Resorption of Soaps. — As to the further question in what form the fat is most readily resorbed, it was formerly the tendency to assume that soap solutions introduced into an intestinal loop are particularly suited for resorption. How- ever, experiments made in the Zuntz Institute 22 have shown that in dogs with Thiry-Vella fistulas of the upper portions of the intestine where a prompt resorption of fat emulsions takes place, soap solutions are not resorbed even after bile and pancreatic juice are added. (On the other hand, in case of fistulas of the lower parts of the bowel soaps were readily absorbed.) The author, in collaboration with J. Schütz 23 and with Bleibtreu, 24 has also noted a poor resorption of soap solutions from isolated intestinal loops. 20 G. Rossi, Arch, di Fisiol., 4, 429, 1909. 21 A. Noll (Physiol. Instit. Jena), Arch. f. Anat. u. Physiol., 1907, 349; Physioiogentag Würzburg, 1909 ; Centralbl. f . Physiol., 23, 290, 1909 ; Pflüger's Arch., 136, 208, 1910; cf. also Literature: E. H. Starling, Handb. d. Biochem.. 3", 226-228, 1909. --W. Croner (Lab. of N. Zuntz, Berlin), Biochem. Zeitschr., 23, 97, 1909. 23 O. v. Fürth and J. Schütz, Hofmeister's Beitr., 10, 462, 1907. 81 M. Bleibtreu, Deutsch, med. Wochenschr., 1906, 1233. 358 DIGESTION AND RESORPTION OF FATS One might be disposed, perhaps, to use this as an argument against the above stated theory of hydrolytic fat cleavage in the intestine. But from the present status of the general question the author would prefer to interpret these observa- tions as indicating that it makes a distinct difference whether the neutral fat is hydrolyzed little by little and immediately elaborated, or whether the intestine is flooded beyond physiological possibilities by large and presumably not in- different quantities of soaps. For the rest, mixtures of fatty acids are apparently at times absorbed with more difficulty than their correspond- ing sodium salts. 25 The rapidity of resorption of neutral fats depends in large measure upon the melting point of the fat concerned, the fats with high melting points (as tallows) being taken up more slowly and less fully, than oily and lard-like fats, this explaining to some degree why the fats passed with fecal matter show a higher melting point than the corre- sponding fats in the food. 26 Method of Study With Isolated Intestinal Loops. — Be- fore proceeding further, some reference should be introduced here upon the methods applicable in experiments upon fat absorption. Some years ago we were accustomed to regard the method of the "isolated intestinal loops" very highly in all sorts of absorption experiments, and fancied that we were working under precise "physiological" conditions when employing this method. When, several years ago, the author, in association with Julius Schütz, 27 undertook to study fat resorption from isolated loops high hopes were centred in the experiments, particularly because the tech- nic employed was a very distinct improvement upon that 26 S. Levites (Instit. of Exper. Med., St. Petersburg), Zeitschr. f. physiol. Chem., 53, 349, 1907. 28 J. Munk, F. Müller, Aruschnik, Levites (1. c), F. Tangl and A. Erd%i (Budapesth), Biochem. Zeitschr., 34, 94, 1911. * O. von Fürth and J. Schütz, 1. c. INFLUENCE OF PANCREATIC JUICE 359 of their predecessors. 28 In order to diminish, as far as possible, the untoward effects of chilling which is unavoid- able in intestinal operations, the experimenters made use of a warm operating table made of a large tin box provided with a heating coil, this enclosing the whole narcotized ani- mal (cat) ; the exposed intestine was spread out on com- presses wet with warm physiological salt solution, kept moist with the latter, and replaced immediately after application of the ligatures and injection of the fluid under investigation, with the usual further steps. In spite of all their effort and care the resorptive efficiency of an intestinal loop thus prepared was so low in comparison with the physiological effectiveness of the normal intestine, that the writer has lost all confidence in studies based on the method of tying off intestinal loops, and can only regret the heca- tombs of animals which the method has cost and, in spite of this warning, is likely to cost in the future. To-day, now that Pawlow and London have shown us how we can arrange our resorption experiments under practically physiological con- ditions by means of the polyfistula method, this pseudo- physiological procedure has lost all justification for its continuance, in the opinion of the writer. INFLUENCE OF THE PANCREATIC JUICE AND THE BILE UPON FAT DIGESTION We come now to the presentation of the important ques- tion of the part taken by the biliary and pancreatic secre- tions in fat digestion. That these two secretions are actu- ally concerned in the process was proved as early as 1852 by Bidder and Schmidt, and is further indicated from the observations of Claude Bernard, who found in rabbits (in which the pancreatic duct opens some distance below the common biliary duct in the small intestine) that after a diet 28 H. J. Hamburger, Arch. f. Anat. u. Physiol., 1900, 433 ; H. von Tappeiner, Zeitschr. f. Biol., 45, 222, 1908; T. Hattori, F. Hercher (Bleibtreu's Lab.), Inaug. Dissert., Greifswald, 1905, 1907. 360 DIGESTION AND RESORPTION OF FATS rich in fat the lymph vessels begin to show injection with the milk-white chyle from that level of the bowel where these two secretions become mixed with the intestinal con- tent. Dastre made a very similar observation in the dog, in which animal he ligated the bile duct and opened the gall bladder by a fistula into the middle of the small intestine, below which point mixture of the chyme first took place with the bile, the commingling of the pancreatic secre- tion occurring, of course, normally. A long series of obser- vations on animals and man, in which after exclusion of one of the two secretions or of both the utilization of fat was manifestly reduced, proved the fact that normal digestion of fat presupposes the combined effect of both secretions. 29 According to the clinical observations of T. Brugsch acute or chronic degenerative pathological processes in the pan- creas of man impair the fat absorption to a very consider- able degree (50-60 per cent.) ; but if in addition to the disturbance of the pancreatic secretion there is associated a cessation of the normal biliary flow the loss of fat may amount to as much as 80 to 90 per cent. — that is to say, the bulk of the fat leaves the intestine unabsorbed. It may be readily understood why by artificially furnishing bile and pancreatic juice one may succeed in favorably influencing disturbances occasioned by the loss of these secretions. So, too, the addition of bits of pancreas to the food may appre- ciably improve the utilization of fat, according to the state- ments of Sandmeyer. 30 28 C. Voit, 1882; F. Röhmann, 1882; F. Müller, 1887; J. Munk, 1890; Minkowski and Abelmann, 1890; A. Dastre, 1891; Sandmeyer, 1894; Harley, 1895; Hedon and Ville, 1897; Rosenberg, 1898; Albu, 1900; A. Schmidt, 1905; F. Umber and T. Brugsch, Arch. f. exper. Pathol., 55, 164, 1906; T. Brugsch (Umber's Clinic), Zeitschr. f. klin. Med., 58, 518, 1906. Literature; upon the Importance of the Bile and Pancreatic Secretion for Fat Digestion: J. Munk, Ergebn. d. Physiol., 1, 323-325, 1902; O. Prym, Handb. d. Biochem., 3", 116- 118, 1909; E. H. Starling, ibid., pp. 228-230; A. Magnus-Levy, Handb. d. Pathol, d. Stoffwechsels, 2d., 1, 32-39, 1906. 30 W. Sandmeyer (Physiol. Instit., Marburg), Zeitschr. f. Biol., 81, 12, 1895; Inouye and T. J. Sato, Arch. f. Verdauungskr., 17, 185, 1911; M. Adler (Sena- tor's Clinic), Zeitschr. f. klin. Med., 66, 302, 1908. EXTIRPATION OF PANCREAS 361 Influence of Extirpation of the Pancreas Upon Fat Re- sorption. — Taking up here the question of the mode of action exercised by the bile and pancreatic secretion on fat resorption, brief reference may be appropriately made first to the ideas which U. Lombroso has evolved as to the in- fluence of the pancreas. These rest upon the statement made by a number of authorities, that it is by no means the same thing for proper utilization of the fat whether we merely ligate the pancreatic ducts or completely extirpate the whole gland. Thus Rosenberg after the first procedure noted no striking disturbance of fat utilization in dogs ; but as soon as he entirely ablated the gland (already markedly degenerated in sequence to the ligation), marked disturb- ance ensued. 31 Observations of the same sort were repeat- edly made by U. Lombroso, 32 R. Fleckseder 3S and P. C. P. Jansen. 34 The first of these suggested the theory (with reference to the fact that after total extirpation of the pancreas there may at times be observed fatty degeneration, crowding of the fat depots of the body and even fat secretion into the intestine) that the pancreas may be involved in in- terruption of the natural process of fat metabolism not only by loss of the mixture of its external secretion with the intes- tinal contents, but also by an internal secretion which is given off into the circulating blood and by which it governs the fat transformation in the same way that it regulates the carbohydrate metabolism. 35 To properly interpret this idea one should keep especially in mind that total pancreatic ex- 81 S. Rosenberg, Pflüger's Arch., 10, 371, 1898. 82 U. Lombroso (Turin), Arch. Ital. de Biol., Iß, 336, 1904; Pflüger's Arch., 112, 531, 1906; Arch. f. exper. Pathol., 56, 357, 1907; 60, 99, 1908; Arch, di Fisiol., 8, 209, 1910. 33 R. Fleckseder (H. H. Meyer's Lab., Vienna), Arch. f. exper. Pathol., 59, 407, 1908. 34 P. C. P. Jansen (Physiol. Instit., Amsterdam) , Zeitschr. f. physiol. Chem., 12, 158, 1911. 85 O. Gross (Steyrer's Clinic, Greifswald), Deutsch. Arch. f. klin. Med., 108, 106, 1912, has also recently accepted with Lombroso that the steatorrhea met in pancreatic affections is due to a loss of the internal secretion of the pancreas. 362 DIGESTION AND RESORPTION OF FATS tirpation is a very serious interference which, results in an inexpressible wasting of the body. It might be asked very properly whether, when "everything is upset" in metabo- lism in sequence, it really is a matter of much wonder that the fat digestion should also become disordered. Minkowski noted in dogs with only a remnant of pancreatic tissue transplanted under the skin of the abdomen, the secretion of which escaped externally, that the resorption of fat was not materially reduced provided the dog could lick up the secretion ; when the secretion, however, was with- held, interference with resorption at once manifested itself. 36 Off-hand there does not seem to be any insistent reason for ascribing to the internal secretion of the pancreas, besides its dominant part in the metabolism of sugar, a second analogous cardinal function in relation to the metabolism of fats. Disturbances of the latter process, after complete pancreatic extirpation, can be fully explained in the first place on the basis of the failure of its external secretion, and in the second place by the general disturbance of the economy which severe pancreatic diabetes brings with it. There will probably be noted a contradiction in the author's first statement that the combined influence of the pancreas and the bile is essential to the proper procedure of fat digestion and the subsequent point to the effect that ligation of the pancreatic ducts need not necessarily give rise to any very important disturbance of fat resorption. This is, however, probably only a superficial contradiction. If under normal conditions the bile and the pancreas co- operate in connection with the resorption of fat (which is undoubtedly the case from the decisive observations of Claude Bernard and Dastre upon the chyle), this does not preclude the possibility of the economy taking advantage of vicarious means of compensating in some measure for the loss of pancreatic secretion. We know, for instance, that 38 G. Burkhardt (Minkowski's Clinic, Greifswald), Arch. d. exper. Pathol., 58, 252, 1908; cf. also the observations of Abelmann and others. ACTIVATION OF PANCREATIC STEAPSIN 363 fat cleavage in the digestive tract is not accomplished ex- clusively by the pancreatic steapsin, but that in addition lipases of the gastric juice, of the intestinal secretion and of the vast numbers of microorganisms in the intestine are also concerned in the process. Activation of the Pancreatic Steapsin by the Salts of the Biliary Acids. — In further effort to properly appreciate the manner in which the pancreatic secretion and the bile influ- ence the digestion of fat, we at once are met by the important fact that the fat-splitting ferment of the pancreas is greatly augmented in its effectiveness by the bile. The first statements bearing upon the reinforcing influ- ence of the bile upon the pancreatic steapsin are probably to be ascribed to M. Nencki. These were confirmed later by a number of observers. 37 The general fact in itself thus seemed settled; but until recently it was not known to which of the components of the bile this characteristic and (for the physiological action of the bile) undoubtedly important property was to be referred. Contrary to the statements of Hewlett, who believed he was in position to ascribe its most important role in fat cleavage to the lecithin of the bile, the author, in collaboration with Julius Schütz, was able to show that the effect (at least for the most part) is due to the salts of the bile acids (glycocholic and tauro- cholic acids). Although these results were regarded as en- tirely conclusive, it was unquestionably a matter of satisfac- tion that R. Magnus 38 determined further that the sodium salts of synthetically formed biliary acids are also capable of acting as powerful activating agents. "In the synthetic production of the two biliary acids," says Magnus, "which was done by starting with cholalic acid and producing suc- cessively the ethylester, the hydrazid and the azid of this "Dastre, 1891; Knauthe, 1898; G. C. Bruno, 1899; Babkin, 1903; K. Glässner, 1904; A. W. Hewlett, 1906; cf. Literature: 0. v. Fürth and J. Schütz, Hofmeister's Beitr., 9, 28, 1906. 38 R. Magnus, Zeitschr. f. physiol. Chem., 48, 376, 1906; cf. also E. F. Teorrine (Lab. of the College of France), Biochem. Zeitschr., 23, 440, 1910. 364 DIGESTION AND RESORPTION OF FATS acid, there were so many and such interfering procedures (prolonged boiling with acids and alkalies, treatment with hydrazine hydrate, sodium nitrite, etc.) required that it seemed quite impossible that any substance active in small amounts could remain intact and maintain itself in practi- cally unchanged quantity through all these processes. . . . The augmenting influence of the bile upon fat cleav- age by the pancreatic juice depends upon its containing biliary salts of alkalies. The economy in this respect works with precisely the same means as the technical chemist, who splits fats today with ferments and activates the latter by small quantities of manganese sulphate." Question of the Complex Nature of the Pancreatic Steapsin. — Here arises the further question of just how this powerful effect (for the fat splitting action of the pancreatic steapsin can be increased more than tenfold at times by the addition of small amounts of bile) is to be interpreted. The author's pupil, Hedwig Donath, whom he induced to take up this question seriously, answers it by supposing that we are here dealing with the conversion of an inactive zymogen or proferment into an efficient enzyme, a slow transformation being also possible ' ' spontaneously, ' ' but greatly accelerated by the catalytic action of the bile salts. From this it is apparently easy to appreciate why steapsin preparations may spontaneously undergo changes of such a character that add to their direct effectiveness but diminish their capacity for activation by cholic acid. Activation of the pancreatic ferment by cholates results in increasing the ferment activity only within certain limits in proportion to the quantity of cholic acid employed ; from this limit forward further addi- tion of cholic acid is not followed by any increase in efficiency. 39 This discovery is of general interest from the fact that in many ways it suggests certain analogies which 39 H. Donath, Hofmeister's Beitr., 10, 390, 1907, conducted under direction of 0. v. Fürth; cf. also 0. Rosenheim, Jour, of Physiol., JfO, Proc. Physiol. Soc, 1910 — Separation of the Pancreatic Lipase from Its Coenzyme. SYNTHETIC PRODUCTION OF FAT 365 ferments present with toxines. It has been especially sug- gested, too, that enzymes, just as toxines, are perhaps com- posed of two parts, one a thermostable "amboceptor" and the other a thermolabile ' ' complement. ' ' Observations like those of H. Donath, of the possibility of partially reactivat- ing pancreatic steapsin which had been inactivated by ex- posure to a temperature of about 60° C. by a thermolabile agent present in normal horse serum, invariably make it seem very likely that the pancreatic steapsin is of a complex nature. It should be added, too, that appearances of this and similar nature fall in very well with the ideas of Victor Henri, who, on the basis of ultramicroscopic observations, has brought into the reach of possibility an explanation of ' ' complement effects ' ' from purely physical factors. 40 Synthetic Production of Fat by Reverse Ferment Action of Lipase. — The fermentative kinetics of pancreatic lipase has been closely studied from a number of standpoints 41 ; but it is impossible here to enter into the details of this phase of the subject, which belong properly to the sphere of physical chemistry. Reference may be made merely to one point, because of its special physiological interest, that is, the fact that the lipases concerned in digestion, coming from the pancreatic and intestinal secretions, are not only capable of separating fat into glycerol and fatty acids, but also of bringing about in a reverse way a synthetic produc- tion of fat from its components. We are concerned here with a true reversible enzyme action, Gylcerol + Fatty acids < > Neutral fat, which, according to conditions, can take place in either direction, and which brings somewhat closer to our under- 40 V. Henri, Congress of Physiologists, Heidelberg, Aug., 1907, Cenfralbl. f. Physiol., 21, 477, 1907. 41 Kastle and Löwenhart, H. Engel, J. Lewkowitseh and J. J. K.. Macleod, Taylor, A. Kanitz, Zeitschr. f. physiol. Chem., 46, 482, 1905; E. F. Terroine, Biochem. Zeitschr., 28, 404, 1910, and others. Cf. therein the Literature. Cf. also O. Rosenheim and collaborators, Jour, of Physiol., 40, Proc. Physiol. Soc, 1910. 366 DIGESTION AND RESORPTION OF FATS standing the fact that fat which has undergone cleavage in the lumen of the intestine is regenerated synthetically in the wall of the bowel. 42 Just as fat cleavage is activated by the salts of the biliary acids, so, too, is the fermentative synthesis of fat. 43 Cleavage of Fat in the Intestine in the Absence of Pan- cretic Secretion. — Since, as above pointed out, the absorption of fat, at least for the greater part, can be completed only after complete cleavage according to the more recent views, and the latter is very much augmented by the influence of the bile, disturbance of fat resorption might at first glance seem to be thoroughly explained after loss of the bile and pancre- atic secretion. On closer consideration, however, this ex- planation is seemingly subverted by the fact that even in a case of this sort the fat which appears in considerable quanti- ties in the faeces is not (as might have been expected) an in- tact neutral fat, but, on the contrary, seems to be practically all split up into its components. This has confused our con- ceptions of the true nature of fat resorption all the more, be- cause, as E. H. Starling 44 very properly remarks, we over- looked the point that we should know not only whether the fat has been split in the digestive tract at all, but whether the cleavage occurred at the right place, that is, in the upper portion of the intestine. It may take place (probably true in case of diminutions of the pancreatic secretion) through the agency of microorganisms only in the lower parts of the intestine, where it is apparently too late, and where the intestine is unable to deal with the products of cleavage. In view of the fact that resorption of fat is apparently much more seriously interfered with when the flow of bile is stopped along with that of the pancreatic fluid, than when 42 Hanriot, J. H. Kastle and A. S. Löwenhart, O. Mohr, H. Pottevin, Ann. Instit. Pasteur, 22, 901, 1906; H. Donath, 1. c; A. E. Taylor, Jour, of Biol. Chem., 2, 87, 1906-1907; W. Dietz, Zeitschr. f. physiol. Chem., 52, 279, 1907. 43 A. Hamsik (Cech. Univ., Prague), Zeitschr. f. physiol. Chem., 59, 1, 1909; 65, 232, 1910; 11, 238, 1911. 44 E. H. Starling, Handb. d. Biochem., S", 230, 1909. SOLVENT POWER OF THE BILE 367 the latter alone is stopped, it is important to call attention to the point that the bile acts as an important activator not only upon the lipase of the pancreatic secretion but also upon that of the unmixed intestinal juice. Solvent Power of the Bile. — Since, following what has been said above, the generally acknowledged powerful effect of the bile on fat digestion does not seem thoroughly ex- plained by its influence upon fat cleavage, earnest efforts have been made to discover other explanatory possibilities. One suggestion has brought into prominence the solvent power of the bile for the higher fatty acids, and lipoids of all sorts. If, for example, an opaque, milky suspension of lecithin be treated with a solution of the biliary salts, the fluid be- comes clear at once, and in the ultramicroscopic field the suspended particles may be watched disappearing from sight. 45 The combination of salts of the biliary acids, lecithin, Cholesterine and mucin in the bile form with the soaps a physical-chemical system in which (as we have learned from the studies of Moore and Rockwood, Pflüger and Rossi 46 ) fatty acids are soluble to so great an extent that, as a matter of fact, the largest amounts which could possibly be regarded as within bound of physiological oc- currence are converted to a water soluble form. This is the more important, as soaps in the intestinal contents very readily undergo hydrolytic dissociation from the influence of carbonic acid, fatty acids being observable as a conse- quence in free state even in the presence of notable amounts of sodium carbonate. 47 Whether, in addition to this im- portant point, the favoring influence (at one time looked upon as important) of the alkali contained in the bile upon emulsification of fat, and the reduction of the surface ten- sion of the intestinal contents by the salts of the biliary 45 L. Kalaboukoff and E. F. Terroine, C. R. Soc. de Biol., 66, 176, 1909. 46 G. Rossi (G. Fano's Lab., Florence), Arch, di Fisiol., 4, 429, 1907, cited in Centralbl. f. Physiol., 21, 811, 1907. 47 G. Rossi, 1. c. 368 DIGESTION AND RESORPTION OF FATS acids, 48 or whether finally an increase of the absorbing activity of the intestinal epithelial cells caused by the bile, should be taken into consideration, may remain where they are. LIP^MIA Passing a step further, we may next consider the route by which the fat passes into the blood stream, and the form which the fat taken up out of the intestine assumes in the blood. Resorption Path of the Fat. — That a part of the fat finds its way by the lymph paths and thoracic duct may be seen by direct anatomical study of an animal killed after a meal rich in fatty substances. Many observations, as those of Munk and Eosenstein on a girl with a thoracic duct fistula, indi- cate that at times the bulk of the fat follows this route. Without question, however, a portion of the fat goes directly into the blood stream. J. Munk and Friedenthal observed after ligating the thoracic duct and feeding freely on food rich in fat (cream) that the blood sometimes contained up- wards of six times the normal amount of fat, so that, after preventing coagulation by the addition of ammonium oxal- ate, the blood on sedimentation became covered with a thick layer of cream. 49 In the writer 's opinion a study which has attracted but little notice, made in Bottazzi's laboratory in Naples, 50 should be regarded as of special importance to the question of the route of fat resorption. From animals during fat digestion two blood specimens are taken at the same time, one from the portal vein and one from the jugular vein; and these are compared for their fat content. Did the principal stream of fat pass from the thoracic duct into the jugular vein obviously one should always expect that the amount of fat in the blood of the latter would exceed that of the portal blood ; actually, however, the opposite was 48 Cf. G. Billard, C. R. Soc. de Biol., 61, 323, 1906. 43 J. Munk and H. Friedenthal, Centralbl. f . Physiol., 15, 297, 1901. 50 G. d'Errico (Bottazzi's Lab.), Arch, di Fisiol., k, 1908. H^EMOCONIOSIS 369 found to be the case. It has not been possible to recover from the lymph of the thoracic duct, collected during the period of resorption of a known amount of fat, more than sixty per cent, of the fat which disappeared from the intes- tine. H. J. Hamburger 51 has satisfied himself that the re- sorption of soaps from loops of the small intestine of the dog continues even after the recognizable lymph vessels have been ligated. Hatmoconiosis. — In what form does the resorbed fat ap- pear in the blood? After free fat diet the plasma, and, too, the serum expressed from the clotted blood, looks milky; and on standing a cream-like layer may be formed on the surface by the collection of the fat globules. Kreidl and Neumann 52 have observed in ultramicroscopic study of the blood at time of fat resorption the appearance in the dark field of great numbers of shining, very minute granules ("haemoconiaB") which are not seen in the blood of the fast- ing human being or of one on fat-free diet. If such exami- nations of the blood are repeated at short intervals after the ingestion of a meal rich in fat, and the quantity of the particles visible in the field be estimated each time, one can recognize a gradual accession of the fatty particles, and after a maximum has been reached a gradual recession suc- ceeding. About twelve hours after the last meal the serum of healthy human beings is clear and free from haemoconiaa. In cats and rabbits the highest point of resorption is reached in about four hours, in man in about six hours. 53 These blood-dust particles seem to belong exclusively to that part of the fat which reached the blood by way of the thoracic duct. Solution in the blood cannot be said to take place. The particles seem to disappear from the blood vessels 51 H. J. Hamburger, Arch. f. Anat. u. Physiol., 1900, 554. 82 A. Neumann, Centralbl. f. Physiol., 21, 1907; Wiener klin. Wochenschr., 1.907; A. Kreidl and A. Neumann, Sitzungsber. der Wiener Akad. Mathem. Naturw. Kl., 120, III, February, 1911. 63 A. Kreidl and A. Neumann, 1. c; E. Neisser and H. Bräuning (Stettin), Zeitschr. f. exper. Pathol., 4, 747, 1907. 24 370 DIGESTION AND RESORPTION OF FATS by traversing the capillary wall and are taken up in corpuscular form, as are other suspended particles in cer- tain cell groups, by the tissues, especially the liver, spleen and bone-marrow. 54 Masking of the Fat in the Blood. — Information obtained in another way indicates, however, that in the disappearance of the fat from the blood that there are processes of a very different character which we must also consider. W. Conn- stein and Michaelis came to the conclusion that there exists a lipolytic function of the blood. If, to be specific, an emulsion of fat be mixed with blood and air conducted through it, one will notice an appreciable loss in the ethereal extract. That an ester-splitting ferment discovered by Hanriot in the blood has no part at all in the disappearance of the fat from the blood was shown by careful studies long ago by Arthus, and also by Doyon and Morel. The latter authors were able to show that the reduction in the ethereal extract obtained from blood containing fat does not coincide in the least with cleavage of the fat into fatty acids and glycerol. 55 Eecent investigations, among which those of Mansfeld, 56 of Budapesth, are prominent, have shown the unexpected fact that in this puzzling disappearance of the fat from the blood (aside from the above mentioned escape of the blood dust particles from the blood vessels through the walls of the capillaries into the cells) we are not dealing with either a cleavage process or with one of destruction, but rather with a process of masking. It was mentioned above that fat in contact with albumin (as shown in Fano's laboratory) 54 S. Biondi and A. Neumann, Wiener klin. Wochenschr., 1910, 734; E. Nobel (S. Exner's Lab., Vienna), Pfliiger's Arch., 134, 436, 1910; J. Leva (Berlin), Berliner klin. Wochenscbr., 1909, 961. 55 Connstein and Michaelis, Hamburger, Weigert, Arthus, Doyon and Morel; cf. the Literature: W. Connstein, Ergebn. d. Physiol., 3', 210-223, 1904; cf. also the statements of P. Rona and L. Michaelis, Biochem. Zeitschr., 31, 345, 1910, as to the existence of a tributyrin-splitting ferment in the blood. 68 G. Manäfeld (Pharmacol. Instit., Budapesth), Magyar Orvosi Arch., 9, cited in Jahresber. f. Tierchem., 1908, 84; Pfliiger's Arch., 129, 46, 63, 1909. MASKING OF FAT 371 may escape detection by osmic acid staining. From Mans- f eld's studies it would appear that the resorbed fat enters in part into some sort of combination with the protein in the blood, and thus becomes insoluble in ether. We are compelled, therefore, to distinguish in the blood between the free fat and the combined fat. Only the free fat can be supposed capable of passing through the capillary walls and entering the tissues. Possibly, too, changes of lipoids may play some part in the apparent disappearance of fat in the blood. 57 Relation of the Masking of Fat to Fatty Degeneration. — That crude interferences like heat coagulation, the influence of alcohol or peptic digestion are capable of breaking the delicate combinations between fat and protein (at first sup- posed necessarily to be physical but later regarded as chem- ical in character) 58 is entirely obvious. Mansfeld is, how- ever, of the opinion that changes of a more intricate type are also capable of destroying this union and that fatty degen- eration and fat metastasis in pathological conditions (as in phosphorus- and acid-poisoning, starvation, etc.) are closely connected with the process. Since infusion of dilute lactic acid under proper conditions sets free the fat in combina- tion with protein in the blood and enables it to pass out through the capillary wall, Mansfeld regards it probable that the fatty degeneration of phosphorus poisoning is related with the accumulation of lactic acid in the blood. Such an assumption would, however, seem justified only after proof by careful quantitative experiments that the amounts of lactic acid which appear in the blood in phosphorus poisoning 51 L. Berczeller (F. Tangl's Lab., Budapesth), Biochem. Zeitschr., 4 J h 193, 1912. 58 Boggs and Morris, Jour, of Exper. Med., 11, 563, 1909, who have observed a distinct lipamia in animals after repeated bleeding, found that the milky serum is not cleared up by shaking with ether, but is readily cleared after adding ammonium oxalate. It would require special study to determine whether the removal of calcium is the essenial point in this procedure and whether we are not rather dealing with some kind of disturbance of the physical- chemical equilibrium. 372 DIGESTION AND RESORPTION OF FATS are capable of breaking up the combinations between fat and protein in the blood, although subject to instant neutraliza- tion by the blood alkali. As long as we lack this proof the fact that in phosphorus poisoning the relation between free and combined fat in the blood is altered so that the former predominates, is open to a number of other interpretations. Pathological Lipcemias. — Coming to the pathological lipsemias, 59 quite a number of pathological conditions are known in which an abnormal accumulation of fat often occurs in the blood. This is true especially of starvation, of the various forms of anaemias and cachexias, of diabetes (both the severe human type and experimentally produced pancreatic and phloridzin diabetes), of chronic alcoholism and long continued narcoses, of phosphorus poisoning and a number of other toxic conditions. It is scarcely possible, in the present state of our knowl- edge, to consider these different lipsemias from a single standpoint. But one may at least endeavor to clear up a group of physiological factors which are open to consider- ation in this connection. Lipcemia from Mobilization of Fat Deposits. — As a very important element in many of these lipaBmias we may safely consider a compensatory regulative effort on the part of the economy enabling it to mobilize its supply of fat in case of need, as in starvation. The importance of this fat mobilization, which manifests itself anatomically in the loss of the fat accumulations in various positions, may be at once appreciated, if we recall that the body in long-continued starvation, as soon as its glycogen has been used up, calls upon fat combustion to cover at least nine-tenths of its de- mand for energy. That this may become possible, however, the fat must lend itself to ready movement. The liver is apparently frequently the first objective point, and may in many pathological states be found the seat of an excess of fat (as, typically, in dogs with pancreatic diabetes). Here 68 Literature on Lipaemias : A. Magnus-Levy and L. F. Meyer, Handb. d. Biochem., h' , 459-470, 1909. DIABETIC LIPiEMIA 373 there may be manifested a certain antagonism between glycogen and fat, the site left vacant by the disappearance of the glycogen coming to be occupied by the fat (not so much in the anatomical sense as functionally, in the view taken by G. Eosenf eld) . This point will be again taken up in connection with fatty degeneration. The classical example of this type of fat mobilization is seen in the lipaemia of the Ehine salmon (during their migration without food) ob- served by Miescher. In starving mammals the time of li- paemic occurrence perhaps may be regarded as correspond- ing with the time when the supply of carbohydrate has become completely exhausted. 60 Diabetic Lipaemia. — It is not easy to see why a mobiliza- tion of fats should not be assumed also for the lipaemia in phloridzin diabetes, in that due to removal of the pancreas, 61 and in protracted diabetic coma. 62 Diabetic coma seems in the vast majority of cases to be associated with lipaemia, in which the amount of fat in the blood may reach an enormous grade of twenty per cent, or more; the blood assuming an appearance not unlike cream and chocolate, and on ophthal- moscopic examination the retina showing directly a milky turbidity. According to G. Klemperer in such cases we are really dealing with a lipoidaemia rather than a lipaemia, the bulk of the ethereal extract consisting, not of fat, but of Cholesterine and lecithin. For this reason he would in- terpret diabetic lipaemia not so much as a phenomenon of mobilization of fat deposit as of the cellular lipoids and as evidence of increased cellular disintegration. 63 Other ob- servers do not express themselves upon this point in by any means as dogmatic a manner. Thus an examination of the blood in a case of pancreatic diabetes showed that the 60 F. N. Schulz, Pflüger's Arch., 65, 299, 1897; W. Daddi (W. Aducco's Lab.) , Lo Sperimentale, 52, 43, 1898. C1 L. Lattes (Turin), Arch. f. exper. Pathol., 66, 132, 1911, and earlier investigators. 62 L. Schwarz (Prague), Deutsch. Arch. f. klin. Med., 76, 233, 1903; G. Klemperer and H. Umber, Zeitschr. f. klin. Med., 61, 145, 1907; 65, 340, 1908. 03 G. Klemperer, Deutsch, med. Wochenschr., 1910, 2373. 374 DIGESTION AND RESORPTION OF FATS lecithin was, as a rule, increased, but the amount of Choles- terine was variable; 64 another observer on the contrary believes that in the lipaeniia of human diabetes we are deal- ing more with a cholesterinaemia than with a lecithinaemia. 65 The writer, too, regards it as more than doubtful whether an accumulation of as much as half a kilogram or more, as esti- mated by Magnus-Levy, 66 in the circulating blood in nu- merous coma cases, can be properly referred to the lipoids arising from cellular disintegration. As there is every rea- son to regard the acetone bodies to be due to changes in the neutral fat, it would appear not at all inappropriate to refer the coincident lipaemia to the same basic cause. That tissue cells do undergo disintegration and that their lipoids gain access to the circulating blood under such circumstances should not, it seems, be a matter of doubt. Lipcemia from Narcosis. — Beicher's suggestion that in the lipaemia which is sometimes seen in long continued anaes- thetization we are dealing with an escape of lipoids from the cells (due to entrance of fat solvents in the blood) is contradicted by the fact that lipaemia is sometimes also seen in morphine narcosis where such an outpouring cannot be considered. 67 On reflection, moreover, it must be confessed that in- creased passage of fat into the blood stream does not en- tirely explain the pathological lipaemias. It should be recalled that even the greatest concentration of fat in the blood, as that which takes place after ingestion of fatty food, disappears under normal circumstances in the course of a few hours, the fat leaving the blood stream. It was formerly believed to be due to the tissue cells taking up the haemoconial particles ; yet it may be presumed that at least in part the fat passes through the capillary walls in dis- 64 J. Seo (Minkowski's Clinic, Greifswald), Arch. f. exper. Pathol., 61, 1, 1909. 05 M. Adler (Univ. Polyclinic, Berlin), Berliner klin. Wochenschr., 1909, 1453. 86 A. Magnus-Levy and L. F. Meyer, 1. c, p. 464. eT Cf. A. Magnus-Levy and L. F. Meyer, 1. c, p. 463. PASSAGE OF FAT OUT OF BLOOD STREAM 375 solved state. The liver undoubtedly performs an important role in working up the fat which passes out of the circulat- ing blood. 08 Observations of K. Glässner and G. Singer on animals with biliary fistulas indicate that the fat of the food passes to the liver by way of the blood, is fixed there for a brief period and may also be in part excreted in the bile. 69 Passage of the Fat Out of the Blood Stream. — But when it is realized that in pathological lipaemia large quantities of fat continue in concentration in the blood, the logical con- clusion must necessarily be that for some reason or other the fat must be hindered in its passage out of the circulating blood. There is not the least basis for accepting the idea that the parenchymatous tissues because of loss of oxidizing power have been deprived of their ability to consume fat in their normal manner. As Magnus-Levy properly suggests, it is impossible to consider anything except some added diffi- culty of passage of the fat out of the capillaries, either from change in the permeability of the capillary walls or from delay in the occurrence of the changes which are necessary in the fat itself in order to enable it to pass through the capillary wall. 70 What the nature of such changes is it is impossible to say at present. Brief reference may here be made to the question as to what conditions are required for the passage of the fat from the blood into the urine. There is no doubt that in condi- tions of concentration of fat in the blood small amounts of the former may pass over into the urine. 71 Both lipuria and albuminuria have been induced coincidently by infusion of cream intravenously in the dog, the fat appearing in the urine as a persistent cloud-like emulsion. 72 Massive occur- 68 F. Ramond, Jour, de Physiol., 7, 245, 1905. 69 K. Glässner and G. Singer (E. Freund's Lab., Vienna), Med. Klinik, 1909, No. 51. 70 A. Magnus-Levy and L. F. Meyer, 1. c, p. 465. 71 B. Schöndorff (Physiol. Instit., Bonn), Pflüger's Arch., 117, 291, 1907: cf. therein the Literature. "A. Magnus-Levy and L. F. Meyer. 1. c.. p. 469. 376 DIGESTION AND RESORPTION OF FATS rence of fat in the urine ("chyluria") is only observed, as indicated beyond doubt by recent studies, 73 when there happens to be an abnormal communication between the chyle vessels and the urinary passages. Fetal Lipcemia. — An interesting form of lipaemia has been observed in fetal guinea pigs by A. Kreidl and his col- laborators. It seems that the blood of developed guinea pig fcetuses is crowded with ultramicroscopic particles of fat. Their presence is in no way due to corpuscular fat contained in the maternal blood ; the placenta prevents the corpuscular fat in the maternal circulation from passing to the foetus, and vice versa. In the end, of course, the foetus obtains its required fat from the mother animal, and it must be assumed, therefore, that the component fatty acids and glycerol are taken from the maternal blood, synthesized by the placenta into fat and transmitted as such to the foetus. The placenta in this matter seems to fulfil a function similar to that which belongs to the secreting mammary gland 74 (v. infra, Chapter XVII). In human beings during preg- nancy the blood becomes richer in fatty substances (espe- cially Cholesterine compounds and neutral fat). 75 The proportion of lipoids in the placenta is maximal in the early stages of pregnancy and diminishes during the course of the period. 76 Peculiar features in the migration of fat may be met in many fish, as in the torpedo, the female of which, according to Reach, does not take food during her pregnancy, her rich supply of hepatic fat passing into the yolks to serve as the most important source of energy for the embryos. 77 73 Carter, Franz and Steyskal, Magnus-Levy ; Literature : A. Magnus-Levy and L. F. Meyer, 1. c, 469-470. 74 Oshima (under direction of A. Kreidl, Vienna), Centralbl. f. Physiol., 21, No. 10, 1907; A. Kreidl and H. Donath, ibid., 2k, No. 1, 1910. 76 E. Herrmann and J. Neumann (S. Fränkel's Lab., Vienna), Wiener klin. Wochenschr., 1912, No. 12, and Biochem. Zeitschr., k3, 47, 1912. 78 B. Bienenfeld (S. Fränkel's Lab. and F. Schauta's Clinic, Vienna), Biochem. Zeitschr., JfS, 245, 1912, and Monatschr. f. Geburtsh., 36, 158, 1912. 77 F. Reach, in collaboration with V. Widakowich (A. Durig's Lab., Vienna) , Biochem. Zeitschr., 40, 128, 1912. PAVY'S THEORY 377 Diet Lipcemia. — In detailing the different forms of lipaemia mention must finally be made of mastlipasmia, seen, for instance, in geese sometimes after forced carbohydrate feeding and after fatty diet. 78 It may, perhaps, be imagined that when there is too great a transformation of carbohy- drates into fat, the fat depots become overfilled to snch an extent that finally they can no longer accommodate it, and that then it accumulates in the blood. It may be, too, that the lipaemia of many obese alcoholics can be referred to a similar mode of causation (v. infra, p. 387). Pavy's Theory. — Pavy 79 observed that if rabbits are fed upon material rich in carbohydrates but poor in fat, as oats, the intestinal chyle vessels afterwards become promi- nent from their milk-white injection. On this fact he based his theory (diametrically opposed to all of our views) that the intestinal villi are able to change carbohydrate into fat, and that this happens with the most of the resorbed carbo- hydrates so that they do not enter the blood as sugar. Gr. von Bergmann and K. Keicher were, however, able to prove that, on feeding with oats from which the fat had been carefully extracted, no fat appears either in the intestinal villi or in the lymph vessels, and that therefore Pavy's hypothesis is not correct. 80 The path to knowledge is paved with mistakes ; this has always been so and always will be. Whoever has devoted himself for any time to experimental study leams from his own failures to be lenient toward the failures of others. And yet one cannot help feeling depressed to see, as in this instance, some new theory brought before the world which upsets all existing opinions and brings about a lot of con- fusion, when even a simple and obvious control examination would have been sufficient to prevent its birth. 78 Bleibtreu, Pflüger's Arch., 85, 345, 1901. 79 F. W. Pavy, Ueber den Kohlehydratstoffwechsel, Leipzig, Engelmann, 1907. 80 G. v. Bergmann and K. Reicher (F. Kraus's Clinic, Berlin), Zeitschr.. f. exper. Pathol., 5, 760, 1909. CHAPTER XVI FAT METABOLISM. OBESITY Having discussed the manner in which the food fat is taken up from the intestine, how it enters the blood and is distributed with the latter in the body, we may next endeavor to gain some insight into the methods by which, having accumulated in the economy, it is transformed in metabolism. We should start with the fact that fat serves as an important source of energy and is a substance well suited to limit the demand upon other materials. In contrast to protein it is strikingly distinguished by its capacity for being stored as a reserve substance in times of abundant supply; while, as is well known, we are not directly in position to in- sure a new formation of organized body-protein by an abundant proteid food supply. Dependence of Protein Destruction Upon Supply of Fat. — When protein and fat are ingested at the same time the amount of protein required can be kept down by the latter. While (following the teachings of the Voit school) in a dog fed on proteid material alone the nitrogen balance can be maintained only by supplying three and one-half times the amount of protein exchanged in inanition, the nitrogen balance may be successfully maintained, if fat be also ex- hibited, with but one and a half or two times the amount of protein metabolized in starvation. At first glance the state- ment based on Voit 's authority that in a starving dog, whose fat deposits have not as yet been consumed, administration of several hundred grams of fat scarcely influences the protein exchange at all, seems surprising. The explanation of this rather remarkable peculiarity is simply that in star- vation fat is in demand also ; as already stated, the starving body draws mainly upon fat to supply its requirements for production of energy. If, therefore, a starving organism, which still has a supply of fat, be flooded with fat, this added fat will be surely burned in place of the tissue fat, but will 378 PARENTERAL FAT ABSORPTION 379 not otherwise make much change in the relative transforma- tion processes. Above all, however, the wear on the cor- poreal mechanism will be manifest in a nearly constant nitrogenous output. 1 Importance of Lipoids in Nutrition. — Is fat a vitally nec- essary food? We formerly were disposed to deny this ques- tion without hesitation, because it was well known that dogs can be readily kept alive and strong for a long time on meat diet alone. However, recently W. Stepp, in. investigations, undertaken under direction of Franz Hofmeister, has brought out the remarkable fact that mice invariably die if their appropriate food be freed of all fatty substances by thorough alcohol-ether extraction. If pure neutral fat (tri- palmitin, tristearin or triolein), or lecithin or cholesterol or even butter, be added to the degreased food, the animals con- tinue to die; apparently it is not the well-defined fats but certain " lipoids" which are essential for maintenance of life. This is only another example of the peculiar things which one is sure to encounter in the course of metabolism studies. 2 Parenteral Fat Absorption. — If it is desired to introduce large amounts of fat into the body apparently the only way is by the bowel, and, perhaps, too, by intraperitoneal intro- duction. Experimental subcutaneous injection of olive oil, and, similarly, stained and iodized fat, has invariably shown that only very small amounts of such substances (at best no more than a few grams per diem) can be absorbed from the subcutaneous tissue, the bulk otherwise remaining unchanged in situ. On reflection it may be readily appreciated that the injected fat in all likelihood (just as the fat of the organized depots) can be absorbed only after it has been changed to a soluble form by some lipolytic ferment. However, a large 1 Literature: Graham Lusk, Ernährung and Stoffwechsel, 2d ed. (German Translation by L. Hess), pp. 149-154, 1910. 2 W. Stepp (Hofmeister's Lab., Strassburg), Biochem. Zeitschr., 22, 452, 1909; Zeitschr. f. Biol., 57, 135, 1911. Cf. also W. Stepp (Med. Clinic, Giessen, Voit), XXIX Kongr. f. innere Med., Wiesbaden, 1912, p. 610. 380 FAT METABOLISM. OBESITY amount of fat injected under the skin presents, of course, a relatively small surface for attack by such ferments, and for this reason it seems possible to very materially improve the resorption by introducing the fat in the form of a fixed emul- sion. In spite of the fact that in occasional experiments it has seemed possible to distinctly prolong the life of starving animals by subcutaneous injections of fat, the hopes which were entertained by clinicians of being able to feed by sub- cutaneous injection of fat, because fat contains in the smallest volume the largest amount of energy (measured in calories), have for the present at least entirely failed of realization 23 (v. infra, Chapter XX). Fat Storageinthe Body. — The amount of fat stored in the economy is very considerable even under normal conditions. In a healthy human being it has been estimated at about 18 per cent, of the body-weight. The importance of the energy thus accumulated can be readily appreciated when one thinks of the long periods of fasting and the diseases which can be endured in which the intake of food seems to be reduced to a minimum. A study made in Pflüger 's laboratory 3 showed in a well nourished dog a fat content equivalent to more than one-fourth of its body weight ; about one-half of the fat was obtained from the skin and subcutaneous tissue, and one- third from the musculature, so that only a comparatively small proportion was distributed in the other tissues. In a fattened animal as much as a third or even a half of the live weight may be represented by fat. The large quantities of glycogen which the liver ordinarily contains during full car- bohydrate diet, are supplanted by fat in case of forced feeding with the latter, thus bringing out the antagonism 2 aW. v. Leube, 1895; E. Koll, 1898; Hofbauer, 1903; H. Winternitz, 1903 and 1906. Literature : A. Magnus-Levy and L. F. Meyer, Handb. d. Biochem., If, 448-449, 1909; and E. Heilner (Munich), Zeitscbr. f. Biol., 5-' h 54. 1910. J. Henderson and E. F. Crofutt (Yale Med. School) , Amer. Jour, of Physiol., Ik, 193, 1905. 3 K. Möckel, Pftiiger's Arch., 108, 189, 1905. DEPOSIT OF FOREIGN FAT 381 which was above mentioned between glycogen and fat. 4 Pflüger found in a dog which had been fed solely upon large quantities of fat for a month, that all of the glycogen had been replaced in the liver by fat, which comprised almost half of the dried hepatic substance. 5 From the readiness with which the economy is able to draw upon its fat supply in case of need, it is remarkable with what tenacity a residuum of the fat is retained. According to F. N. Schulz 6 the external appearance alone of a starved dog affords no basis for judging the amount of fat in its economy, and mere long continuance of starvation is not sufficient to actually make an animal free of fat. Deposit of Foreign Fat. — We may now take up more fully the question of the manner in which the economy makes use of the food fat. A long series of experiments have proved that the body can lay down foreign types of fat in its own depots. It has been shown that oil of rape, linseed oil, oil of sesame, mutton suet and cow's butter are not only deposited in the system, but that foreign types of fat may also find their way into milk, eggs and the coccygeal secretion of birds. 7 Iodized and bromized fats can also be laid down in the body, as shown by the studies of Winternitz, Coronedi and others. 8 For a long time no more doubt has been entertained as *A. Magnus-Levy, Noorden's Handb. d. Path. d. Stoffw., 2d ed., 1, 177, 1906, regards the antagonism between glycogen and fat accumulation in the liver as not an absolute one; he states that he has frequently found in typical examples of fois gras of Strassburg geese very large deposits of glycogen along with enormous quantities of fat. Cf. also Pflüger and E. Junkersdorf, ibid., 131, 225, 1910. "F. N. Schulz (Pflüger's Lab.), Pflüger's Arch, 66, 145, 1897. 7 Radziejewski, Lebedeff, J. Munk, Leube, G. Rosenfeld, Winternitz, Cas- pari, Zaitschek, Röhmann, Henriques and Hansen; Literature: G. Rosenfeld, Ergebn. d. Physiol., 1", 673-678, 1902; A. Magnus-Levy, Noorden's Handb. d. Pathol, d. Stoffw., 2d ed., 1, 178, 1906. 8 G. Coronedi and R. Luzzatto, Arch, di Farmacol., 12, 343, cited in Cen- tralbl. f. Physiol., 21, 122, 1907; cf. G. Coronedi (Parma), VIII Intern. Physiol. Congr., Vienna, Sept., 1910; G. Coronedi and F. Dematheis, Boll, della Societä med. di Parma., July, 1911. 382 FAT METABOLISM. OBESITY to the reality of assimilation of foreign fats. However, it is not well known even at present to what degree the body changes this acquired fat, and, as it were, impresses upon it the stamp of its own individuality. We know, it is true, that the fats of different genera of animals present nearly con- stant characteristics. As far as the fat which is formed within the body (probably from carbohydrates) is con- cerned, there is nothing remarkable in this. But in case of the fat which is acquired directly from the food, this would demand a constancy in the food which certainly does not always obtain (providing the fat does not undergo secondary changes). G. Eosenfeld found, as a matter of fact, that a dog which had been stuffed with mutton suet continued a month after interrupting the fat diet to show a pure mutton fat in his tissues ; and he was of the opinion that a panther, for instance, if he would devour nothing but sheep would necessarily put on sheep fat. He found, too, that gold fish and carp when fed mutton suet deposit this form of fat; and comparison of the fats of various marine forms with the fats of their customary foods showed a widespread similar- ity between them. The same seems to be true, too, for vegetarian animals, although in these, besides the fats con- tained in the food, there is also that which is formed in the economy from carbohydrates. "Thus we find," says G. Eosenfeld, 9 "that the herbivorous animals have a firm fat, poor in oleic acid, while the graminivora have a soft fat. The fat of the food shows almost precisely the same char- acteristics ; green food has a firm fat, seeds contain a soft oil. If a horse becomes fat from feeding on oats, his fat will be fluid; if he has been fattened on hay, his fat is much firmer. The similarity of the tallow from the ox, sheep, roe and hart is referable to the similarity of food, i.e., grasses." Transformation of Fat in the Body. — Be these discov- eries as illuminating as they may, we have, however, no sub- stantial reason for doubting the ability of the body to adapt 9 1. c, p. 676. SCLEREMA NEONATORUM 383 the acquired fats to its own requirements by certain changes. Such changes may include more rapid absorption of oleic acid, elimination of volatile fatty acids, change of saturated into unsaturated fatty acids or the reverse, or of the latter into oxy acids, or separation of the long carbon chains into shorter groups, etc. Thus, for example, after cramming a dog with butter which is characterized by its high proportion of volatile fatty acids, the Reichert-Meiszl figure (which is indicative of the amount of the latter substances) was not found higher in the fat of the animal than under normal conditions. 10 As an example of such an adaptation process may per- haps be pointed out that in infancy the proportion of oleic acid in the dermal fat, as stated by Knöpfelmacher and LehndorfT, 11 increases from month to month, and that the buccal fat (which acts as a support to the buccinator muscle and prevents aspiration of the poorly developed muscle be- tween the jaws in the oral negative pressure in suction) seemingly is poorer in oleic acid, and therefore more resis- tant than the general subcutaneous fat tissue of the same individual. The skin fat of children fed upon human milk is always richer in unsaturated fatty acids, than that of artificially fed infants. When an infant undergoes emacia- tion the proportion of oleic acid of its fat decreases, and it appears, according to Knöpfelmacher 's studies, that the in- flexibility of the fat tissue thus induced, which is poor in oleic acid, is related with the condition which is well known by podiatrists under the name sclerema neonatorum. 12 Comparing the iodine combining power of yolk fat and that of the fat of the embryo chick in course of development 10 W. v. Leube, Verhandl. d. Kongr. f. innere Med., IS, 424, 1895. 11 W. Knöpfelmacher and H. Lehndorff, Zeitschr. f. exper. Path., 2, 133, 1906; H. Lehndorff (Abt. Knöpfelmacher), Jahrb. f. Kinderheilkunde, 66, 286, 1907. "Literature upon Sclerema: Cf. F. Luithlen, Die Zellgewebsverhärtung der Neugeborenen, Vienna, Alfred Holder, 1912; G. Rommel, in Hand. d. Kinderheilk., 1, 423-425, 1910. 384 FAT METABOLISM. OBESITY within the egg, the fact has been noted that the former gradually grows poor in unsaturated acids, corresponding with the greater mobility and resorption of oleic acid (in contrast to stearic and palmitic acids). On the other hand, the gradual increase in the iodine value of the organized fat of the embryo chick indicate that saturated acids in the embryo are undergoing conversion into unsaturated acids (v. inf.). 13 Depot Fat and Cell Fat. — According to studies of Abder- halden and Brahm it would be well in the study of fat metabolism to distinguish between depot-fat and cell-fat. In order to determine whether not only the former but also the fat directly involved in the construction of the body cells is dependent upon the character of the food fat ingested, dogs were fed for a long time with mutton suet or with rape-seed oil and then killed. The readily extracted depot-fat is then removed by ether. In order to make it possible to extract the cell-fat, however, the tissue to be subjected to ether is digested or broken up by dilute hydrochloric acid. It is interesting to note from this procedure that the proper cell-fat in contrast to the depot-fat is independent of the character of the ingested food. 14 This difference between depot-fat and cell-fat acquires special significance when it is recognized that a considerable fraction of the ethereal extract from the liver, heart, kidneys and other tissues does not consist of ordinary neutral fat but of phosphatides of many types (v. Vol. I of this series, pp. 171-172, Chemistry of the Tissues), which contain be- sides the typical high fatty acids even a higher proportion of unsaturated fatty acids of the linseed oil and linolic acid series. 15 There is perhaps some connection between the fact 13 E. C. Eaves ( Instit. of Physiol., Univ. College, London ) , Jour, of Physiol., 40, No. 6, 1910. u E. Abderhalden and C. Brahm, Zeitschr. f. physiol. Chem., 65, 330, 1909. * 5 Rubow, Heffter, Henriques and Hansen, Erlandsen, Hartley, Leathea and Kennaway and others. Literature: J. B. Leathes, Ergebn. d. Physiol., 8, 366-370, 1909. OXIDATIVE FUNCTION OF LIVER 385 that these acids undergo oxidation very readily in the air with loss of iodine-binding power and the further fact that Röhmann 16 and his collaborators found the acetyl propor- tions of the mixture of fatty acids obtained from the liver extracts distinctly higher than those of the fat from adipose tissue; the acetyl figure (the number of milligrams of potassium hydrate which are combined with the amount of acetic acid in one gram of acetylized fat after saponification with alcoholic solution of potassium hydrate) is a well-known measure for the amount of free hydroxyl groups in a fat. It seems that unsaturated acids are to be found in tissues not only in the form of lecithids and Phosphatids but also in the form of simple glycerides. Oxidative Function of the Liver in the Catabolism of Higher Fatty Acids. — These results acquire special signifi- cance from the observations of G. Joannovics and E. P. Pick, and, too, of Loathes and Hartley, indicating that the liver upon access of fat with the food subjects it to an oxidation catabolism with formation of high unsaturated acids. The first mentioned investigators were able to reduce this oxida- tion power in the living animal by narcotics. It might well be imagined, perhaps, that by the introduction of new double combinations into the long carbon chain the fatty acids would be prepared for their oxidative destruction, these double combinations forming points of lessened resistance at which the chains may be separated into shorter fragments, the lat- ter then in turn undergoing further disintegration. 17 The essential features of the subject are not proven, however, as yet. In this connection the passage of fat to the liver might from one standpoint be interpreted on the supposition that 1S F. Röhmann, with W. Lummert, Y. Nukada, Pfliiger's Arch., 11, 176, 1898; Biochem. Zeitschr., ll h 419, 1908. 17 G. Joannovics and E. P. Pick (Instit. Paltauf, Vienna), Wiener klin. Wochenschr., 1910, 573; Pfliiger's Arch., UfO, 327, 1911; J. B. Leathes, 1. c; J. B. Leathes and L. Meyer- Wedell, Journ. of Physiol., 38, Proc. Physiol Soc. XXXVIII, 1909; P. Hartley (Lab. of J. B. Leathes), Jour, of Physiol., 88, 353, 1909; H. Mottram, Jour, of Physiol., 3S, 281, 1909. 25 386 FAT METABOLISM. OBESITY the higher fatty acids (as Nasse has suggested) are there prepared for further elaboration; on the other hand, as Magnus-Levy believes, the accumulation of fat in the liver may be an expression of a duty ascribed to this organ of hold- ing in readiness reserve material available for any suddenly introduced increase of metabolic exchange. " It is evident that finely divided fat can be passed when needed into the blood much more readily and quickly from the liver with its full vascular arrangement, than from the fat globules of the adipose cells of the subcutaneous tissue, which present so small a surface in proportion to their contents. According to this view the liver with its deposits both of glycogen and of fat would have the office of providing material for combus- tion for the body at any time when there is a sudden increase of demand put upon it. " 18 Formation of Fat from Sugar. — A matter of much impor- tance to the physiological procedure of metabolism is the formation of fat from sugar. This subject may now be somewhat more fully considered as the reverse process, the formation of sugar from fat, has been discussed above (v. supra, p. 240). The fact that carbohydrate is converted into fat in the economy seems today quite axiomatic to us ; it is in fact one of the few scientific points in physiological chemistry which has passed into popular knowledge. Every layman realizes in these days that people who want to avoid becoming too obese must guard against eating too much bread and made dishes. And yet it was at the expense of long continued and difficult metabolic study that the fact of the formation of fat from sugar was finally removed from any doubt. 19 The 18 A Magnus-Levy, Noorden's Handb. d. Pathol, d. Stoffwechs., 2d ed., 1, 177-178, 1906. "Literature upon Fat Formation from Carbohydrates: G. Rosenfeld, Ergebn. d. Physiol., 1, 666-671, 1902; R. Tigerstedt, Nagel's Handb. d. Physiol. 1, 513-516, 1905; A. Magnus-Levy, Noorden's Handb. d. Pathol, d. Stoffwechs., 2d ed., 1, 165-168, 1906; A. Magnus-Levy and L. F. Meyer, Handb. d. Biochem., 4, 449-450, 455-456, 472-474, 1909. TRANSFORMATION OF FAT INTO CARBOHYDRATE 387 absolute proof was attained by forced feeding of various experiment animals, like pigs, sheep, dogs, and geese, on fat- free food, poor in protein but rich in carbohydrates, after a previous fasting. Careful equilibrium experiments showed that large amounts of carbon are retained in the body. That this was not retained in the form of protein carbon was proved by calculating the retained nitrogen. Even granting that a high proportion of glycogen could be accepted as accumulated by the process, there still remained a large over- plus of carbon which could not be accounted for save as representing new-formed fat. The conversion of carbohydrate into fat is shown in respiration experiments by a marked rise of the respiratory quotient. Sugar being rich in oxygen and fat poor in oxygen, transformation of the former into the latter will set free considerable oxygen which may be used to burn carbon into carbonic acid. This will lead to an increase in the numerator of the ratio between the exhaled carbonic acid and the inhaled CO oxygen, — 2 • the amount of C0 2 eliminated increases with- out increase of the oxygen inhaled. Thus Bleibtreu noted, in case of geese stuffed with carbohydrates, and Pembrey in marmots, which were preparing for hibernation and taking on a large supply of fat by gorging with carbohydrates, in order to enable themselves to pass the winter a respiratory coefficient which reached nearly 1.4. This amounts to almost double the proportion to be met in a normally fed individual. Transformation of Fat into Carbohydrate in the Vege- table Economy. — For the most part in these lectures it is unfortunately impossible to bring forward the analogies which exist between the metabolism of animals and plants. In the present connection, however, attention should be called to the fact that the plant economy is likewise able to change carbohydrate into fat and vice versa. While, for example, unripe oily seeds contain much starch and mannite, as they ripen the carbohydrate is converted into oil. However, the 388 FAT METABOLISM. OBESITY reverse takes place when oil-bearing seeds come to sprout, 20 the oil being converted in greater part into carbohydate, going directly as it disappears from the reserve stock into the formation of material from which the cell walls of the young plants are constructed. Many bulbs and, too, ever-green leaves, and even wood, may contain reserve fats. In many trees starch disappears from the wood in winter, consider- able amounts of fat taking its place ; in the spring there is a return of the fat into carbohydrate, and the process may be to a certain degree reversed by artificial chilling. Characteristics of Fat which is Formed de novo from Carbohydrates. — The fat formed de novo in the animal body from carbohydrate is characterized by its low proportion of oleic acid and its peculiar firmness. G. Eosenfeld found that nine-tenths of the fat of fasting geese consisted of oil, with only a trace of solid fat in it; the fat of geese which had been fed freely on potatoes was, however, of a distinctly firmer consistence (very like lard), which did not become soft at full summer heat and when tried out consisted for the most part of crystals of palmitic acid and stearic acid glycerides, with which only a small amount of fluid fat was mixed. In mirror carp, too, which were freely fed on seeds in a bowl, the engorgement with carbohydrate led to a fat poor in oleic acid. 21 If the same conditions obtain in man, there ought to be a distinct difference observable between the adipose layer of Esquimaux, who assimilate the fluid fat of the north polar animals, and that of a negro or China- man in whom free rice diet has helped to produce a well developed panniculus adiposus; and this may well have some physiological significance. For instance, it may be readily thought that an individual with fat containing con- siderable oleic acid would be in position to more rapidly mobilize his fat supply than one in whom the firm fats pre- vail. Perhaps there is some difference whether a child as- 30 Literature: O. v. Fürth, Hofmeister's Beitr., 4, 430, 1903. 21 G. Rosenfeld, Ergebn. d. Physiol., V, 670, 1902. PLACE OF FAT FORMATION 389 similates the fluid fat of codliver oil or the firm fat of cow's butter. We have little positive information on these points ; but it would undoubtedly be worth while for the podiatrists especially to devote some attention to the subject. Refer- ence has already been made to the observation that the wasting of young individuals leads to an impoverishment of the subcutaneous fat in oleic acid. May not the well- proved favorable influence of codliver oil, aside from its very real food value, be possibly dependent upon the fact that it, in the absence of fat depots, serves to supply fluid and easily absorbed fatty acids? Place of Origin of the Fat from Carbohydrates. — An- other important question is, in what tissues is the fat formed from carbohydrates. One is, of course, first likely to think of the liver. Gr. Rosenfeld, however, concludes from results obtained by Bleibtreu with geese stuffed with carbohydrate food, in some of which the blood remained poor in fat, that if the carbohydrate fat is actually formed in the liver and thence worked into the subcutaneous depot the migration of the fat would necessarily always be recognizable by an in- crease in the amount of fat in the blood. Arguing in this wise Rosenfeld finds himself forced to the hypothesis that fat is constructed from carbohydrate at the place of its deposit, therefore especially in the fat cells of the sub- cutaneous tissue. 22 "While normally the adipose tissue of rabbits is free from glycogen, it has been shown that after free carbohydrate nutrition microscopic examination will reveal a marked amount of glycogen in this situation ; but after continued forced feeding of carbohydrates the glyco- gen after a time disappears from the fat cells. 23 Magnus- Levy believes that the interpretation that this is an evidence 22 G. Rosenfeld, 1. c, p. 671. 23 Gierke (Freiburg), Verb., d. Pathol. Gesellscb., 1906, 182; Best, ibid., 184. 390 FAT METABOLISM. OBESITY of formation of fat from carbohydrate is probably correct, even though further study is necessary. 24 Chemistry of Fat Formation from Sugar. — The problem of the chemical transformations by which sugar is converted into fat has as yet not passed beyond the hypothetical stage. The fact that the high fatty acids concerned in the synthetic production of fat show an even number of carbon atoms sug- gests the idea that groups, each containing two carbon atoms, may, perhaps, be concerned in the construction of the long chains. In review of hypotheses of Nencki and Hoppe- Seyler, Magnus-Levy suggests 25 that the formation of fat from sugar may take place along the same lines as {v. supra., p. 347) the bacterial butyric acid fermentation, that is, by way of lactic acid and acetaldehyde. 26 As is well known, two molecules of lactic acid may be derived from one molecule of sugar, C 6 H 12 6 = 2 C 3 H 6 3 ; the latter, then, by oxidation CH3 CH, gives rise to acetaldehyde, CH.OH + O = COH + co 2 + H,o. COOH This under certain condensation conditions is changed into CH, CH 3 + CH, = CH.OH a four chain group, aldol : \ ^ ^ ^ H • It has been COH proved, moreover, that two molecules of this latter substance may be condensed in vitro into an eight-carbon atom group : CH, CH, CH.OH + CH.OH = CH,-CH(OH)-CH,-CH(OH)-CH,-CH(OH).CH,.COH. CH 2 CH 2 COH COH The conversion of this last in the laboratory into n. octylic acid, CH 3 .CH 2 .CH 2 .CH 2 .CH 2 .CH 2 .CH 2 .COOH, is easily ac- 24 A. Magnus-Levy and L. F. Meyer, 1. c, p. 456. 25 A. Magnus-Levy and L. F. Meyer, 1. c., pp. 472-474. 20 A. Magnus-Levy, Verhandl. d. physiol. Ges., Berlin, March 15, 1902, etc. DISINTEGRATION OF FAT 391 complished. 27 Finally it is undoubtedly rational to suppose that the aldol by taking up one atom of oxygen may change CH3 CHj CH.OH + O = CH.OH . into /?-oxybutyric acid : l . This, however, CH, CH S COH COOH undoubtedly plays an important role in the physiological chemistry of the fatty acids, to which further reference will be made in a succeeding lecture. However, it may well be that the route from sugar to fat may be quite different, and may not lead through lactic acid and aldehyde. Even to- day it is still impossible to speak at all positively in this connection. In precisely the same way as the upbuilding of the long carbon chain of the fatty acid molecule is shrouded in dark- ness, so, too, is its catabolism unknown. As no opportunity seems open in the study of animal metabolism to solve this mystery the writer has endeavored by two experiments in the field of plant physiology to force an access. Experiments upon the Disintegration of Fat by Plants. — In the first place the writer, at the suggestion of his teacher, Franz Hofmeister, took up the behavior of the fat of oil- bearing seeds at time of sprouting (v. supra, p. 387) in order, if possible, to obtain from the study of their fat catabolism some basic information applicable to the chemistry of the conversion of fat in the animal body. In spite of all the care with which large numbers of sun-flower and castor-bean seedlings were reared in the basement of the Strassburg Institute, it was impossible to find in them any trace of intermediate products between fat and sugar, or to de- termine in a single instance the new formation of unsatur- ated acids or oxy-acids of the fatty acid series. 28 27 Raper, Jour. Chem. Soc, 91, 1831, 1907; Proc. Chem. Soc, 23, 235, 1907, cited in Chem. Centralbl., 1908', 223. 25 0. v. Fürth (F. Hofmeister's Lab.), Hofmeister's Beitr., l l , 430, 1903. 392 FAT METABOLISM. OBESITY There was not much more success when later with his friend, Carl Schwarz, the writer undertook to study the disintegration of fat by low forms of plant life (mould- fungi, bacillus fluorescens liquefaciens, proteus). 29 The microorganisms were watched in their growth and develop- ment upon inorganic media containing as their sole organic substance high fatty acids, and in their abstraction of their carbon requirements from these fatty acids ; but the investi- gators never succeeded in obtaining any catabolic product. The only point that was learned was that this type of fat catabolism cannot be regarded, as is occasionally done, as parallel to any of the known fermentation processes. To all appearances it is rather an intracellular oxidation process. Catabolism of the Fatty Acids in the Animal Body. — The most available points of attack for the type of catabolism of the higher fatty acids which occurs in the economy are pre- sented from the studies of F. Knoop 30 on the one hand, and by those of Gr. Embden 31 and his associates on the other, somewhat as follows : The catabolism of the saturated ali- phatic fatty acids takes place by oxidation at the ß-carbon atom with cleavage of the two carbon atoms from the car- boxyl end : CHz— CH 2 — CHr- CH 2 — CH 2 — COOH CHj— CH 2 — CHo— CH(OH)— CH 2 — COOH CH 2 — CH 2 — CH 2 — COOH CH 2 — CH(OH)— CH 2 — COOH CH a — COOH. Embden held that, in case the process actually takes place in the manner depicted, it would be necessary to expect that eventually from the higher fatty acids, after the long carbon atom chain is again and again shortened by throwing off two 28 O. v. Fürth and C. Schwarz (Festschr. f. Giulio Fano) , Archivio di Fisiol., 7, 441, 1909. 30 F. Knoop, Hofmeister's Beitr., 6, 150, 1905. 31 G. Embden, H. Salomon and Fr. Schmidt, Hofmeister's Beitr., 8, 129, 1906; G. Embden and A. Marx, Hofmeister's Beitr., 9, 318, 1908. CATABOLISM OF FATTY ACIDS 393 carbon atoms, we would obtain butyric acid and from this ß-oxybutyric acid, diacetic acid and acetone : BUTYRIC ACID /S-OXYBTJTYRIC DIACETIC ACETONE ACID ACID CH 3 CH 3 CH 3 I CHOH 1 CH 3 CH 2 CO 1 — >• 1 — * CH 2 CO + co 2 CH 2 CH 2 CH 3 COOH 1 COOH COOH There follows now the very interesting fact that if various acids are added to the blood perfused through the living excised liver of a dog, the acids with even number of carbon atoms (butyric acid, C 4 ; caproic acid, C 6 ; caprylic acid, C 8 ; capric acid, C 10 ) give rise to a notable acetone formation; while in case of the acids with uneven number of carbon atoms the amounts of acetone formed are no greater than when the liver is perfused with normal blood. Embden 32 properly says that the supposition that fatty acid catabolism takes place in the manner described has been made much more probable by these experiments. It should be definitely understood that, provided the oxidation of a carbon chain in the body takes place at the /?-carbon atom (as we have every reason for assuming, following Knoop) we can never obtain from a normal fatty acid with an uneven number of carbon atoms /3-oxybutyric acid and acetone ; for example : CH, CH, CH 2 CH, CH,.OH CH, CH 2 — ►• CH.OH — >- CH 2 — >■ CH 2 CH 2 CH 2 COOH COOH. I I COOH COOH Eeference will again be made to these points, which argue a direct relation between the acetone bodies and fat disinte- gration in the economy, when the former are discussed. Con- sideration of the occurrence of the lower fatty acids in milk * 3 G. Embden and A. Marx, 1. c. 394 FAT METABOLISM. OBESITY will also give occasion to take up the matter of fatty acid catabolism. It should only be added that Dakin, 33 who hoped to imitate the oxidation reactions in the body by the action of hydrogen peroxide, observed besides the oxida- tion at the ß-carbon atom the same thing at the a-atom and the catabolism of the ß-oxybutyric acid into not only diacetic acid and acetone but also into acetic acid and formic acid: CHj.CHCOEO.CH, j.COOH jS Sk CH,.CO.CH 2 .COOH CH,.COOH \ \ CHj.CO.CO, + CO a H.COOH + CO, \ C0 2 + H 2 0. Whether an analogous disintegration, as this just described, is also to be thought of as physiological, is, however, at pres- ent not known. Nature of Obesity. — Here may be taken up briefly what is known from the standpoint of the biochemist of the nature of obesity. Common experience indicates that many persons who are " disposed" toward corpulence accumulate fat even though they do not ingest any more food apparently than do other individuals ; in such cases one might suspect that there exists a lowering of material exchange. A whole series of studies upon metabolism in obesity are now at our disposal for consideration. 34 Many authorities, as Jaquet and Svenson, found, as a matter of fact, in corpulent persons a distinctly lower increase in gas-interchange after ingestion of food 33 H. D. Dakin (C. A. Herter's Lab., New York), Jour, of biol. Chem., k f 77, 91, 227, 1908. "Thiele and Nehring, 1896; Stuve, 1896; A. Magnus-Levy, 1897; C. von Noorden, 1900; Jaquet and Svenson, 1900 ; Rubner, 1902; Reach, 1904; E. A. v. Willebrandt, 1908; R. Stähelin, 1909; G. v. Bergmann, 1909; F. Umber, 1909. Literature on Metabolism in Obesity: C. v. Noorden, Die Fettsucht, Nothnagels Handb., Part 7, 1900; v. Noorden's Handb. d. Pathol, d. Stoffw., 2d ed., 2, 189-211, 1907; A. Jaquet, Ergebn. d. Physiol., 2', 553-554, 1903; F. Umber, Lehrb. d. Ernähr, u. d. Stoffwechselkr., 73-117, 1909; G. von Berg- mann, Handb. d. Biochem., 4" 208-237, 1910. NATURE OF OBESITY 395 than in normal individuals ; the reaction, too, was of abnor- mally short duration, the gas interchange sinking to normal within two or three hours after taking food. They regarded the conclusion justifiable that the obese individual must necessarily as a result of diminished interchange be storing his combustible material. Observation of Reach, Stähelin and Gr. v. Bergmann also indicated that at least many corpu- lent persons differ from normal persons particularly in that their curve of interchange does not rise as high and descends less sharply to a level after intake of food, in such manner that a ' ' trailing out of metabolism ' ' is suggested. However, these positive statements are completely contradicted by those of other authorities. Thus Rubner 35 compared with the greatest care the metabolism of two brothers, both child- ren and differing only a single year in age, one of whom was corpulent, the other spare. Calculated according to the square surface, the exchange in the two was exactly the same, and there could not be considered any specific diminution of metabolism. We cannot off hand, therefore, ascribe to all obese persons a diminished protoplasmic disintegration energy; on the contrary we may be forced to assume, fol- lowing Rubner, apparently for the majority of them just as much utilization of energy in relation to their body mass as would correspond to an equivalent normal individual. 36 A. Loewy and F. Hirschfeld also find that there are normal, in fact lean, persons in whom the maintenance exchange is so low that it may be of the same level at least as the interchange determined in individual cases of obesity. Per- haps it would be best to foresee, if the expression may be permitted, that the methods of investigation now at our command are not good enough to allow us to say that there is a true and constant abnormality in the metabolism of all obese individuals ; which does not say, however, that such fault does not exist. 35 Rubner, Beiträge zur Ernähr, im Kindesalter, Berlin, 1902. 3 * Cf . F. Umber, 1. c, pp. 78 and 84. 396 FAT METABOLISM. OBESITY Corpulence and Overfeeding. — For the rest, it should be understood that we can explain very well (even in persons who are not customarily large eaters) an accumulation of fat by the summation of very small amounts of food beyond the maintenance requirements. Carl von Noorden 37 illus- trates this point by the following proposition: A healthy man weighing 70 kilograms, who uses 40 calories for each kilogram per diem, in all, therefore, 2,800 calories, will gen- erally regulate his intake of food involuntarily according to the actual requirements of his body ; and is able thus, even with entirely free choice of food, to keep himself in average state of nutrition for many years. A small daily excess of food of only 200 calories will result, if we presume that it is converted into fat and deposited as such, in a yearly gain in weight of eleven kilograms. One would be surprised to learn to how small a quantity of food these 200 calories corre- spond : 1/3 liter of milk, or 4/10 liter of light beer, or 90 grams of rye bread, or 25 grams of butter, or 100 grams of fat meat. What fat man would dare then to disclaim the possibility with quiet conscience that at least at times he has not been guilty of overeating? Moreover, if this is to be properly appreciated, we must also take into account the relation between "ballast" and "live substance" in the body. "The relation between bal- last and live substance, ' ' says H. Friedländer, ' ' is variable in individuals at different periods of age. . . . Abnor- mally fat animals, like whales and seals, as well as ab- normally fat men, contain to the unit of weight less active substance than lean animals of the same age period; the complaint of many fat persons can be understood when they declare that they continue to put on weight with an absolutely lower ingestion of food. In ratio to their small proportion of live substance the ingested food is frequently not small, and a portion of it is still available for weight increment. ' ' 3S 37 C. von Noorden, Handb. d. Pathol, d. Stoffw., 2d ed., 2, 190, 1907. 38 H. Friedenthal (Nicholas Lake in Berlin), Centralbl. f. Physiol., 23, 437, 1909. ANTIFAT TREATMENTS 397 Antifat Treatments. — It is impossible to enter into the various antifat treatments 39 beyond mentioning that in the majority of cases they are essentially fasting treatments, with different variations. Thus in the so-called Banting treatment there are given, mostly in form of meat, only about 1100 calories to a person instead of the 2800 calories due him ; the Ebstein bill of fare with about 1300 calories gives preference to fat ; that the intermittent restricted milk diet in limited amounts is equivalent to actual fasting goes with- out saying. The same thing is true of the Rosenfeld potato treatment (with about 1200 calories). OertePs treatment is based on the restriction not only of the number of calories but also of the amount of fluid allowed; but it should be remarked that the losses in weight obtained with this last method, for which the patient must pay with the torments of thirst, are partly only apparent and are due to the loss of water. We have learned to realize, too, that the patient should not be allowed to be weakened in the course of an antifat treatment. It is important that modern methods of dietetic reduction of fat be so planned, like that of G. Gärtner, that the subjective sensation of hunger be kept down by full use of bulky food rich in cellulose, of low caloric value, like green vegetables and fruits. It is of value, too, to further the process of reduction by increasing the mus- cular activity. Employment of mineral waters containing sodium sulphate (Marienbad, Karlsbad, Neuenahr, Tarasp) often is of service by lowering the complete utilization of the food. ' ' The combined action of all the factors favoring the reduction of fat in special baths," says F. Umber, "the regulation of diet (which is frequently, for example in Ma- rienbad, in Kisch's regulations, administered in a bill of fare similar to that of the Banting system), the drink treat- ments, the bath treatments, the systematic muscular exer- cise, and not the least the removal of the patients from their 38 For details cf. E. H. Kisch, Entfettungskuren, Berlin, 1901 ; F. Umber, Lehrb. d. Ernährung u. Stoffwechselkr., 1909, pp. 94-114. 398 FAT METABOLISM. OBESITY every-day surroundings, all these are elements which make for success in such an establishment for the cure of the obese. First and foremost the result is that these obese people, who either are unwilling or cannot use the necessary energy and precaution at home to deal with their trouble, amuse themselves with the annual outcome of their Marien- bad or Kissingen sojourn and make excuses for their per- sistent failings." The thyroid gland medication in the obese depends upon an entirely different principle. Gr. von Bergmann 40 holds that an increase of calory-exchange of from 25 to 50 per cent, can be obtained by administration of thyroid sub- stance, with protection of the protein store; and believes that (if we exclude direct or indirect toxic influences, espe- cially upon the nervous system, which may give rise to contraindication for this method of treatment) thyroid gland substance is "from a purely metabolic standpoint the ideal means of reducing fat." As has been previously stated (Vol. I of this series, p. 450, Chemistry of the Tissues), we cannot fully share this view, and are more likely to accept the idea (on the basis of the statements of Magnus-Levy, Ebstein, v. Noorden, Umber and others) that the antifat treatments by administration of thyroid not only are want- ing in every practical but also every physiological justification. Fattening. — In conclusion of the present discussion we may point out what principles are involved if the opposite of the above depicted effect is sought to be obtained, namely, putting on fat. Probably nothing better can be done than to follow herein the lines of thought which C. von Noorden has developed in his remarkable monograph concerning hyper- nutrition. 41 40 G. v. Bergmann, Handb. d. Biochem., 4", 230-237, 1910. a C. von Noorden, " Die Ueberernährung," Handb. d. Pathol, des Stoffw., 2d ed., 1, 548-577, 1906. FATTENING 399 A sharp difference must be drawn between fattening and putting on flesh. There are undoubtedly cases in which a true protein up- building takes place ; this is seen, for example, in the period of growth, during pregnancy, in functional hypertrophy of the muscles from exercise, and above all in convalescence after sickness and undernutrition. In correspondence to an increased intake of food, which (in contrast to the normal of 40 calories per diem for each kilogram of body weight) may reach as much as 70 calories in the first weeks, the energy exchange, for example, in a convalescent from typhoid fever may be found tremendously exaggerated (30 to 50 per cent.). The respiratory quotient remains generally in the neigh- borhood of 1, as the energy expenditure is chiefly made good by combustion of carbohydrates, while the fat and protein are not burned but accumulate in the economy. The usual fattening, as, for instance, that which is prac- tised in such large measures by animal raisers, is truly a putting on of fat. The production of well-muscled animals with large amount of meat is not possible by forced feeding, but only by selective breeding (a proposition which has suffered scarcely any check from occasional observations which have been made upon nitrogen retention after feed- ing abnormally large amounts of protein). Besides fat, water may also contribute to an important degree to the on-put of weight of fattened individuals. This is observed, for example, in the very marked increase of weight seen at first in forced feeding, followed, however, by a pause; at first in such cases there is a marked accumulation of water in the tissues, which is thereafter gradually, with rise of diuresis, replaced by fat. If we ask, further, what type of food may be expected to be followed most quickly by increase of fat, the answer would be that protein seems least suited, as an excess of calories provided in protein form is usually not fixed. The carbohydrates are much more favorable; there is 400 FAT METABOLISM. OBESITY always lost about one-fourth of their energy value in passing from the intestine to the fat depots, in which, according to the findings of N. Zuntz and Eubner, not only the work of di- gestion but also the heat abstraction which takes place in the passage of carbohydrate into fat, are included. "The most favorable substance is fat" says Carl von Noorden. "This requires but little expenditure of force on the part of the digestive organs and is deposited as fat with almost no loss of energy. Practical medicine still hesi- tates to make effective use of fat as a fattening medium, and generally gives preference to the carbohydrates. I have often pointed out that this should be abandoned, and that large, and even enormous amounts of fat (neglecting here certain pathological changes of the stomach and intestines) are excellently borne, and bring about results which can scarcely be equalled, not to say surpassed, by extra adminis- tration of carbohydrates. ' ' Alcohol, too, may find a place among the adjuvants in fattening, because of the large energy-value it possesses, and because its combustion serves to protect other material. However, because of its toxic features it can be considered only in very limited sense as a food material; and is, of course, in no wise indispensable (as has been thoroughly established). The writer is, however, not so naive as to fancy that the many fellow beings whom alcohol serves as a fattening agent in an empiric way not worthy of imitation will allow themselves to be at all disturbed in the use of this substance by considerations of a physiological-chemical nature. Now and then we realize to our surprise that practice does not always direct its measures as theory believes is right. This holds good, unfortunately, not only for alcohol, but also for many other measures looking to the advance of human culture. CHAPTER XVII FAT-SPLITTING TISSUE FERMENTS. FAT FORMATION FROM PROTEIN. FATTY INFILTRATION AND FATTY DEGENERATION. ORIGIN OF MILK-FAT. FAT-SPLITTING TISSUE FERMENTS From the general knowledge we possess as to the fate of fat in the living body there is no doubt that even outside the intestinal tract a voluminous catabolism of neutral fat takes place. We see a flood of fat pouring into the blood after ingestion of fat-bearing food, and after a time dis- appearing; we see that in wasting processes the fat disap- pears from its depots in cells and tissues ; we notice in the processes of fat-migration of every type how the fat is mobilized in the compass of the large depots. There is certainly probability in the assumption of a close relation between fat mobilization and fat cleavage and in the belief that, just as the stable glycogen passes into solution as soon as the economy requires it, the same is true of fat. From what has been said it might be supposed that the fat, which according to Pflüger 's opinion can pass from the intestine only after cleavage, can also only pass through the wall of the blood capillaries when in fully split state, as soap. Many authorities have earnestly tried to attribute it to the cleav- age of fat in the blood and the tissues. 1 Cleavage of Fat in the Blood. — We next ask ourselves what is there in the proposition of fat cleavage in the blood. It has been previously stated that Hanriot, when engaged upon the subject, came to the conclusion that there exists in the blood a lipolytic ferment; while the opponents of this view 2 were of the opinion that at most there is an 1 Literature upon Fat-splitting Tissue Ferments: W. Connstein, Ergebn. d. Physiol., S, 223-226, 1904; H. M. Vernon, Intracellular Enzymes, London, John Murray, pp. 53-60, 1908; C. Oppenheimer, Die Fermente, 3d ed., 5-24, 1909; F. Samuely, Handb. d. Biochem., 533-537, 1909; A. Magnus-Levy and L. F. Meyer, ibid., 4', 457, 1909. *Arthus, Doyen and Morel. 26 401 402 FAT-SPLITTING TISSUE FERMENTS estersplitting ferment present, but no lipase. We have al- ready seen, too, that the apparent disappearance of fat from the blood, as may be observed in vitro (v. supra, p. 370) , is due to a masking of the fat but not to a lipolysis. In the course of the last few years P. Eona and L. Michaelis 3 have reverted to the question of cleavage of esters and fat in the blood. They start out from the fact that observation of the surface tension of aqueous solutions of glycerol esters permits one to follow the cleavage of these substances with unusually marked clearness ; the glycerolesters of the lower fatty acids (in contrast to their cleavage products) belong to the sub- stances having very marked surface activity which sharply lower the tension of water. They were able to recognize, using Traube 's drop counter, the presence not only of Han- riot 's monobutyrin-splitting ferment but also of a tributyrin splitting ferment in the blood. The velocity of ester cleav- age was approximately in proportion to the quantity of fer- ment. 4 The authors named speak of the tributyrin-splitting ferment as a "true lipase," a statement which is perhaps entirely correct from the standpoint of physical chemistry. Whether, however, this lipase is of importance from a physiological point of view, and whether it is actually to be considered as involved in the cleavage of true fat can scarcely be regarded as proved. An observation of Abder- halden and Eona is of interest here, indicating that after feeding large amounts of specifically foreign fat and, too, in starvation, an exaggeration of the ester-splitting power be- comes noticeable in the blood of dogs. 5 That the serum lipase cannot be held to be simply resorbed pancreatic lipase is shown by the points that it is not materially influenced by removal of the pancreas, and that it does not seem to be at s P. Rona and L. Michaelis (Hospital at Urban, Berlin) , Biochem. Zeitschr., SI, 345, 1911. 4 P. Rona, Biochem. Zeitschr., 33, 413, 1911; P. Rona and J. Ebsen, ibid., 89, 20, 1912. 5 E. Abderhalden and P. Rona, Zeitschr. f. physiol. Chem., 75, 30, 1911; E. Abderhalden and A. E. Lampö, ibid., 78, 396, 1912. CLEAVAGE OF ESTERS IN THE TISSUES 403 all activated, as the pancreatic lipase is, by the salts of the biliary acids. 6 Lipase of the Leucocytes. — In the cellular elements of the blood, too, the existence of a lypolytic ferment has been demonstrated, not only by ester-splitting but also by a plate method, somewhat like the Müller-Jochmann method. Just as in the latter method the tryptic power of cells is recog- nized by the formation of depressions in a gelatine plate, in this case the formation of delles in a wax plate is ob- served. Preparations, consisting mainly of lymphocytes, especially tuberculous pus, show fine examples of delle- formation dependent upon fat-cleavage. Myeloid cells ap- parently do not contain a lipase. If capillary tubes filled with yellow wax be introduced aseptically into the peritoneal cavity of living animals examination will show after one or two days that at the open ends of the tubes the wax will have disappeared, and will have been replaced by a whitish ma- terial consisting principally of mononuclear white blood corpuscles. 7 In this connection it is very suggestive that, according to observations of Edmund Nirenstein, infusoria (par- amcecia) are not only capable of ingesting large amounts of fat from an emulsion of oil, but also of digesting it within the food vacuoles (that is, decomposing it into components soluble in water). 8 Cleavage of Esters in the Tissues. — As far as the exist- ence of lipolytic ferments in tissues is concerned, a number of statements are to be found in the literature concerning the cleavage of different esters (as monacetin, monobutyrin, ethylbutyrate, tribenzoin and amylsalicylate). P. Saxl, who 8 C. L. von Hess (Carlson's Lab., Chicago), Jour, of Biol. Chem., 10, 381, 1911. T S. Bergel (Surgical Clin., Berlin), Münchener med. Wochenschr., 1909, H. 2; N. Fiessinger and P. L. Marie, C. R. Soc. de Biol., 67, 1909. 8 E. Nirenstein (II Zoöl. Instit., Vienna), Zeitschr. f. allgemein. Physiol., 10, 137, 1909 ; cf. on the contrary the different interpretation of W. Staniewicz, Bull, de l'Acad. de Cracovie, 1910, 199, cited in Centralbl. f. Physiol., 24, 855, 1910. 404 FAT-SPLITTING TISSUE FERMENTS tried out these statements at the request of the author, found that the first three esters above named are split in only very slight but always measurable degree by short exposure (an hour at room temperature) to the influence of minced tissues (but salicylic acid amylester was split by almost all the tis- sues studied). On longer exposure to the same influence usually a marked access of acidity could be recognized titri- metrically, brought about not only by the cleavage of the esters but also by formation of acid by autolysis of the tis- sues. Saxl came to the conclusion that none of the recom- mended methods is adapted to quantitative study of ester cleavage, and that all the statements dealing with quantita- tive variations of lipase in the tissues are without proper foundation. 9 P. Rona has shown that tracing the change in surface tension of a solution of monobutyrin or tributyrin may also be employed in the recognition of ester-splitting ferments in aqueous extracts of tissues. 10 But this method has not been properly elaborated as a quantitative one any more than the rest. The greatest problem in this connec- tion not as yet solved, and perhaps not capable of solution, is that of quantitative extraction of the lipases from a tissue. Studies upon animal and plant lipases especially have raised reasonable doubt as to whether the lipases are at all soluble in water and whether they are separable from organ- ized cytoplasm. 11 With this should be associated the strik- ing observation that the ester-splitting power of freshly prepared tissue extracts becomes enormously augmented when they are preserved on ice. 12 All in all, this matter is »P. Saxl (under direction of 0. v. Fürth), Biochem. Zeitschr., 12, 343, 1908; consult therein the older Literature: Hanriot, P. Th. Müller, Kastle and Loevenhart, R. Magnus, N. Sieber; cf. also L. B. Mendel and Leavenworth, Amer. Jour, of Physiol., 21, 95, 1908. 10 P. Bona, Biochem. Zeitschr., 82, 482, 1911. "Astrid and Euler, Hoyer, Armstrong, Nicloux, cited in H. M. Vernon, 1. c, p. 60; L. Breczeller (Tangl's Lab.), Biochem. Zeitschr., 34, 170, 1911. " M. C. Winternitz and R. Meloy ( Johns Hopkins Univ. ) , Jour. Med. Research, 22, 107, 1910. FAT-SPLITTING TISSUE FERMENTS 405 by no means, to the writer's way of thinking, satisfactorily removed from the plane of uncertainty. Fat-splitting Tissue Ferments. — This feeling of uncer- tainty grows when we come to consider the true lipases, that is, those tissue ferments which bring about cleavage of neu- tral fats into their components. There are a few references in literature to this type of ferments. 13 P. Saxl, 14 in follow- ing these up in control tests, obtained results indicating that neutral fat, both that contained in the tissues and that added thereto undergoes but a slight degree of cleavage during postmortem autolysis (excluding action of bacteria). The author's usual contention, however, here applied to Saxl's negative findings, would rank them as of less relative value than the positive results of other authors, who, like Umber and Brugsch, 15 Pagenstecher 16 , and Berczeller, 17 conducted their experiments carefully and added the fat in the form of an emulsion to the tissue pulp. In experiments of this sort it is likely, too, that activation of zymogens plays an important role. Thus in the adipose tissue of freshly killed hens practically no lypolytic power can be detected; but it appears after they are kept for a long time. 18 It is im- possible here to enter in detail into the ferment-kinetics of the lipases ; only a few more features seemingly essential to a sketch of their characteristics, may be briefly mentioned. 13 Lüdy; Ramond (1889), ]904; N. Sieber, Zeitschr. f. physiol. Chemie., 55, 177, 1908. "P. Saxl, 1. c; cf. also G. Comessati, Clin. Med. Ital., 46, 417, cited in Jahresber. f. Tierchem., 38, 179, 1908. 15 F. Umber and Tli. Brugsch, Arch. f. exper. Pathol., 55, 164, 1906. 16 A. Pagenstecher (Heidelberg), Biochem. Zeitschr., 18, 285, 1908; cf. also A. Juschtschenko (St. Petersburg), Biochem. Zeitschr., 25, 49, 1910 ; G. Izar (Catania), ibid., 40, 390, 1912. 17 L. Berczeller (F. Tangl's Lab., Budapesth), Biochem. Zeitschr., 44, 185, 1912. 13 M. E. Pennington and J. S. Hepburn, Jour. Amer. Chem. Soc, 34, 210, 1912; United States Dept. of Agriculture, Bureau of Chem., Circular No. 75, 1911, cited in Centralbl. f. d. ges. Biol., 13, No. 5, and 156, 1912. 406 FORMATION OF FAT FROM PROTEIN Dakin's observation that the racemic esters of mandelic acid, | are asymmetrically split by the lipase CH(OH).COOH' J f J f from hog's liver has been interpreted to mean that the enzyme itself is an optically active substance. 19 It is worth noting, too, that the tissue lipases, just as the pancreatic lipase, may be activated by biliary salts. 20 More- over, the fact that minute amounts of acid serve to activate a tissue lipase of vegetable nature, the lipase of ricinus, has proved of very wide practical value in industrial fat cleav- age, based on the studies of Connstein, Hoyer and Water- berg. 21 The same ferment, which brings about cleavage of neutral fat in the presence of an excess of water, works in the reverse fashion, that is, to cause synthetic formation of neutral fat, when allowed to act upon a mixture of fatty acids and glycerol in a medium poor in water. 22 FORMATION OF FAT FROM PROTEIN. FATTY DEGENERA- TION AND FATTY INFILTRATION At this point we turn to a difficult and complicated problem which has had a prime position for the past half century in the interest of metabolic physiologists and pathol- ogists, the question of formation of fat from protein. The doctrine of a metamorphosis of protein into fat takes its starting-point on the one hand from E. Virchow's micro- scopic observations upon fatty degeneration of tissues, and on the other from metabolic investigations. 23 In the years from 1862 to 1871 Carl Voit in association >9 Dakin, Jour, of Physiol., 32, 199, 1905. -°A. S. Loevenhart (Johns Hopkins Univ.), Jour, of Biol. Chem., 2, 391, 415, 1907. 21 E. Hoyer, Zeitschr. f. physiol. Chemie, 50, 414, 1907. 22 Cf. A. Welter, Zeitschr. f. angew. Chem., 24, 385, 1911, cited in Chem. Centralbl., 1911', 1258. 23 Literature upon Pat Formation from Protein in Metabolism : G. Rosen- feld, Ergebn. d. Physiol., 1, 655-699, 1902; R. Tigerstedt, Nagel's Handb. d. Physiol., 1, 511-512, 1905; A. Magnus-Levy and L. F. Meyer, Handb. d. Biochem., //, 451-453, 1909. FORMATION OF FAT FROM PROTEIN 407 with Pettenkofer in a series of extensive studies developed the theory that protein is the principal source of fat in the living body. For decades metabolic physiology remained under the domination of this doctrine, supported as it was by the great authority of its founders, until it was over- thrown by the ponderous opposition of E. Pflüger. ' ' These celebrated experiments of Voit and Pettenkofer," wrote Pflüger at the beginning of the nineties, ' ' prove nothing for the formation of fat from protein. For the computations of these investigators, as here involved, are really the result of a mistaken assumption as to the elementary composition of lean meat, which Voit adopted, not after analysis, but from his personal judgment, in contravention of analyses of other investigators generally regarded as reliable, and in fact con- trary to the results of his own analyses. It is on such a foundation that the modern superstructure of metabolism rests for the majority of physiologists. ' ' In answer to these ideas the Voit school set about energetically to defend itself; and in particular E. Voit, M. Cremer and M. Grruber brought forward new arguments for the retention of a carbon residium after meat consumption which did not seem covered by the carbohydrate in the food, and which, therefore, was held to indicate a formation of fat from protein. Pflüger invariably returned to the fray with new objections, of the correctness of which different opinions might be, and in fact were held. To-day the whole question has shifted; since, as has been stated, we must necessarily accept the formation of sugar from protein as a settled fact, and cannot possibly doubt that fat is formed from sugar. The logical conclusion, therefore, follows that it must be granted that in the living body fat may be formed from protein. It is another ques- tion, of course, whether under practical conditions this possibility becomes an actuality. When very large amounts of protein are fed one would probably always contemplate such a result. Magnus-Levy, as well-informed in the funda- mental points as he is an objective observer in the subject of 408 FORMATION OF FAT FROM PROTEIN metabolism, believes that really the formation of fat from protein does not occur to any important amount, although the possibility of such process must be maintained. Nor is it absolutely essential that the route should be through a fully formed sugar; for if we may presume that groups of six carbon atoms, combined from the "building stones" of the protein molecule, unite to the formation of the sugar mole- cule, we have as much right to fancy that eight or nine such groups, if need be, can enter into the construction of the long fatty acid chains. When, however, would this be needed? "When carbohydrates and fat are available in metabolism there is no occasion for further production from protein. Whenever, in case of deficiency of carbohydrate, important amounts of sugar are formed from protein, it seems to be required for the immediate needs of the body, unless, as in diabetics, it is excreted. As we cannot recognize in animal experiments that in case of exclusive protein diet any exten- sive formation of fat takes place, so there is no reason for holding that the process plays any important part under natural conditions of life in the carnivorous individual. ' ' 24 On the contrary, only recently E. A. Bogdanow has con- cluded from his studies on the fattening of pigs that fat for- mation from protein is at least probable. 25 Tangl and Farkas 26 have found an increase in fat con- tent of trout-eggs in the course of development which was held to be due to transformation of protein into fat because the undeveloped egg does not contain any important supply of either glycogen or sugar ; although the possibility of gly- coproteid with its carbohydrate group in combination in pro- tein, should be considered. It is the same old story : the great conflict over the for- mation of fat from protein in metabolism has, if the expres- M A. Magnus-Levy and L. F. Meyer, 1. c, p. 453. M E. A. Bogdanow (Moscow, 1909), cited from the Russian in Jahresber. f. Tierchem., 89, 585, 1909. * F. Tangl and Farkas (Budapesth), Pfliiger's Arch., 10 > h 624, 1904. FAT PHANEROSIS IN AUTOLYSIS 409 sion be allowed, stalled in the sand — more exactly stated, has found its own natural solution. It would be difficult to fore- go the opportunity to add here a moral reflection that, al- though in the determination of private differences it is not always possible to get along without disturbances of temper, it should always be a rule to deal with differences of scientific opinion with equanimity and without feeling ; in confidence that, with the progress of knowledge, that which is right is bound to win recognition entirely of its own strength. The writer might, however, in an objective manner equally add that here, as well as elsewhere, it is much easier and more convenient to give good advice to others than to practice it personally. Thus far, we have, however, been dealing with but one side of the problem of formation of fat from protein, that which is manifest to us in metabolic experimentation. The question, however, has many other sides for consideration in turn. A historical development of the question from the beginning may be dispensed with, the writer contenting him- self with an explanation of its present status. Fat Phanerosis in Autolysis. — Probably it will be best to consider first the simpler phenomena and then pass on to the more complicated, taking up first the subject of "fatty degeneration" of tissues occurring in extracorporeal auto- lysis. Here there is at no time a possibility of the fat being introduced by the blood stream and deposited as an infiltra- tion. Since, as has been noted by numerous authors, 27 in tissue autolysis there are histological pictures reminding one fully of fatty degeneration, two possibilities are here pre- sented : either fat is being formed de novo through f ermen- tative changes or else fat which was previously present, but which was not directly visible and was not demonstrable by the usual methods of staining, is being made appreciable to us by the autolytic process. This last has recently been ex- 27 Cf . the older Literature upon Fatty Degeneration in Autolysis : G. Rosen- feld, Ergebn. d. Physiol., 2, 89-94, 1903. 410 FORMATION OF FAT FROM PROTEIN pressed by the very appropriate term "fat phanerosis." A large number of careful investigations, especially those (con- ducted under direction of P. Hofmeister) of F. Kraus 2S and of F. Siegert, 29 as well as those of Gr. Rosenfeld, 30 and A. Slosse 31 and a number from the Medical-chemical Insti- tute in Tokio, 32 have shown that the formation de novo of the higher fatty acids is impossible in autolysis without bac- terial contamination. A few statements adverse 33 to this view, in the author's opinion, prove absolutely nothing, as the technique involved is by no means above objection. It is especially unfair to account, for such purpose, only the bare ethereal extract as fat; the tissues must be completely broken down by an intense saponifying process (as in the methods of Liebermann and of Kumagawa and Suto) 34 and their higher fatty acids, poorly soluble in water, estimated as fat as well. There is not the least contribution to the solution of the situation in the attitude of Waldvogel, who instead of simplifying the problem by reverting to the iso- lated higher fatty acids introduced into the question "hepatic protagon" and "jecorin" (more than problematic in its chemical individuality), and tried to make the latter an intermediate product between protein and fat, for which assumption there was not even the shadow of proof. From the fact that in prevailing opinions, as will be dis- cussed later, we are, in the production of a fatty liver from 28 F. Kraus (F. Hofmeister's Lab., Prague), Arch. f. exper. Pathol., 22, 174, 1897. 29 F. Siegert (F. Hofmeister's Lab., Strassburg), Hofmeister's Beiträge, 1, 114, 1902. ?0 G. Rosenfeld, Ergebn. d. Physiol., 2, 90, 1903. £1 A. Slosse (Brussels), Arch, internat. de Physiol., 1, 384, cited in Biochem. Centralbl., 3, No. 711, 1904. S2 Kohshi Ota, Biochem. Zeitschr., 29, 1, 1910; N. Shibata, ibid., 31, 321, 1911. "Kotsowski, Waldvogel, Leathes; cf. the criticism of J. Meinertz (Thier- felder's Lab.), Zeitschr. f. physiol. Chem., U, 371, 1905. 84 In reference to the Literature upon the more modern Methods of Fat Analysis: Cf. the articles by Röhmann, Rosenfeld, and by Kumagawa and Suto, in Abderhalden^ Handb. d. biochem. Arbeitsmethoden, 5', 477-488, 1911. FAT PHANEROSIS IN AUTOLYSIS 411 phosphorus poisoning, dealing with an immigration of fat from the blood stream, that is, with a fatty infiltration, one must necessarily be intensely interested in the statement of Mavrakis 35 that there may be found the well-known his- tological appearance of fatty degeneration in specimens when an aqueous suspension of yellow phosphorus has been injected into a branch of the portal vein of a liver which has been excluded from the circulation and then left otherwise unmolested. Under the author's direction his student, P. Saxl, 36 has repeated this experiment. He was able to fully convince himself that under the experimental condi- tions named tissue changes may be produced which are histologically entirely comparable to the picture of fatty degeneration. Exact analyses, however, indicated that even in this case there is no actual new formation of fat by con- version of the protein of the cellular protoplasm, but that because of the increased tissue autolysis there is a his- tological manifestation of fat previously present but in- visible. Here, too, should be mentioned the experiments of Ernst Weinland, in the course of which he believed he found evidence of an autolytic new formation of higher fatty acids "absolutely in minute but relatively in large amount" in the expressed juice of the larvas of certain flies (calliphora) ; which new formation, following the Voit traditions, he inter- preted as a formation of fat from protein. As a matter of fact, however, with general recognition of the care involved in these studies, it seems probable to the author that only the more recent experiments of Weinland 37 have been conducted with a reliable method of estimating fatty acids, and that his absolute results are quite too small and variable to permit one to recognize them as proving the position (fat disintegra- 35 C. Mavrakis (Athens) , Arch. f. Anat. u. Physiol., 190 k, 94. S6 P. Saxl (under direction of O. v. Fürth), Hofmeister 's Beitr., 10, 447, 1907; L. Hess and P. Saxl (v. Noorden's Clinic, Vienna), Virchow's Arch., 202, 148, 1910; cf, also A. Krontowski (Kiev), Zeitschr. f. Biol., 54, 479, 1908. 37 E. Weinland (Munich), Zeitschr. f. Biol., 52, 1909; cf. also Biol. Cen- tralbl., 29, 565, 1909. 412 FORMATION OF FAT FROM PROTEIN tion also takes place in the larval pulp, and there is even said to be a periodicity between increase and loss of fat in the ground-up maggots). We may conclude, then, that there is no proof of the new formation of higher fatty acids from protein in autolysis, that in fact this is highly improbable, and that all of the microscopic observations which apply here may be interpreted very satisfactorily as instances of "fat phanerosis." Nature of Fat Phanerosis. — Are we actually in position to offer a precise chemical interpretation of the phenomenon of fat phanerosis? In the first place, are we dealing here with a process of a chemical or of a physical nature? In the writer's opinion it partakes of both. It is essential that we conceive of the fat substances in the cells of the tissue for the most part, not as neutral fat, but as made up of a variety of Phosphatids. We are, moreover, undoubtedly fully justi- fied in supposing, as Friedrich v. Müller suggested years ago, that changes which involve these Phosphatids in the course of autolysis are connected with fat phanerosis. Yet even without such an assumption it is possible to fancy that the cells, as the result of autolytic processes, have lost their ability of maintaining fat in a state of solution, 38 as perhaps in connection with cellular swelling, coagulation and acid changes. Then, too, we may think that possibly true chem- ical combinations between fatty acids and proteid bodies {"lipoproteids") exist, which may undergo cleavage in the autolytic process. In this aspect it is of interest to recall that amido-com- binations between high fatty acids and aminoacids (as those synthetically reconstructed in the first place by Bondi 39 and again by Abderhalden, 40 and their co-workers) in contrast to free fatty acids are not soluble in ether, do not take up the 38 Cf. V. Rubow (Copenhagen), Arch. f. exper. Pathol., 52, 174, 1905. 39 S. Bondi, in association with T. Frank! and F. Eissler ( J. Mauthner's Lab., Vienna), Biochem. Zeitschr., 17, 1909, and 23, 1910. 40 E. Abderhalden and C. Funk, Zeitschr. f. physiol. Chem., 65, 61, 1910. HOFFMANN'S EXPERIMENT 413 fat-staining reagents, and are split into their components by the ferment action of autolyzing tissue (but not by trypsin). Having in some degree come to an appreciation of these points, a further step may be taken and attention given to other examples of the supposed formation of fat from protein. Formation of Higher Fatty Acids by Microorganisms. — We at once encounter here the fact, of importance to appre- ciation of the general problem, that we must acknowledge without question the power of forming fat from protein as belonging to the lower vegetable organisms (a power which, as far as the animal organism is concerned, if not absolutely contested, can be recognized only conditionally and with much reservation with the preformation of sugar from pro- tein in mind). A long time ago Emmerling concluded that higher fatty acids are produced in culture of staphylococcus pyogenes aureus on egg albumin. The experiments of several American authors (Beebe and Buxton) 41 seem particularly significant in this connection. They have shown that if bacillus pyocyaneus be cultivated on media free from sugar and from fat, such quantities of fatty substances are formed that they become evident on the culture surface in the form of microscopic needle-shaped crystals. Hoffmann's Experiment with Fly-maggots. — The ability of microorganisms to construct fat out of protein explains other points ; particularly the famous maggot experiment made by Franz Hoffmann in the early seventies. This in- vestigator determined the fat-content of a portion of a num- ber of fly-eggs and then allowed the remainder to develop upon defibrinated blood. It was proved at the close of the experiment that the fat-content of the larvae exceeded by tenfold the sum of the egg-fat and of the fat in the blood. Long ago Pflüger 's acumen suggested the apparently perti- nent interpretation for this experiment, which was later 41 S. P. Beebe and B. H. Buxton (Cornell Univ., New York), Amer. Jour, of Physiol., 12, 466, 1905. 414 FORMATION OF FAT FROM PROTEIN repeated with unsatisfactory result by Otto Frank, that the enormous numbers of bacteria present in the cultures were to be suspected of bringing about the trick of building up fatty acids from protein. Adipocere. — Another puzzling natural phenomenon, which is constantly used as an example of the formation of fat from protein in the animal economy is that of the produc- tion of cadaveric wax* 2 As is well known adipocere is formed when corpses or parts of corpses are buried in moist places or are kept in contact with water, the muscles and soft parts being replaced by a mass made up of a mixture of free fatty acids along with palmitates and stearates of mag- nesium, calcium and ammonium. There was formerly a common disposition to regard the process as one of a direct production of fat from protein. "We are at present inclined to assume that we are dealing only with the fatty acids which were previously present in the structures ; that through the influence of lipases and putrefying bacteria the neutral fats are split into their components, the fatty acids rendered soluble by combining with the ammonia produced by putre- faction, and the soap solution then permeating and percolat- ing through the soft parts ; that thereafter by replacement of the ammonia of the soaps by calcium and magnesium salts there results the formation of relatively insoluble precipi- tates. There are, however, statements to the effect that the total quantity of fatty acids in the formation of adipocere undergoes a definite increase. In view of the gross tech- nical errors involved in the older analyses it is very difficult to properly evaluate this contention. It would appear, how- ever, that even if an actual formation de novo of the higher fatty acids be recognized when adipocere is formed, the agency of bacteria may be looked upon as largely explanatory. 42 Literature upon Adipocere: G. Rosenfeld, Ergebn. d. Physiol., V , 659- 664, 1902; H. G. Wells, Chemical Pathology, 1st ed., 342-343, 1907; cf. therein the work of Kratter, Ermann, Zillner, E. Voit, Lehmann, Salkowski ; cf. also C. Ipsen (Innsbruck), Ber. der Univ., 1909, cited in Jahresber. f. Tierchem., 1910, 891. PHOSPHORUS POISONING 415 Formation of Fat in the Ripening of Cheese. — Precisely the same is true in case of the old problem of the formation of fat in the ripening of cheese. 43 It may be accepted as proved that there actually is an increase in the ethereal ex- tract when cheese is ripened. Yet recently M. Nierenstein 44 has called attention to the point that this increase need not necessarily be referred to an increase in fat alone, as in the ethereal extract in addition to fat and cholesterol there are also present putrescine, cadaverine, etc. But where actual new formation of higher fatty acids is taking place (and there is scarcely basis for doubting this) there is reason to at least suspect it to be due to the activity of micro- organisms. Accumulation of Fat in the Liver in Phosphorus Poison- ing. — Having progressed with reasonable assurance thus far, we may venture with confidence upon a question which in a certain measure is the central point of the whole problem, namely, that of intravital fatty change of the tissues. 45 "Fatty degeneration of the liver" in phosphorus poison- ing has been held up as a classical example of such change for the longest time; for this reason the process should be con- sidered next in turn, the more so because its essential nature seems at present to be satisfactorily understood. While the older pathologists permitted themselves to be so much im- pressed by the microscopic picture of the fatty degenerated liver as to have no doubt whatever as to the occurrence here of a transformation of protein into fat, we know to-day that the bulk of the fat in a phosphorus liver has really come as a fatty infiltration into the organ. This has been proved with thoroughly satisfactory exactness. In the first place, contrary to the older statements to the 43 Literature upon Fat Formation in the Ripening of Cheese : G. Rosen- feld, Ergebn. d. Physiol., V , 663-664, 1902. 44 M. Nierenstein (Bristol), Proc. Roy. Soc, Series B, 83, 301, 1911, cited in Centralbl. f. d. ges. Biol., 1911, No. 2087. 45 Literature upon Vital Fatty Degeneration of Tissues : G. Rosenfeld, Ergebn. d. Physiol., 2, 64-86, 1903; R. Tigerstedt, Nagel's Handb. d. Physiol., 1, 510-511, 1905. 416 FORMATION OF FAT FROM PROTEIN opposite, 46 complete proof has been furnished by a series of studies 47 showing that the total amount of fat in animals poisoned by phosphorus does not undergo any increase; there is merely a change in the fat distribution, in conse- quence of which there is more fat accumulated in a number of tissues, above all in the liver. Besides, this has been very strikingly confirmed by the recognition that the fat of the phosphorus liver corresponds in its composition with depot-fat, and that if the fat storage- sites be filled with fat of a type foreign to the body and phos- phorus poisoning is then induced, this foreign form of fat will accumulate in the liver. This was shown first by Lebe- deff, employing linseed oil, then by G. Rosenfeld, who used mutton-fat and cocoa-butter. Similar experiments carried out with iodized fat by Gideon Wells 48 gave an uncertain result; while the same experiment by Schwalbe 49 resulted positively. Finally G. Rosenfeld, undertook another crucial experi- ment, and showed that the fatty change does not occur in phosphorus poisoning if animals which are very poor in fat be used. The outcome of this experiment proves how fatty change comes to involve the liver in phosphorus poisoning: were we to assume that it is due to a breaking down of protein into fat, then necessarily phosphorus should invariably pro- duce a fatty liver, because the assumed mother substance of the fat, protein, is always present. If the change, however, is the result of a passage of the depot-fat into the liver, the condition must fail to appear if no depot-fat is available for immigration. 50 Actually, too, under these circumstances, it does not appear. ** Leo, Polimanti. "Athanasiu (E. Pfliiger's Lab.), Taylor, 1899; F. Kraus and Sommer, 1902; J. Barro, Jahresber. f. Tierchem., 31, 75, 1902; Boruttau, Arch, de Fisiol., 2, 26, 1904; Shibatu Negamachi, Biochem. Zeitschr., «37, 345. 48 H. G. Wells (Chicago), Zeitschr. f. physiol. Chem., Jf5, 412, 1905. 49 Schwalbe (Heidelberg), Verh. d. deutsch, pathol. Gesellsch., Kassel, 1903, 71. 80 G. Rosenfeld, Ergebn. d. Physiol., 2', 68, 1903. ROSENFELD'S THEORY 417 Fatty Infiltration in Other Pathological Conditions. — Whatever is true of the origin of the fatty liver in phos- phorus poisoning seems, from all we know thereof, to be also applicable in case of the fatty livers which follow poisoning from arsenic, antimony, chloroform, alcohol and many other poisons. There are many other pathological conditions in which at times a typical fatty liver is met, as for example, starvation, phloridzin diabetes and pancreatic diabetes 51 and overheating. (It was customary for a long time in France in producing fatty livers in geese to confine them in small, warm coops.) We have no substantial reason for doubting that in such instances, as well as in the so-called "liver of pregnancy," 52 we are dealing with the phenomena of typical fatty infiltration. Rosenfeld's Theory. — Are we able to offer any explana- tion for the fact that the same process, fatty infiltration of the liver, is common to pathological conditions of the most diverse type? G. Rosenfeld, basing his views upon the fact that in the different fatty livers due to intoxications the organ is generally devoid of glycogen and that, for example, in phloridzin intoxication the production of a fatty liver may be inhibited by free administration of sugar, meat 53 and other substances which go to form glycogen, has formulated the following line of thought : "If cells are brought under the influence of any sort of noxious agent . . . they in- crease their resistive power by oxidation of every bit of carbohydrate of which they may be possessed (for which rea- son the liver of the animal with phosphorus poisoning is free of glycogen). ... If there is no reserve material or an amount insufficient to protect the cellular protein, the cells take recourse to their last adjuvant; they attempt to re- store their supplies for production of resistive power by 51 H. Lattes, Arch. Scienze med. Torino, 33, cited in Jahresber. f . Tierchem., 40, 816, 1910. 62 J. Hofbauer (Königsberg), Arch. f. Gynäkol, 93, 405, 1909. 53 G. Rosenfeld (Breslau), Berliner klin, Wochenschr., 41, 1268, 1910. 27 418 FORMATION OF FAT FROM PROTEIN taking up an increased amount of fat. Res redit ad triarios: the last reserves enter the fray if the legionaries are de- stroyed. If the cell then succeeds in mastering the poison, the victory is through the aid of a fatty regeneration ; if even this be of no service, the cell dies the death of a hero ; in spite of the fatty infiltration degeneration ensues. ' ' 54 Association of Fat Phanerosis in the Phenomena of Fatty Degeneration. — Probably, however, it is not correct to end the matter by attributing solely to fatty infiltration all of the appearances which the older pathologists recog- under the name ''fatty degeneration." To the author's mind it is entirely logical to assume as well a superimposi- tion of the process of fat phanerosis. We have had under discussion the fact that when phosphorus is injected into the portal vein the picture of a fatty degeneration may be pro- duced even in a liver removed from the body. Besides in case of the isolated, artificially perfused hearts of warm-blooded animals it has been shown that the same noxious factors (as incomplete nutrition and poisons) which cause fatty de- generation in the living body, give rise to "fatty change" also of the isolated heart, in which there can be no sus- picion of the entrance of fat from any of the fat depots. 55 Di Christina, for example, ascribed to phosporus two entirely distinct influences, one a necrosing, the other a steatogenous action (the latter in the sense of a mobilization of fat in the depots). 50 We have, too, every reason for assuming that an intoxication, such as phosphorus poisoning, does not leave the protein material of the liver undisturbed ; according to a study made in KossePs laboratory 57 catabolism of the pro- tein molecule is likely to result in a spliting off of compounds rich in bases with a residue of material poor in bases and nitrogen. That such a process of disintegration cannot be 64 G. Eosenfeld, 1. c, p. 84. 68 A. Cesaris-Demel (Pisa), Arch. ital. de Biol., 51, 197, 1908. M Di Christina, Virchow's Arch., 181, 509, 1905. "A. J. Wakeman (A. Kossel's Lab., Heidelberg), Zeitschr. f. physiol. Chem., M, 335, 1905. FATTY CHANGE OF THE KIDNEY 419 thought of as unattended by serious change of the physical and chemical characters of the hepatic proteins, goes with- out saying. The view indicated by Mansfield therefore is decidedly plausible, this writer from his observations upon fat combination (v. supra, p. 370) believing it quite char- acteristic of phosphorus poisoning that there should exist a loss in the power of the haemic and tissue proteins to fix fat. 58 The author would readily believe that the assumption of such a change in the ability to fix fat would make it possible to regard the features of fat phanerosis and those of fat migration from a common point of view. The fundamental cause which destroys the union between the fats and the cells of the fat depots, and brings about mobilization of the latter as well as fatty infiltration of the liver, may very well be the same as that which breaks up the bond between the tissue- fat and the tissue-cells, and which in this way induces fat phanerosis (that is, brings into view fat which was previ- ously invisible). In this sense we may, therefore, perhaps look upon fatty infiltration and fat phanerosis as different phases of one and the same process. " Fatty degeneration" is by no means necessarily con- fined to the liver, but may involve other structures as well, as the cardiac and skeletal musculature, the kidneys, the lungs and the epithelium of the intestinal tract. 59 Fatty Change of the Kidney. — A few remarks at this point in reference to fatty change of the kidneys may not be inappropriate. Kosenfeld and several other investigators had concluded that the histological observations of a fatty change of a kidney could not be regarded as having any real reference to the actual amount of fat in the organ ; that, although a kid- ney may appear to be "fatty degenerated," actually its fat 68 G. Mansfeld ( in collaboration with E. Hamburger and F. Verzär, Phar- macolog. Instit., Budapesth), Pflüger's Arch., 129, 46, 1909. "J. and S. Bondi (v. Noorden's Clinic and R. Paltauf's Lab., V 7 ienna), Zeitschr. f. exper. Pathol., 6, 254, 1909. 420 FORMATION OF FAT FROM PROTEIN content may be decidedly less than normally. 60 In opposi- tion to this idea K. Landsteiner and V. Mucha 61 have applied themselves with great confidence. As a matter of fact if the whole kidney is not analyzed but the cortex only be used (thus excluding the fat situated about the renal pelvis, which has nothing whatever to do with pathological processes), the microscopic determination and chemical analysis ap- parently agree fairly well. Precise analyses conducted in E. Ludwig 's laboratory indicate that while the cortical sub- stance of the kidney may normally contain at most as much as eleven per cent, of fat, this proportion may be much in- creased in phosphorus poisoning. According to Landsteiner it is possible to distinguish two types of renal fatty change, the one a true fatty infiltration (as in the diabetic kidney), and another form in which the fat deposit is associated with distinct cellular destruction. This brings into prominence again the old distinction between fatty infiltration and fatty degeneration. With Gr. Klemperer 62 one may believe that in the latter process fat phanerosis may also be granted a part. However, sufficient has probably been said to indicate that there is no room left in modern thought for the old accepta- tion of fatty degeneration, that is, that it involves a direct conversion of cellular protein into fat. The ability of the kidney to construct fat from its com- ponents, it may be remarked in passing, has been proved by Fischler in the Heidelberg Pathological Institute. He per- fused a living kidney with blood to which soap and glycerine were added, with the production of typical pictures of fatty change of the renal epithelium. 63 eo G. Rosenfeld, 1. c, pp. 78-81; A. Orgler (Salkowski's Lab.), ibid., 176, 413, 1904; E. Kuznitsky (Ribbert's Lab.); Baum and Rosenfeld (Breslau), Berliner klin. Wochenschr., 1909, 629. 61 K. Landsteiner and V. Mucha (Lab. of Weichselbaum and E. Ludwig, Vienna), Centralbl. f. allgem. Pathol, u. pathol. Anat., 15, 18, 1904. 62 G. Klemperer (Moabite Hosp., Berlin), Deutsche med. Wochenschr., 1909, 89; cf. also Löhlein (Pathol. Instit., Leipzig), Virchow's Arch., 180, 1, 1909. 83 F. Fischler (Arnold's Lab., Heidelberg), Virchow's Arch., Ill}, 338, 1903. CHOLESTEROL-ESTER STEATOSIS 421 Cholesterol-ester Steatosis. — The problem of the process of fatty change would be incompletely dealt with were the author not to mention an entirely new phase, that of choles- terol-ester steatosis. As previously stated (v. Vol. I of this series, p. 324, Chem- istry of the Tissues) the esters of cholesterol with the higher fatty acids are of interest both from a physiological and a pathological point of view. These esters have been demon- strated in wool fat, in epidermal scales by Salkowski, 64 in the cutaneous fat by Unna, 65 and in the blood serum by Hiirthle. 66 It has been shown in Eöhmann's laboratory 67 that in the acetic-ether extract of the liver, along with free cholesterol, a certain amount of cholesterol esters is present, and that the liver also contains an enzyme capable of split- ting these esters. What is of especial interest to us, how- ever, is the fact that a crystalline, doubly refractive sub- stance found in fatty kidneys has been determined by Pan- zer, 65 in Ernst Ludwig 's laboratory, as an ester of cholesterol with an unsaturated higher fatty acid. This discovery has been fully confirmed by Windaus 69 by his digitonin method of determining cholesterol ; and we are justified in assuming that the cholesterol esters actually take an important part in the formation of doubly refractive substances in fatty tis- sues. The pathologist Aschoff has in fact seen fit to regard cholesterol-ester fatty change as being a form of fat metab- olism of at least equal importance and of equivalent origin to glycerol-ester fatty change; and he intrusted his pupil, M E. Salkowski, Arb. a. d. Pathol. Instit., Berlin, A. Hirschwald, 1906; Biochem. Zeitschr., 23, 362, 1910. co Unna and Golodetz, Biochem. Zeitschr., 20, 469, 1909. 66 Hiirthle, Zeitschr. f. physiol. Chem., 21, 331, 1895. « T K. Kondo (Röhmann's Lab., Breslau), Biochem. Zeitschr., 26, 238, 243, 252, 427, 437, 1910. °T. Panzer (E. Ludwig's Lab., Vienna), Zeitschr. f. physiol. Chem., 48, 519, 1906; 54,239, 1907. 08 A. Windaus (Freiburg), Zeitschr. f. physiol. Chem., 65, 110, 1910; cf. also J. Pringsheim, Biochem. Zeitschr., 15, 52, 1907. 422 FORMATION OF FAT FROM PROTEIN Kawamura, 70 with the task of determining by means of a great number of morphological methods (determination of their behavior with neutral red, nile-blue, and a number of other staining reagents, their refraction in glycerol, etc.) whether the cholesterol-esters can be definitely distinguished from the glycerolesters and the other lipoids. The author contrasts (unnecessarily introducing new terms for previ- ously known conditions) steatosis (fatty infiltration) with myelinosis (fat phanerosis), and divides the former into glycerol-steatosis, cholesterol-steatosis and lipoid steatosis. It is very clear that only thorough chemical studies in close coordination with morphological observations could possibly determine the value of such classifications. It is, however, quite a matter for congratulation that morphologists are also largely being brought to recognize that differentiation by staining methods is really nothing more than a very special form of chemical or physical-chemical reaction, and that it is really desirable that they supplement this by other and better defined chemical methods. The tremendous enlargement of the sum total of scientific knowledge is constantly leading to the rise of new specializations and changes of limitations. Unfortunately in view of the limited receptive power of the human brain this cannot be avoided. Yet the chemist, idling between his tubes and his jars, and ignoring designedly and stubbornly everything which he cannot boil, extract and distill, fits just as sadly in the field of modern science as does the morphologist, to whom nothing else seems worth bother- ing about, and who regards nothing as important as his stained sections. Freer vision will come only to the ap- pointed, to him for whom the thickets of the lowland are too confined and who strives upward to the heights. 70 R. Kawamura, Die Cliolesterinesterverfettung (Pathol. Instit., Freiburg, i. B.), Jena, G. Fischer, 1911; cf. also F. M. Hanes (Columbia Univ., New- York), Johns Hopkins Hosp. Bull., 23, 77, 1912. ORIGIN OF MILK FAT 423 ORIGIN OF MILK FAT As the final subject of this section of our study of fat metabolism, the origin of the fat in milk may be presented for consideration. 71 In bringing this subject forward in direct connection with the chapter upon the processes of fatty change, a historical rather than a logical relation is being followed. The older pathologists looked upon the origin of the milk fat as an example of supposed conversion of protein into fat. Virchow was fully convinced that both the fat globules and the other constituents of milk arise from a fatty degeneration and disintegration of the cells of the mammary gland. Then, later, Haidenhain modified this view by supposing that only that part of the gland cell next to the lumen sets free the fat droplets as it undergoes disintegration. C. Voit, in connec- tion with his above mentioned doctrines, advocated the idea that protein breaks down in nursing animals in such manner that its nitrogen is excreted as urea and the residue, rich in carbon, is secreted as fat with the milk. That the doctrine of the protein origin of the milk fat was firmly impressed by the weight of the great and overpowering authority of these men upon the minds of physiologists is, of course, not in the least remarkable ; and it has required a great deal of labor to evict it. Among others, the noted Heidelberg pathologist, J. Arnold (to whom I refer as one of my teachers with special appreciation), not long ago undertook to reinvestigate the subject by a thorough histological study of the mammary glands of women and female animals. He came to the con- clusion that, contrary to the assumption of Virchow, even the freest secretion of milk may take place without any degener- ation of the cells of the mammary glands. The fat appears in the interior of the cells, especially in the basal portion of the cytoplasm, that away from the lumen of the gland, and "Literature upon Milk Fat: K. Basch, Ergebn. d. Physiol., 2', 366-373, 1903; R. W. Raudnitz, ibid., 259-264, 1903; A. Magnus-Levy and L. F. Meyer, Hand. d. Biochem., .'/', 468, 1909; L. Kalabaukoff, Biologie mgdicale, Jan., 1909. 424 ORIGIN OF MILK FAT no fat is to be seen in the neighborhood of the secretory cells. Arnold concludes from what he saw and with the prevailing status of metabolism in mind, that the component parts of the fat are brought to the lactating cells from without in a water-soluble form, and that the fat is newly constructed from such material within the cellular protoplasm by func- tionating vital processes. What then do we really know of the origin of the fat in milk? In the author's opinion it is necessary to recognize a three-fold origin. It arises in part from the fats of the food, in part from those of the fat depots of the body, and in part, finally, from the carbohydrates which are undergoing trans- formation in the economy into fat. Passage of the Fat of the Food into the Milk. — As far as concerns the subject of passage of the fat of the food into the milk, a large number of investigations 72 should be noted, in which studies were made with all sorts of extraneous fats (as cottonseed oil, sun-flower oil, peanut oil, cocoa butter, lindseed oil, oil of sesame, almond oil, palm oil, goose fat, mutton suet, iodized and brominized fats) and which have removed all doubt from the subject. The ques- tion is of interest not only from a general scientific view- point, but has, too, a very decided bearing upon medical practice. The general composition of the milk-fat of a nurse, as pointed out by Engel 73 from systematic studies in the Dresden Infants' Home, depends upon the character of the fats of the food ingested. The importance of the details of diet for maintenance of health of the normal human being has unquestionably been very greatly exaggerated by the laity generally ; and it is of more immediate importance to "Willy, 1889; Stellwag, 1890; Heinrich, 1891; Klien, 1892; Lehmann, 1896; Winternitz, 1897; Rosenfeld, 1898; Baumert and Falke, 1898; Hen- riques and Hansen, 1899; Caspari, 1899; Engel, 1906; Gogitidse, Zeitschr. f. Biol., 45, 353, 1904; J t 6, 403, 1905; J t l, 4:15, 1906; W. Caspari and H. Win- ternitz, Zeitschr. f. Biol., 49, 558, 1907; cf. Literature in K. Basch, 1. c, and A. Magnus-Levy and L. F. Meyer, 1. c, p. 468. 73 Engel (Schlossmann's Clinic), Arch. f. Kinderheilk., .',3, 194, 1906; Zeitschr. f. physiol. Chem., U, 352, 1905. MILK FAT FROM CARBOHYDRATES 425 the healthy adult individual that he should really have some- thing to eat (a postulate which unfortunately in this best of all worlds is apparently very incompletely satisfied) than that the food, provided in a general way it is palatable, should have any particular fixed composition. How often has the author been amazed at the patience shown by his col- leagues in active medical practice toward the very silly questions about diet with which old ladies of both sexes are in the habit of plying them in their anxiety for their relatives or from pure love of asking questions. But it is a very dif- ferent matter when we are dealing with the feeding of in- fants. Here there is room for the most painstaking care and attention from a practical standpoint. And it is par- ticularly important that the physician keep constantly before him the fact that the infant's supply of fat is directly depen- dent upon the food which is ingested by the nurse and upon the character of her fat deposits. This is no "nursery tale"; it is a scientifically proved fact. Thus, often the trouble met in changing wet nurses may in the last count be connected with the biochemistry of fat ; and there is jus- tice in the demand that in the first place the milk of nursing women be kept constant by selection of an appropriate fat- mixture, and again that by proper feeding of cows a milk be produced with fat similar to the fat of human milk. 74 In spite of the enormous extent of milk literature, the details of which cannot possibly be entered into here, a great deal of work remains to be done before we can clearly appreciate how far race, heredity, mode of nutrition, sexual activity, season, calory-requirements of the growing body, etc., affect the composition of milk. 75 Origin of Milk Fat from the Carbohydrates of Food. — A portion of the fat in milk doubtless comes from the carbo- hydrates of food. Thus an experiment upon a cow fed for three months on material poor in fat (hay and grain-food 74 Engel and Plaut, Wiener klin. Wochenschr., 1906, No. 898. 75 Cf. G. von Wendt, Skandin. Arch. f. Physiol., 21, 89. 1909. 426 ORIGIN OF MILK FAT from which the oil had been removed) showed that in this period the animal produced in her milk about fifty pounds more fat than she consumed with her food. And yet the cow had become much fatter; the actual amount of newly formed fat must, therefore, have been much more than this. The extent of protein decomposition, determined by the nitrogen output, was at the same time far from being suf- ficient to explain in any degree the new formation of fat. The bulk of the latter was certainly derived, therefore, from the carbohydrates of the food. 76 Lower Fatty Acids in Müh. — Besides the typical higher fatty acids (palmitic, stearic and oleic acid) there are also small quantities of the lower fatty acids in milk, the presence of which is the more interesting because it offers a very im- portant and, as far as the author knows, hitherto little con- sidered suggestion as to the nature and the method actually followed by physiological catabolism of the higher fatty acids in the economy. It is certainly not a matter of accident that only normal fatty acids with unbranched chains and an even number of carbon atoms are to be found along with the higher fatty acids in milk, namely, myristic acid, C 14 ; lauric acid, C 12 ; capric acid, C 10 ; caprylic acid, C 8 ; caproic acid, C e , and butyric acid, C 4 . 77 Elsewhere, too, we find precisely these same normal fatty acids with paired carbon atoms (considering here the acids with more than three carbon atoms) emerging in all sorts of places from the sea of metabolism. Thatwe once ina whilemeet withisobutyric acid, CH 2V , . . . . . . CH 3X >CH— COOH, and isovalerianic acid, >CH— CH 2 — COOH, CH 3 / CH/ is not of any special moment, because these may be regarded 78 W. Jordan and C. G. Jenter, New York Agric. Exp. Station Bulletin, 1897. " The only seeming exception to this rule known to the author, a statement CH 3 v of Chevreul as to the occurrence of isobutylacetic acid, _>CH — CH 2 — CH 2 — COOH, and of isovalerianic acid in cow's butter, may not be held as of very great importance when we remember the great age of the observation, almost a century. Cf. W. E,. Raudnitz, 1. c, p. 260. SEBACEOUS GLANDS AND COCCYGEAL GLAND 427 as protein derivatives (from the branched chain of leucin, CH JN CH— CH 2 — ch.nh 2 — COOH)^ In the author's opinion the oc- currence of the normal acids, C 4 , C 6 , C 8 , C 10 , C 12 , C 14 , in the milk is very suggestive of the possibility that they may be due to an oxidation reduction from the typical higher fatty acids, the long chain of the latter being gradually short- ened, each time by two carbon atoms by oxidation at the /?-position, following Knoop's idea (vide supra, p. 392) : CH» CH 2 CH 2 I CH 2 CH 2 COOH CH 2 CH 2 I CH 2 CH.OH CH 2 I COOH CH 2 I CH 2 I CH 2 I COOH CH 2 CH.OH CH 2 I COOH CH 2 COOH. Sebaceous Glands and Coccygeal Gland. — In attempting to understand the function of the mammary glands the idea that they are really modified sebaceous glands is not without significance. A number of the lower mammals (as the monotremes) have instead of the mammary glands a large number of small dermal glands and the offspring lick the surface of the abdomen instead of sucking. It is interesting, too, to know that in the material secreted by ordinary sebaceous glands casein has been recognized. The coccygeal gland of water fowl corresponds, too, in its development, structure and function, to a modified sebaceous gland. Its fatty secretion, however, according to Röhmann, is not ordinary neutral fat, but consists for the most part of esters, combinations of fatty acids with octadecylalcohol (derived by oxidation from oleic acid and stearic acid). Fats TS Literature upon The Occurrence of Fatty Acids in the Body: W. Glikin, Handb. d. Biochem., 1, 95-102, 1909. 428 ORIGIN OF MILK FAT foreign to the birds' economy may, just as in milk, pass into the secretion of the coccygeal gland. 79 Haptogenic Membranes. — In conclusion a few words may be devoted to the old question as to the form in which the fat exists in milk and the nature of the much discussed haptogenic membranes which surround the fat globules. The contention on this subject took its inception in the observations of Ascherson in 1840 ; and it is not settled to- day. The physicist Quincke took the position that the cover- ing of the fat globules consisted merely of a layer of protein condensed about the globules; by surface tension ; V. Storch and a number of other observers (as, recently, W. Völtz 80 and H. Bauer 81 ) held that the haptogenic membrane is of a gelatinous, relatively firm character, often compared to the stroma of red blood cells. Völtz refers with emphasis to the fact that if milk globules passed through a column of water are skimmed off from the surface of the water and centrif- ugated, invariably in a short time the coverings of the milk globules are largely precipitated and collect as a firm (not a gelatinous) substance at the bottom. When the fat is re- moved by use of a Soxhlet apparatus the haptogenic mem- branes remain behind. Under the microscope the isolated coverings show very irregular appearances. Analysis and hydrolysis (a few decigrams may be obtained from a liter of milk) show that they contain a very large proportion of ash along with protein, and consist not only of casein but also of calcium salts of the fatty acids. 82 All this does not in the author's opinion prove that they are organized, preformed structures in the milk. It may easily be fancied that a layer ™~F. Röhmann (Breslau), Hofmeister's Beitr., 5, 110, 1904. 80 W. Völtz (Zoöteeh. Instit., Agric. High School, Berlin), Pfluger's Arch., 102, 373, 1904; Handb. d. Biochem., 3', 394, 1910. 81 H. Bauer (Instit. of Dairying, Agric. High School, Vienna), Biochem. Zeitschr., 32, 362, 1911. 62 E. Abderhalden, and W. Völtz, Zeitschr. f. physiol. Chem., 59, 13, 1909; Bredenberg (N. Zuntz's Lab.), Abhandl. d. Agrikulturwissenschaftl. Ges. in Finnland, H. 4, Helsingfors, 1912, cited in Centralbl. f. d. ges. Biol., 1912, No. 2011. HAPTOGENIC MEMBRANES 429 of protein, condensed about a fat globule by surface tension, may become still more condensed by these very processes of separation (passing through a layer of water, centrifuga- tion), rendering it a firm and separable structure. That coagulative phenomena may take place in the adsorbed surface layer of a colloidal solution has been repeatedly emphasized, and by Kreidl and Lenk for milk especially. 83 These latter writers have, too, proposed a very simple experiment, which in the author 's opinion distinctly contra- dicts the idea of an organized nature for the haptogenic mem- branes. It is well known that the theory of the haptogenic membrane is based on the fact that fat is not separable from cow 's milk by merely shaking with ether, but can be accom- plished only after first treating the milk with potassium hydrate solution. This has been interpreted as indicating that the potash solution dissolves the "membrane" which encloses the fat globule and protects it from the penetration of the ether. Kreidl and Lenk, 84 however, have found that when a drop of milk is placed on Lösch carton it divides into three concentric zones as the result of capillary adsorption ; in the central zone the suspended fat remains behind, in the middle zone the imperfectly dissolved casein, while the water and fully dissolved materials (as sugar) pass furthest and are to be found in the outer ring. If the milk be rich in fat there remains in the centre a large proportion of a butter- like material, which is readily soluble in ether (particularly apropos to the matter in hand). To the writer's mind this seems altogether at variance with the assumption of an organized nature of "milk-stromata" or "haptogenic mem- branes." It would be impossible to think that they are stripped away from the individual fat globules in the course of distribution of the milk in the Lösch paper. The facts probably are that the insolubility of the milk fat in ether is connected with the presence of ultra microscopic particles of 83 A. Kreidl and E. Lenk (Vienna), Pflüger's Arch., 1^1, 558, 1911. 84 L. c, pp. 543-549. 430 ORIGIN OF MILK FAT casein in the milk. According to K. Liesegang the addition of the ether, by precipitating the casein emulsion, produces the formation of the membrane as an artefact. When potash solution is added the ether does not meet an emulsion of casein, but a solution of casein, and therefore cannot induce membrane formation. It has been long known that the fat of human milk, in contrast to that of cow's milk, is directly soluble in ether; this, according to Kreidl and Lenk, 85 de- pends on the fact that casein is not present in human milk in the form of ultramicroscopically demonstrable particles, but in solution. 85 A. Kreidl and E. Lenk (Verhandl. d. morpholog-physiol. Ges., Wien), Centralbl. f. Physiol., 25, No. 12, 1911. CHAPTEE XVIII ACETONE BODIES That the acetone bodies are introduced here in immedi- ate connection with our studies of fat metabolism is because of the fact that these mysterious substances, which appear in an apparently unregulated way, now here, now there, upon the surface of the tide of metabolism, and which at one or other time were referred now to one, now to another of the principal types of foods, must, in consonance with the pres- ent status of science, be regarded, at least principally, as catabolic products of fat. In the group of acetone bodies we recognize, as is well known, ß-oxybutyric acid, acetoacetic acid and acetone, the relative chemical connection of which seems to be that in- dicated in the following schema : /9-OXTBUTYRIC ACID ACETOACETIC ACID ACETONE CH, CH, CH, CH— OH CO i: _^ | > CO H2 — H2 CH2 — C02 I CH 8 . COOH COOH These accumulate in the body in certain pathological con- ditions, notably in diabetic coma. Without entering into the historical development of the subject of the acetone bodies, the author may with pro- priety in introduction briefly outline the basis for assum- ing that there is a connection between them and the breaking down of the higher fatty acids in the body. 1 Relation of the Formation of Acetone Bodies to the Cor- poreal Fat and to that of the Food. — It may be stated first 1 Literature upon the Relation of the Acetone Bodies to Fat Destruction in the Economy: A. Magnus-Levy, Noorden's Handb. d. Pathol, d. S'toffw., 2d ed., 1, 184-188, 1906 ; A. Magnus-Levy and L. F. Meyer, Handb. d. Biochem., 4, 483-484, 1909; 0. Porges, Ergebn. d. Physiol., 10, 8-11, 1910; C. Oppenheimer and L. Pincussohn, Handb. d. Biochem., k, 697-702, 1911. 431 432 ACETONE BODIES that no parallelism has been recognized between the excre- tion of acetone bodies and the reduction of the body protein (reckoned from the nitrogen output), but that such paral- lelism has been noted in case of reduction of the body-fat in starvation, diabetes, cancer, phosphorus poisoning, and other pathological conditions. Thus Brugsch observed abundant excretion of acetone bodies in case of a profes- sional faster who in spite of the low nutrition to be expected in his calling, had a magnificent fatty panniculus ; whereas in a woman in extreme emaciation, who had not the slightest visible trace of body fat left, no evidence of ' ' acidosis ' ' could be noted. 2 The striking inhibitory influence manifested upon the excretion of acetone bodies by exhibition of carbo- hydrates is naturally explained by the consequent diminu- tion of the destruction of the body fat. Magnus-Levy noted in a case of diabetic coma the excretion of such large amounts of acetone bodies (more than one-third of a kilogram esti- mated as oxybutyric acid, in the course of three days) that even if the total carbon of the coincident protein destruction were converted into oxybutyric acid (which naturally was not the case) it would not have been equivalent. This ob- servation alone would permit scarcely any other interpreta- tion than that the acetone bodies originate from the fat. 3 Another and for us an important fact is that frequently the pathological excretion of acetone bodies is associated with a high grade of lipaemia, in evidence of mobilization of the fat from the depots. It was pointed out above that the blood may be so rich in fat in diabetic coma as to look like chocolate and cream. Origin of the Acetone Bodies from the Lower Fatty Acids with Even Carbon Atom Chain. — There are a number of observations of an increased excretion of acetone bodies 2 Brugsch, Zeitschr. f . exper. Pathol., 1, 426, 1905. * A. Magnus- Levy, 1. c, p. 184. ORIGIN OF ACETONE BODIES 433 after ingestion of dietary fat. 4 That this sequence does not evince itself in a more striking manner is, in the author's opinion, due to the fact that we are not always able to insure an increased utilization of fat by increasing the ingestion of fat, as the excess is generally simply deposited. Twice in the course of these lectures (Chapter XVI, p. 392 and Chap- ter XVII, p. 427) occasion has been taken to refer to the important discoveries of F. Knoop and G. Embden which indicate (with great probability, in the author's opinion) that the catabolism of the normal fatty acids proceeds with a two-by-two loss of carbon atoms from the carboxyl end. It has been pointed out above that there are no essential difficulties in the belief that, beginning with the higher fatty acids, by a continuous shortening of the carbon chain we finally obtain butyric acid, from which we get oxybutyric acid, and from this then diacetic acid and acetone. CH, H 2 CH S i CH 2 CH, CH, I I I CH 2 CH 2 CH 2 CH, — > CH 2 — >■ CH, I I I CH, CH 2 COOH CH, COOH CH, 1 CH, i CH.OH 1 CO CH, i CH, — > CH, — 1 1 > CO COOH COOH CH, i OOH It is very well worth noting and is established by many observations on diabetic human beings and animals 5 that administration of butyric acid (C 4 ) and caproic acid (C 6 ) * Geelmuyden ; L. Schwarz; E. P. Joslin (Harvard Univ., Boston), Jour, of Med. Research, 12, 433, cited in Biochem. Centralbl., 3, No. 828, 1904; E. Allard (Minkowski's Clinic, Greifswald), Arch. f. exper. Pathol., 57, 1, 1907; F. Steinitz, Centralbl. f. innere Med., 25, 81, 1904; Forssner (Stockholm), Skandin. Arch., 23, 305, 1910; cf. therein the older Literature. 6 Geelmuyden, Rumpf, L. Schwarz, Lob, Strauss and Philippsohn ; cf . Magnus-Levy, 1. c, p. 185; L. Schwarz (Prague), Deutsch. Arch. f. klin. Med., 76, 233, 1903; J. Bär and L. Blum ( S'trassburg ) , Arch. f. exper. Pathol., 55, 89, 1906; 59, 321, 1908; 62, 129, 1910. 28 434 ACETONE BODIES appreciably increase the elimination of acetone bodies, and, too, in greater proportion than the exhibition of the higher fatty acids. A. Löwy and R. Ehrmann saw a deep and per- sistent coma occur in butyric acid poisoning, while in poison- ing with isobutyric acid, * J>CH— COOH (the branched chain of which is unable to pass over into ß-oxy butyric acid), noth- ing in the least comparable was noted. 6 C. von Noorden recommends that the butter intended for diabetics be thoroughly worked with water in order to re- move the butyric acid. In close consonance with the above conception of fatty acid catabolism is the fact that only the normal fatty acids with an even number of carbon atoms are productive of acetone bodies, as Embden found experimentally by perfu- sion of the liver, and Bär and Blum found in diabetics (vide supra, p. 393). As a-aminoacids in catabolism are first deprived of one member it can be easily understood why a -aminovalerianic acid (but not a-aminobutyric acid or normal a-aminocaproic acid) may be classed with the acetone producers : 7 CHj CH2 CH3 CHj I I I CH, CH 2 CH.OH CH.NH 2 CHj CH 2 COOH COOH COOH. Possibility of Disintegration of the Longer Fatty Acid Chains into Short Parts. — The above presented mode of formation of oxybutyric acid from higher fatty acids indi- cates one possibility of this connection, but not the only one •A. Löwy, Berliner physiol. Ges., Dec. 16, 1910; A. Löwy, together with R. Ehrmann and P. Esser, Zeitschr. f. klin. Med., 72, 496, 500, 502, 1911. 'G. Embden and A. Marx, Hofmeister's Beitr., 11, 318, 1908; J. Bär and L. Blum, 1. c. DISINTEGRATION OF FATTY ACID CHAINS 435 by any means. It is stated that in severe diabetes some- times there are excreted a greater number of molecules of oxybutyric acid than can be accounted for by the number of molecules of fatty acids which are in course of decomposi- tion. As a matter of fact it is very difficult, if at all possible, to estimate correctly the number of the latter in our calcula- tions in metabolic experiments. However, were this the case it would strongly indicate that the long fatty acid chains are not reduced step by step until they come to the four carbon atom stage and are changed into butyric acid; we would have to assume that the chain is first broken into a number of segments, each of which is transformed into butyric acid. 8 Ernst Friedmann 9 found in F. Hofmeister 's laboratory that (of a number of substances with two-armed carbon chain subjected to investigation) acetaldehyde alone was synthesized into diacetic acid in perfusion of the liver. As two molecules of acetaldehyde are very readily condensed into aldol and this in perfusion experiments is capable of transformation into diacetic acid, it is not unlikely (follow- ing the conceptions of Magnus-Levy 10 and Spiro) that the reactions may be represented by the following formulae : ACETALDEHYDE ALDOL CH,.COH + CH,.COH - CH 3 .CH(OH).CH 2 .COH ALDOL Ö-OXYBUTYRIC ACID CH,.CH(OH).CH 2 COH + O = CH 3 .CH.OH.CH 2 .COOH. It might also be imagined that the fatty acid chains are primarily broken up into links with only two carbon atoms each, and that these then become synthesized by way of acetaldehyde into /3-oxybutyric acid. 8 A. Magnus-Levy, Arch. f. exper. Pathol., 40, 433, 1901. E. Friedmann (F. Hofmeister's Lab., Strassburg), Hofmeister's Beitr., 11, 202, 1908. 10 A. Magnus-Levy, Die Oxybutyrsäure und ihre Beziehungen zum Coma diabeticum, Leipzig, F. C. W. Vogel, 1899, p. 78. 436 ACETONE BODIES Again, ethyl alcohol may give rise to diacetic acid in experimental perfusion of the liver (probably by way of ace- taldehyde). 11 Is ß-oxybutyric Acid a Product of Normal Metabolism? — Another important question, which for the present must be accepted as an open one, is whether ß-oxybutyric acid is to be regarded as distinctly a product of normal or only of patho- logical metabolism. The author's early deceased friend, Leo Schwarz, who insured for himself a lasting place in science by his investigations upon diabetes, long since satis- fied himself that ingested ß-oxybutyric acid is metabolized with more difficulty by diabetics than by normal human be- ings (whereas ingested acetone is apparently attacked with equal difficulty by the normal and by the diabetic economy) . Therefore it can scarcely be thought that acetone plays any important role in normal metabolism, as it would other- wise necessarily appear also normally. But one cannot ex- clude the possibility, in case of ß-oxybutyric acid, of its occurrence as an intermediate product of physiological metabolism. If this be true, then an increased excretion of this substance should be due to the fact that its destruction in the economy is diminished under pathological conditions. It might be, too, that in pathological conditions the fault lies in an increased formation of the acetone bodies. Emb- den found in his perfusion experiments that the liver of the dog with pancreatic diabetes undergoes a change in its normal function of forming diacetic acid, in the form of a decided exaggeration. 12 As he further found that the ability of the surviving liver to induce disappearance of diacetic 11 N. Masuda (G. Embden's Lab.), Biochem. Zeitscbr., 45, 140, 1912. From another standpoint alcobol is to be classed with the antiketogens along with other substances, as tartaric acid and glycerol-aldehyde, which, according to experimental conditions, can either serve as a source for the acetone bodies or act to inhibit the formation of acetone. (Cf. G. Embden and K. Ohta, Biochem. Zeitschr., J t 5, 170, 1912; R. T. Woodyatt, Jour. Amer. Med. Assoc, 55, 2109, cited in Jahresber. f. Tierchem., //0, 818, 1912.) 12 G. Embden, and L. Lattes, Hofmeister's Beitr., 11, 327, 1908. DERIVATION OF ACETONE BODIES 437 acid is not abnormally diminished for the depancreatized dog, he thought that the increased excretion of acetone bodies in case of the diabetic individual is not due to a diminished catabolism of these substances but more likely to their increased formation. This result, however, cannot be accepted as definitive, as it is not as yet proved that the loss of diacetic acid noted in experiments with the ground-up hepatic tissue, is consistent with the intravital catabolism of diacetic acid. 14 Derivation of Acetone Bodies from Compounds ivith Branched and Cyclical Chains. — Thus far we have been con- sidering only the relations of the acetone bodies to the nor- mal fatty acids. However, the acetone bodies have also been traced back to compounds with branched and cyclic nuclei, like leucin, tyrosin and Phenylalanin; and the question naturally arises whether these cleavage products of the protein molecule can also serve as a source of the acetone bodies in the living body. The author will attempt to pre- sent the existing status of the problem, which, thanks to the studies of Embden, 14 Bär and Blum, 15 Borchhardt and Lange, 16 Friedmann, 17 and other authors 18 has been con- siderably clarified, as well as he understands it, and requests special attention to the points involved that confusion may be avoided. 13 G. Embden and L. Michaud, Hofmeister's Beitr., 11, 331, 1908, and Biochem. Zeitschr., 13, 262, 1908; cf. also E. Allard (Minkowski's Clinic, Greifswald), Arch. f. exper. Pathol., 59, 380, 1908. 14 G. Embden, in collaboration with Almagia, Kalberlah, Marx, Salomon, Schmidt, Engel, Wirth and Sachs, Hofmeister's Beitr., 8, 129, 1906; 11, 323, 847, 1908; Biochem. Zeitschr., 21, 20, 27, 1910. 15 J. Bär and Blum, 1. c. M Borchhardt and Lange (Weintraud's Clinic), Hofmeister's Beitr. 9, 116, 1907. 17 E. Friedmann (Berlin), Hofmeister's Beitr., 11, 365, 371, 1908. 13 A. Baumgartner and H. Popper (E. Freund's Lab., Vienna), Centralbl. f. Physiol., 20, No. 12, 1906; G. Forssner (Stockholm), Skandin. Arch. f. Physiol., 25, 338, 1911; E. M. Fittipaldi (Naples), Centralbl. f. Stoff w., 5, 161, 1910; cf. also Literature: O. Porges, Ergebn. d. Physiol., 10, 17, et seq., 1910. 438 ACETONE BODIES The fact of the matter is that leucin is recognized as capable of serving in the economy as a source of acetone bodies. As long as it was known only that acetone could be produced from it by perfusion through the liver it could be supposed that this occurred by the separation of the leucin nucleus at the position of branching and the entrance of an atom of oxygen at this point : CH3 CH3 CH CH 3 CH, I \ / ■ CH 2 — >- CO CH.NH, I COOH precisely as we suppose that acetone is formed in oxidation of proteins with peroxide of hydrogen (Vol. I of this series, p. 23, Chemistry of the Tissues). This view, however, was seen to be incorrect after it was recognized that diacetic acid in this case also serves as a forerunner of acetone. It is CH 3 \ now held that isoamylamine, ^CH— CH,— CH,.NH 2 , lsovaler- CH s. CH 3 / aldeyde, >CH— CH 2 — COH, and isovalerianic acid, CH,/ OTT \ >CH— CHj— COOH, can contribute to the formation of CH,/ acetone bodies in the economy, as well as ethylbutyric acid, CH, \ .,..., CH,\ >CH— CH,— COOH, ß-oxjis ovaler iamc acid, >C(OH)— CH, C 2 H 6 / CH,/ —COOH, dimethylacrylic acid, \c = CH— COOH,and crotonic CH3/ acid, CH 3 — CH=CH— COOH. In the first mentioned instances a possible arrangement might be made by which one of the alkyl groups above the position of branching is thrown off and replaced by a hydroxyl radicle : CH, CH, CH, CH CH.OH CH, CH, COOH COOH. CARBOHYDRATE DEFICIENCY AND ACIDOSIS 439 This possible mode of explanation, unless forced, fails, how- . CHa-CHj— CH— COOH, ever, m case of a-methylbutyric acid, i CH3 from which by analogous procedure a-oxybutyric acid should arise. There is particularly no possibility of explaining by this means the fact that a tricarbon complex like glycerol 19 and that certain benzol derivatives, whose ring is subject to disintegration in the animal body, like Phenylalanin, C 6 H 5 - CH 2 - CH.NH 2 - COOH, and tyrosin, / H CeHr- CH*— CH.NHr- COOH , must be classed among acetone forming substances. And as the formation of acetone bodies from these latter can be con- ceived synthetically possible only from groups containing but few carbon atoms, the question at once arises whether in case of the formation of ß-oxybutyric acid from normal higher fatty acids the process may not follow pretty much the line of explanation proposed by Friedmann. This in the author's opinion can be made to fit in, too, with Embden's observation upon the contrast between acids with even and with odd numbers of carbon atoms. It is, however, quite as likely that the development of /?-oxybutyric acid in the economy takes place in different ways : that it may be formed from stearic acid, for example, by "two-atom shortening of the chain, ' ' but from tyrosin by way of synthesis of two- carbon-atom compounds. Carbohydrate Deficiency and Acidosis. — In what relation, next, does the appearance of the acetone bodies stand to the carbohydrate exchange in the economy? Allusion has been made above to the distinct "antiketogenic" influence of carbohydrates, along with the statement that this may be fully explained on the basis of an inhibition of fat decom- position because of the combustible material thus introduced into the body. To the author's way of thinking, this makes all other complicated theories entirely superfluous, espe- U F. Reach (A. Durig's Lab., Vienna), Biochem. Zeitsehr., Ik, 279, 1908. 440 ACETONE BODIES cially such theories as that of Geelmuyden, who first urged the idea that carbohydrates and acetone bodies unite syn- thetically to form a combination which is essential for the further exchange of the acetone bodies, and then undertook to show that acetone bodies are convertible in the body into carbohydrates, and that they constitute a transition stage in the supposed transformation of fat into carbohydrates. It seems more likely that the observed fact that subcutaneous injection of diacetic acid or ß-oxybutyric acid in animals poisoned with phloridzin increases the output of sugar in the urine, is explicable on the supposition of a toxic increase of protein decomposition, rather than upon the assumption of Geelmuyden that there occurs a sugar synthesis from acetone bodies. 20 According to J. Bär simple withdrawal of carbohydrates is followed in normal human beings accustomed to a mixed diet, and also in apes, by an acidosis, that is, by an excretion of acetone, diacetic and oxybutyric acids, along with coinci- dent increase in the ammonia elimination. In the dog, goat and pig acidosis does not show upon mere reduction of carbo- hydrate, but is induced by combining phloridzin intoxication with fasting. 21 Diabetic Coma. — As is well known diabetic coma, a symp- tom complex clearly described many years ago by Kussmaul, has been recognized, especially from the investigations of Stadelmann and the Naunyn school, as an acid intoxication caused by an accumulation in the system of acetone bodies. 22 Magnus-Levy, 23 who took a prominent part in these investi- gations, showed that oxybutyric acid appears in the urine of all severe cases of diabetes, even apart from the coma, 20 H. C. Geelmuyden (Christiania), Zeitschr. f. physiol. Chem., 41, 128, 1904; 73, 176, 1911. 21 J. Bär (Med. Clinic, Strassburg), Arch. f. exper. Pathol., 54, 153, 1905. 22 Literature upon Acetone Bodies in Diabetic Coma: C. v. Noorden, Handb. d. Pathol, d. Stoffw., 2d ed., 2, 69-86, 1907. 23 A. Magnus-Levy, 1. c; Arch. f. exper. Pathol., '/I and Jfö. ALKALINE TREATMENT OF DIABETIC COMA 441 in very large amounts (twenty to thirty grams in the course of twenty-four hours). In cases of coma the forma- tion of this acid rises to an abnormal level, and coincidently there is a lowering of its combustion. Under such circum- stances enormous quantities of the substance may appear in the urine (as much as 160 grams in twenty-four hours). In the body of subjects dead from diabetic coma one or two hun- dred grams of the acid may be found. The coma is regarded as an acid intoxication, in which, it would seem, all the indi- vidual phenomena are dependent either directly or indirectly upon the accumulation of acid. As the supply of alkali which the system keeps ready for the neutralization of the acids permeating it is not large, these acids (oxybutyric acid, diacetic acid) are for the most part neutralized by ammonia, which is thus withdrawn from urea formation. In these cases, therefore, the urinary ammoniacal elimination serves as an approximate index of the acid excretion. 24 Alkaline Treatment of Diabetic Coma. — Oxybutyric acid, the sites of production of which may be looked upon as the liver and muscles and perhaps other tissues as well, pro- duces in case of diabetic coma, as indicated by the observa- tions of Minkowski, F. Kraus and others, a lowering of the alkalescence of the blood and of the amount of carbonic acid fixed thereby in the blood. 25 For this reason it is alto- gether logical that in the coma, which in French's opinion is the most serious menace to the life of the diabetic subject (aside from pulmonary phthisis), efforts be made to an- ticipate it by a prophylactic alkaline treatment. Umber believes that in those cases of diabetes in which persistence of diacetic acid in the urine can be recognized by the ferric chloride reaction, enough alkali should be given daily to give the urine an amphoteric reaction. "This frequently 24 A. Magnus-Levy, Die Oxybuttersäure und ihre Beziehungen zum Coma diabeticum, Leipzig, J. C. Vogel., 1899, p. 83. 25 G. Embden and L. Lattes (Frankfurt a. M.), Hofmeister's Beitr., 11, 327, 190S; H. C. Geelmuyden (Christiania), Zeitschr. f. physiol. Chem., 58, 253,. 1909. 442 ACETONE BODIES requires comparatively large amounts, which the patient often can scarcely master . . . fifteen to one hundred, up to two hundred grams or more, of sodium bicarbonate may be necessary to accomplish the desired end. ... I have never seen any more effect from intravenous adminis- tration of alkali (usually given in the form of three or four percent, soda solutions) than from subcutaneous infiltrations of isotonic sodium chloride solutions ; and for this reason, like Naunyn, I have long ago given up the former method. There is no question as to our ability to save severe cases of diabetes by alkaline treatment in the face of an acidosis of years ' duration, although without this method of treatment this would be a matter of impossibility. When coma is fully developed in adults all effort to stop it is in vain; but in children now and then therapeutic results may be obtained. 26 C. v. Noorden 27 is in the habit of saying of these influ- sions, because of his own rich experience, that any one who has ever witnessed a single time the wonderful result pro- duced by the infusion of a soda solution upon a comatose diabetic subject is bound to accept as a certainty the effec- tiveness of the alkaline treatment. Time and again the patient is seen waking up from the deepest coma while the infusion is being administered. There is no other agency which will do the same thing. Antiketogenic Substances. — In view of the antiketogenic influence of carbohydrates, one should seek in those cases in which acidosis arises after the withdrawal of carbohydrates to give some form of these substances adapted to the diabetic patient. In Umber's opinion these are the cases which should be placed upon v. Noorden 's oatmeal treatment, by which not infrequently it is possible to restore tolerance and remove the impending danger of coma. 28 36 F. Umber, Lehrbuch d. Ernähr, u. d. Stoffwechselkr., p. 231, 1909. 27 C. von Noorden, 1. c, p. 85. 28 F. Umber, 1. c, p. 229. ANTIKETOGENIC SUBSTANCES 443 Other substances which are easily combustible in the economy also have an antiketogenic action, in the same way as carbohydrates. According to Embden it is also possible, in experimental perfusion of the liver, to inhibit the forma- tion of acetone bodies by adding easily oxidizable substances to the perfusion fluid. For example, we may prevent the production of diacetic acid from caproic acid by introducing into the liver valerianic acid (which is not a source of acetone bodies, because of the uneven number of its carbon atoms). 29 It is conceivable that substances of very varied character (as xylose, gluconic acid, mannite, glycerol, alcohol, tartaric acid, lactic acid, propionic acid, citric acid, glycocoll, alanin, glutaminic acid) which readily undergo combustion in the liv- ing body, may at times manifest an antiketogenic power. 30 An especially marked antiketogenic influence is ascribed by J. Baer and L. Blum (from their observations in phoridzin COOH CH: diabetes) to glutaric acid, CH 2 ; it is said its antiketogenic CH 2 COOH influence is all the more definite, the more marked the sugar excretion and the more severe the disturbance of metabolism which manifests itself in the acidosis. In severe acidosis and high grade glycosuria after phloridzin administration the authors mentioned have observed a complete disappear- ance of sugar and of oxybutyric acid, along with marked reduction in the nitrogen elimination. A similar influence is ascribed also to the homologous dicarbooxylic acids with five to ten carbon atoms in their chains, the influence decreas- ■*G. Embden and S. Wirth, Biochem. Zeitschr., 27, 1, 1910. 30 Cf. Literature: A. Magnus-Levy, Noorden's Handb. d. Pathol, d. Stoffw., 2d ed., 1, 184, 1906; G. Satta (Frankfurt a. M.), Hofmeister's Beitr., 6, 376, 1905; J. Bär and L. Blum (Strassburg) , Hofmeister's Beitr., 10, 90, 1907; 11, 101, 1908; Arch. f. exper. Pathol., 65, 1, 1911; O. Neubauer, Münchener med. Wochenschr., 1906, 791. 444 ACETONE BODIES ing as the number of carbon atoms increases. A. J. Ringer has very recently occasioned a great deal of surprise by reporting that in control experiments, conducted under direction of Graham Lusk, he was unable to confirm these findings, and by attributing the above results to fault in the experimental technic of his predecessors. 31 In the author's opinion however, the oft-repeated results of Baer and Blum, assembled with so much care, are not to be put aside in such summary manner; it would seem rather to be an objective requirement to follow up the particular factors which are the ultimate cause of this contradiction. Ammonia Elimination and Acidosis. — As already stated the proportion of ammonia in the urine affords an approxi- mate evaluation of the elimination of oxybutyric acid. How- ever, the ammonia, even if a large quantity is excreted, does not, according to C. von Noorden, ordinarily represent more than twenty to twenty-five per cent, of the total urinary nitrogen. In a case of diabetic coma we may, it is true, meet with not less than forty-five per cent, of the total nitrogen as ammonia. 32 Yet it would be a decided misuse of words to identify, as is often done, two different phenomena like in- creased elimination of acetone bodies and of ammonia, which, of course, often run along parallel, under the collective term "acidosis." There are conceivable cases in which an in- creased ammonia excretion is due to altogether different causes. Disturbances of the synthesis of urea in the liver may be such a cause, as was pointed out above. According to W. Schlesinger 33 an abnormally increased fat cleavage in the intestine and an abnormally high loss of calcium in the fasces, from the formation of relatively insoluble lime soaps, may give rise to alkali impoverishment and to a compensa- tory increase of excretion of ammonia. Then, too, a notable 31 A. J. Ringer (Univ. of Pennsylvania), Jour, of Biol. Chem., 12, 223, 1912. 32 C. von Noorden, Handb. d. Pathol, d. Stoffvv., 2d ed., 2, 82, 1907 ; cf. also W. Camerer, Zeitschr. f. Biol., U, 22, 1903. 33 W. Schlesinger (Vienna), Zeitschr. f. klin. Med., 5J h 14, 1904. INTERRELATION OF ACETONE BODIES 445 increase of the lower fatty acids in the intestine doubtless calls out a lowering of the alkalinity of the body, as the acids appear in the fasces in a practically entirely neutralized condition. 34 Interrelation of the Acetone Bodies. — This discussion of the acetone body problem would be incomplete were there no attempt made to explain the mutual relations of these substances. Formerly this relationship was supposed to consist of a transition of the ß-oxybutyric acid in the economy into diacetic acid, which then under normal conditions was sup- posed to undergo combustion (without forming acetone by cleavage of carbonic acid). It was presumed, moreover, that in severe diabetes this transformation was so disturbed that the oxybutyric acid and diacetic acid were passed off as excretions. This conception must be modified, to be in harmony with modern investigations. Otto Neubauer 35 proposes the fol- lowing schema : BUTYRIC ACID OXYBUTYRIC ACID CH3 CH3 CH 2 CH.OH CH2 CHo COOH COOH C0 2 + H 2 \ ^CH S \ I \ CO CH S CH 2 > CO I I COOH CH 3 DIACETIC ACID ACETONE. According to this the oxybutyric acid, but not the aceto- acetic acid, would be a normal intermediate metabolic prod- 54 Cf. also A. Schittenhelm and A. Katzenstein (Göttingen). Zeitschr. f. exper. Pathol., 2, 542, 1906; L. F. Meyer and L. Langstein (Berlin), Jahrb. f. Kinderheilk., 63, 30, 1906. 35 O. Neubauer, 27th Internat. Kongr., Wiesbaden, 1910, 566. 446 ACETONE BODIES uct. In healthy individuals the oxybutyric acid would be converted by oxidation into carbonic acid and water, the diacetic acid not appearing as an intermediary product. In case of interference with the normal catabolism of oxy- butyric acid, however, a false route would be interposed: oxidation into diacetic acid, and then formation of acetone by splitting off carbon dioxide. It may be thus conceived why in a healthy individual (as contrasted with the diabetic subject) even large quantities of oxybutyric acid do not give rise to diacetic acid production, 36 and why in perfusion ex- periments on the normal liver (in contrast to a phloridzin liver) the amounts of oxybutyric acid decomposed are in absolute disproportion to the newly formed diacetic acid. 37 An example of the saying that when the proper time ar- rives for a truth it falls like a ripe fruit from the tree of knowledge, is to be seen in the fact that by chance E. Fried- mann in Berlin, 0. Neubauer in Munich, H. D. Dakin in New York, and L. Blum in Strassburg, all recognized at the very same time that the passage of oxybutyric acid into diacetic acid is a reversible process, and that the economy is not only able to oxidize the former into the latter, but may in reverse manner reduce diacetic acid into oxybutyric acid : CH 3 .CH(OH).CH 2 .COOH K > CH 3 .CO.CH 2 .COOH.^ How the /?-oxybutyric acid undergoes combustion in the normal body is unknown ; it might be supposed that it breaks down into acetic acid : CH 3 - CH(OH) - CH 2 - COOH + = 2 CH 3 .COOH; but it is by no means certain that it is entirely consumed. It is not impossible that the oxybutyric acid may enter into 36 L. Schwarz, Zeehuyzen, Araki and others. 37 B. O. Przibram (F. Kraus's Clinic), Zeitschr. f. exper. Pathol., 10, 1912. 38 E. Friedmann and Maase (Berlin), Münchener med. Wochenschr., 1910, No. 34; Biochem. Zeitschr., 21, 474, 1910; O. Neubauer, 1. c; L. Blum, Intern. Kongr., 1910, Münchener med. Wochenschr., 57, 1910; H. D. Dakin (New York), Jour, of Biol. Chem., 8, 97, 1910; A. J. Wakeman and H. D. Dakin, ibid., 105. INTERRELATION OF ACETONE BODIES 447 further synthetic changes. A number of authorities, as Minkowski and von Noorden, have thought that it may per- haps be looked upon as an intermediate link between fat and sugar. 39 The statement has been made above that there is at present no convincing basis for Geelmuyden's view that the acetone bodies should be regarded as transition stages in the supposed formation of sugar from fat. There are many arguments against the idea that oxybutyric acid can be in any way concerned with the synthetic formation of fatty acids from carbohydrates. Embden, in his last publications, expresses the belief that the route from sugar to the fatty acids may, perhaps, lie through lactic acid, acetaldehyde and diacetic acid. "Acetaldehyde, as E. Friedmann first showed, in the experimentally perfused liver forms diacetic acid. If acetaldehyde, as would appear, is a product of carbohydrate catabolism which develops in very large amounts, we may perhaps regard this substance, as earlier authors have sug- gested, as the point of attack in the synthetic production of fatty acids from carbohydrates. ' ' 40 Should this line of thought prove correct an interesting relation would be estab- lished between the acetone bodies with both the anabolism and catabolism of the higher fatty acids. Thus far, how- ever, the connection of these substances with fat decomposi- tion in the economy constitutes the best based phase of the whole problem. The fact that enzymic catalysers are known to exist in the tissues, which are capable of oxidizing oxybutyric acid to form diacetic acid, 41 and of converting the latter into acetone by splitting off carbon dioxide, 42 is no proof that this is a process which takes place physiologically. It is impossible to say at present what factors determine 39 Cf. B. O. Przibram, 1. c. 40 G. Embden and M. Oppenheimer, Biochem. Zeitschr., 45, 202-203, 1912. 41 A. F. Wakeman and H. D. Dakin, Jour, of Biol. Chem., 6, 373, 1909. 42 L. Pollak (R. Paltauf's Instit., Vienna), Hofmeister's Beitr. 10, 232, 1907. 448 ACETONE BODIES whether the diacetic acid arising in metabolism is to be excreted unchanged or as acetone. According to Lüthje 43 a portion of the diacetic acid may perhaps be excreted by the kidneys as an alkaline salt, provided sufficient supply of alkali be available; this portion otherwise appearing as acetone. Determination of Acetone and Diacetic Acid. — In con- clusion a few remarks upon the quantitative determination of the acetone bodies are desirable. 44 In the determination of acetone, precedence is still given to the long approved Messinger-Huppert method. This con- sists in distillation of the acetone from the urine, transform- ing it into iodoform by means of iodide of potassium in an alkaline solution, and determining by titration the amount of iodine used in the formation of iodoform. Acetone can also be separated from the distillate by • nitrophynylhy- drazine (according to Eckenstein and Blancksma) as a bright yellow crystalline sediment, and weighed. 45 It may, too, be precipitated by an alkaline solution of mercuric cyanide, the acetone-mercurial precipitate broken up by acids, and the mercury determined by titration (very like Vollhard 's method of determining silver). 46 Finally the fixation of sodium bisulphite by acetone may be used for iodometric estimation, provided a very long period of reaction is given ; 47 while in experiments of short duration hydrolytic dissociation is so marked that besides the fixed sulphurous acid there is always present a considerable fraction in free state. 48 In the de- 43 H. J. Lüthje (Kiel), Therap. d. Gegenw., 51, 8, 1910. 41 Literature upon the Quantitative Determination of the Acetone Bodies : G. Embden and G. Schmitz, Handb. d. Biochem., 3, 906-939, 1910; E. Letsche, ibid., 5, 1, 197-199, 1911; cf. also G. Embden and L. Michaud, Biochem. Zeitschr., 13, 262, 1908 ; Hofmeister's Beitr., 11, 332, 1908. 45 S. Möller (v. Leyden's Clinic), Zeitschr. f. klin. Med., 6/ f , 207, 1907; W. C. de Graaff, Pharmac. Weekbl., U, 555; Jahresber. f. Tierchem., 37, 356, 1907. 46 H. Scott Wilson (Oxford), Jour, of Physiol., Iß, 444, 1911. 47 A. Jolles, Ber. d. deutsch, ehem. Ges., 39, 1306, 1906. 48 J. Mondschein (under direction of O. v. Fürth), Biochem. Zeitschr., Iß, 95-97, 1912. DETERMINATION OF OXYBUTYRIC ACID 449 termination of acetone, if glucose be present in the urine, special care should be taken, because in heating sugar-con- taining solutions substances of the nature of ketones and aldehydes are easily produced which may be confused some- times with acetone. 49 Separation of acetone and cliacetic acid, as elaborated by Embden and Schliep 50 and by Folin, 51 is performed by driv- ing over the preformed acetone in a given amount of urine by careful distillation at very low pressure and temperature (not above 35° C), or by an air current, and estimating it. Another portion of urine is subjected to the Messinger method, after boiling with phosphoric acid, the diacetic acid being thus converted into acetone; by which measure the total of acetone and diacetic acid is obtained. Rowald 52 recommends a combination of Embden 's vacuum distillation with the method of precipitation by nitrophenylhydrazine as the most practicable means of separate determination of acetone and diacetic acid. Quantitative Determination of Oxybutyria Acid. — There are essentially four methods available for quantitative de- termination of oxybutyric acid : Polarimetrie estimation, con- version into crotonic acid, into diacetic acid and into acetone. The Polarimetrie method worked out by Magnus-Levy, depending upon the optical activity of oxybutyric acid and yielding excellent results when large amounts are present, is not reliable when one is dealing with small quantities, be- cause of laevogyrating substances which can be obtained even from normal urine in ether extraction, and because the specific rotating power of ß-oxybutyric acid is not high. 53 Darmstädter proposed a method by which the oxy- 49 Borchhardt (Wiesbaden), Hofmeister's Beitr., 8, 62, 1906. 60 L. Schliep (Embden's Lab.), Centralbl. f. Stoff w., No. 2, 250, 289, 1907. 61 0. Folin, Jour, of Biol. Chem., 3, 177, 1907; J. S. Hart, Jour, of Biol. Chem., k, 473, 1908. 62 J. Rowald, Inaug. Dissert., Giessen, 1908. 63 Embden and Schmitz, Geelmuyden, B. O. Przibram, Zeitschr. f. exper. Pathol., 10, 279, 1912. 29 450 ACETONE BODIES butyric acid is converted into crotonic acid by distillation with sulphuric acid, with separation of water, and the crotonic acid then determined by alkalimetry. This method, however, has proved to be rather inexact in control tests ; 54 but apparently excellent results are obtained, according to Eyffel 55 and B. 0. Przibram, 56 by determining the crotonic acid, not by alkalimetry, but by taking advantage of its ability to fix bromine : OXYBUTYRIC ACID CROTONIC ACID BROMINE ADDITION PRODUCT CH3 CH3 CHj CH.OH CH CH.Br I > II > I CH 2 CH CH.Br COOH COOH COOH. The bromine is added in excess ; the excess of bromine, on the addition of potassium iodide, sets free an equivalent amount of iodine which is estimated by titration with thiosul- phate. It is recommended that the urine be not subjected to direct sulphuric acid distillation, but that, saturated with ammonium sulphate and acidulated with sulphuric acid, it be first extracted for twenty-four hours with ether in Lindt's apparatus and the ethereal extract further employed. A colorimetric method of estimating oxybutyric acid has been proposed, depending upon transformation of oxy- butyric acid into diacetic acid by hydrogen peroxide, and subsequent estimation of the latter optically by the beautiful red color which it yields on addition of ferric chloride. 57 In view of the instability of diacetic acid, however, it is impos- sible to restrain a feeling of distrust for this method. An excellent mode of determination, however, is afforded in Schaff er 's method, 5S which consists of heating to boiling the fluid containing the oxybutyric acid, in the presence of " Embden and Schmitz, B. O. Przibram, 1. c. 55 Ryffel, Jour, of Physiol., 82, Proc. Physiol. Soc, LVI, May 20, 1905. 63 0. F. Black, Jour, of Biol. Chem., 5, 207, 1908. 07 B. O. Przibram, 1. c. 88 P. A. SchafFer, Jour, of Biol. Chem., 5, 211, 1908. DETERMINATION OF OXYBUTYRIC ACID 451 dilute sulphuric acid, and then decomposing it by adding drop by drop a solution of potassium bichromate. The oxy- butyric acid undergoes decomposition according to the fol- lowing formula, into diacetic acid, then into acetone, carbon dioxide and water : CH 3 CH .OH CO I CH COOH CH.OH CO I + O = H,0 + | = H 2 + C0 2 + CO CH2 CH2 CH,. The acetone distilled over is taken up in water and the proportion determined by Messinger 's method with iodine and thiosulphate. J. Mondschein, 59 who worked out a method in the author's laboratory by which the quantitative estimation of oxybutyric and lactic acids is possible together (vide infra), was able to fully satisfy himself of the delicacy of Schaff er 's method. So much may be said of the acetone bodies and their physiological significance. 50 J. Mondschein, 1. e. CHAPTER XIX LACTIC ACID. FATE OF BODY-FOREIGN SUBSTANCES IN THE ECONOMY LACTIC ACID As an addendum to the acetone bodies it seems desirable at this place to examine into the problem of lactic acid, which is of the greatest importance to the comprehension of the metabolic processes. This is one of those questions which are always of special stimulative interest to the writer, not so much because of the wealth of knowledge which comes im- mediately from them (for this for the present can satisfy only very modest demands) but rather because of the feeling that here we are before the entrance to a steep and laborious mountain trail which may lead one, if he be able to surmount its difficulties, to a broad and as yet unknown plateau. Quantitative Estimation of Lactic Acid by the Method of Fürth and Charnass. — The reason that the lactic acid prob- lem has not advanced more rapidly is primarily due to the fact that it has been only recently that a successful method of overcoming the difficulties of exact lactic acid estimation has been attained. The older authors conducted their de- terminations of lactic acid as a rule by extracting it by simply shaking it for a time with ether in a separatory fun- nel, then forming the relatively insoluble zinc or lithium salt of lactic acid and weighing it. As lactic acid is by no means readily taken up by ether from water, and as quan- titative extraction is seemingly possible only by long con- tinued treatment in some such elaborate apparatus as Lindt's rotating extraction apparatus, as, too, the above men- tioned salts are not entirely insoluble in their mother fluids, and impurities can be removed only by repeated crystalliza- tion, it must be evident that the older methods of estimating lactic acid were attended by very serious faults and that 452 QUANTITATIVE ESTIMATION OF LACTIC ACID 453 the results from such methods seem extremely unreliable. Boas had originally recommended that in qualitative de- termination of lactic acid (as in gastric juice) the fluid under examination be distilled with manganese and sulphuric acid and the acetaldehyde, formed according to the equation : CH 3 I CH 3 CH.OH + 0=1 + C0 2 + HjO, COH COOH be determined by transforming it into iodoform with an alkaline solution of iodine. E. Jerusalem having shown in the author's laboratory that oxidation of lactic acid in hot sulphuric acid solution by the addition of drop after drop of permanganate solution may be employed for the purpose of quantitative determination, 1 the author, in collaboration with D. Charnass, 2 worked out a reliable method based upon this principle for its quantitative estimation. They were able to show that oxidation cleavage of aldehyde from lactic acid occurs when certain experimental conditions are main- tained, even if not absolutely quantitatively, at least with such a degree of uniformity that a quantitative method can very well be based upon this process as a foundation. Titri- metric estimation of aldehyde by the iodoform method gives reliable results only when very exact experimental condi- tions are maintained. It is very inferior in practice to the iodometric method of Eipper based on the addition of bisulphite : CHj CH3 I + NaHS0 3 = I /H COH C(-ONa \HS0 3 , as the latter yields almost theoretically correct results. 3 The 1 E. Jerusalem (under direction of 0. v. Fürth), Biochem. Zeitschr., 12, 361, 379, 1908; O. v. Fürth, Emendation to article of E. Jerusalem, ibid., 2k, 266, 1910. 2 0. v. Fürth and D. Charnass, Biochem. Zeitschr., 26, 199, 1910. »Cf. also W. Sobolowa and J. Zaleski (St. Petersburg), Zeitschr. f. physiol. Chem., 69, 441, 1910. 454 LACTIC ACID amount of aldehyde obtained from lactic acid in the v. Fürth and Charnass tests averaged eighty-nine per cent, of the theoretical value; and the constant error is taken into ac- count by introducing a correction factor. In Embden's laboratory 4 several improvements have recently been made in the method, which make it satisfy even far reaching re- quirements. The aldehyde recovery of the original method particularly can be increased by using a very dilute solution of permanganate (n/100 instead of n/10 solution) for oxidizing purposes. Observations in Embden's laboratory show the need which existed for replacing the weighing of the zinc lactate by another and better method. This is done by extracting the lactic acid for about twenty-four hours in the Lindt rotary apparatus (which forces a whirl of very fine ether bubbles through the fluid) and removing the lactic acid from the etheral extract for weighing as zinc lactate. Control ex- periments by the author's method show that in determina- tions of lactic acid from tissue extracts made by weighing the lactate of zinc, the zinc salts contain so many impurities at times that no fixed conclusions can be drawn as to the amounts of lactic acid present by mere weighing. 5 A source of error until recently entirely overlooked in the determination of lactic acid in tissues may arise from the fixation of the free lactic acid by the tissue proteins. J. Mondschein, for example, has determined in the author's laboratory that the previously existing statements as to the amount of lactic acid in muscle are too low, as about one- third of the lactic acid present (which, if the tissue is well boiled, remains fixed in the coagulum because of combina- tions with proteins) escapes estimation. However, the 4 G. Embden, Handb. d. biocbem. Arbeitsnieth., 5, 1255-1259, 1912. 6 S. Oppenheimer (Embden's Lab.), Biochem. Zeitschr., 45, 30, 1912. 6 J. Mondschein (under direction of O. v. Fürth), Biochemie. Zeitschr., 42, 105, 1912. METHOD OF LACTIC ACID DETERMINATION 455 lactic acid in the extract obtained by boiling mnscle can be determined with great accuracy by titration (using Phenol- phthalein as indicator), because all the recognizable lactic acid is present in free form, and the amount of other acid products is of no practical importance ; and the fraction of lactic acid in the proteid coagulum can be released for de- termination by liquefying the coagulum by heating with caustic alkali, the albuminous fluid freed of its protein by addition of saturated salt solution, and finally the lactic acid determined by the method proposed by the author and Charnass in the protein-free filtrate. Determination of Lactic Acid and ß-oxybutyric Acids Together. — Recourse should also be had to a special method for determination of lactic acid in case fluids containing both lactic acid and ß-oxybutyric acids are to be dealt with. In separate portions the two acids may be determined, respec- tively, as aldehyde or acetone (v. sup.), after oxidation with permanganate or with bichromate in a sulphuric acid solu- tion. As in both cases, however, a mixture of aldehyde and acetone distills over, separation of the two substances is requisite. In estimating the lactic acid a modification is in- troduced, whereby in an aliquot portion of the distillate the readily destroyed aldehyde is decomposed by boiling with hydrogen peroxide and potassium hydrate, the acetone re- distilled and titrated according to Eipper. In estimating the butyric acid the distillate (containing acetone and alde- hyde) is freed of its aldehyde by boiling with hydrogen peroxide and caustic potash ; the acetone is redistilled and determined by titration according to Messinger 's method. 7 Ryjfel's Method of Lactic Acid Determination. — Ryffel proposed a method of lactic acid determination which de- pends upon a somewhat different principle. In this the lactic acid in the urine is broken up directly into acetaldehyde 7 J. Mondschein (under direction of 0. v. Fürth), Biochem. Zeitschr., Iß, 91, 1912. 456 LACTIC ACID and formic acid by distillation with fifty per cent, sulphuric acid : CH 5 ^HjOH I COOH CH 3 + H CHjOH - I I I COH COOH. The aldehyde is then estimated colorimetrically by the hand- some red color which it strikes with a rosaniline solution decolorized with sulphurous acid. 8 Postmortem Formation of Lactic Acid. — We may at this point take up again the striking features of the present status of the lactic acid problem. It should be definitely understood that as sources of lactic acid we must keep in mind carbohydrates, proteins, and, too, a substance of unknown nature, which may be characterized, following the suggestion of Embden, as lactacidogen. The chemical relation with carbohydrates may be seen in the very readily occurring decomposition of one molecule of sugar into two molecules of lactic acid : C 6 H 12 6 =2 C 3 H 6 3 ; and for the relation with proteins we may at once refer to the deamidization of alanin: CH 3 .CH(NH 2 ).COOH + H 2 = NH 3 + CH 3 .CH(OH).COOH. (The possibility of an actual intracorporeal transformation of alanin into lactic acid has been demonstrated by the experiments of Neuberg and Langstein upon animals deprived of glycogen.) We may first take up for consideration the postmortem acid change of the tissues. The fact has been known a very long time that this is due to formation of lactic acid, and that the much studied postmortem production of acid in muscle (Vol. I of this series, pp. 145-147, Chemistry of the Tissues) is nothing more than a particular instance of a process equally likely to occur in all the tissues. This acid change is at present regarded as a continuation of an intra- vital process. A great number of investigations have been devoted to the subject of acid production in aseptic and anti- 8 J. H. Ryffel, Jour, of Physiol., 89, Proc. Physiol. Soc, V, 1909. POSTMORTEM FORMATION OF LACTIC ACID 457 septic autolysis, which leave no doubt of the enzymatic nature of the process. The acid met with may be said to be 8-lactic acid. (The observations indicating the occurrence of inactive fermentative lactic acid in autolysis may be fully explained, in the writer's opinion, by the associated influence of microorganisms.) 9 When it is recalled that the amount of lactic acid in a specimen of tissue pulp for a time increases until it reaches a maximum, and thereafter undergoes diminution, and that this decrease (as recently shown among others, by Ssobolew, 10 in the Vienna Physiological Insti- tute) also takes place under experimental conditions surely exclusive of bacterial influence, we seem justified in assum- ing the existence of enzymes which decompose lactic acid, as well as of those which cause the formation of this substance. When a tissue juice contains an insufficient proportion of alkali lactic acid formation ceases, apparently at a certain degree of concentration of hydrogen ions, by autoinhibi- tion. 11 Although even earlier investigations 12 indicated the improbability of a direct connection between the amount of glycogen of a tissue (especially of muscle) and postmortem acid change, the author's pupil, R. Türkei, 13 recently de- ceased, was able to prove that a liver, even if practically free from glycogen and sugar, is capable of forming lactic acid in the course of autolysis, and that, too, in proportions not ma- terially less than is a liver with normal carbohydrate con- 'Mochizuki and Arima, Kikkoji, Inouye and Kondo; cf. Literature: C. Oppenheimer, Die Fermente, 3d ed., 475, 1909; E. Salkowski, Zeitschr. f. physiol. Chemie., 63, 237, 1909; R. S. Frew (Salkowski'a Lab.), ibid., 60, 15, 1909; T. Saito and J. Yoshikawa, ibid., 62, 107, 1909; T. Saiki, Jour, of Biol. Chem., 7, 17, 1910; G. v. Stein, Inaug. Dissert., Berlin, 1911; Biochem. Zeitschr., W, 186, 1912: H. Youssouf (Salkowski's Lab.), Virchow's Arch., 207, 374, 1912. 10 N. Ssobolew (under direction of O. v. Fürth), Biochem. Zeitschr., ^7, 367, 1912. U K. Kondo (G. Embden's Lab., Frankfurt a. M.), Biochem. Zeitschr., ^5, 63, 1912. 12 Cf. Literature: O. v. Fürth, Ergebn. d. Physiol., 2', 594-600, 1903. 13 R. Türkei (under direction of O. v. Fürth), Biochem. Zeitschr., 20, 431, 1909. 458 LACTIC ACID tent. As, moreover, introduction of alanin and inosite gave no definite result, the conclusions reached were in con- sonance with the important observations of Embden u with carbohydrate-free expressed muscle juices, and those of Fletcher 15 with ground muscle, that postmortem forma- tion of lactic acid takes place not at the expense of carbohy- drates, or of inosite or alanin, but at the expense of some unknown predecessor, Embden 's "lactacidogen," which may possibly be thought of as related with the hypothetic phos- phosarcous acid of Siegfried (v. Vol. I of this series, p. 148, Chemistry of the Tissues). The observations upon muscle autolysis thus gave no basis for assuming a connection between lactic acid and the carbohydrates. And as no increase of lactic acid production was obtained by perfusing the posterior extremities of dogs with sugar added to the blood, Asher and Jackson were dis- posed to accept the idea that the lactic acid does not arise from carbohydrates but from the decomposition of protein. 16 Origin of Lactic Acid from .Sugar. — Much more certain evidence of the origin of lactic acid from carbohydrates may be met, however, in the liver perfusion experiments of Emb- den and his associates. 17 These indicated that in perfusing a dog's liver, free of glycogen, with bovine blood no lactic acid is formed de novo; although in perfusing a liver rich in glycogen under similar experimental conditions there could always be observed a very marked new formation of S-lactic acid. A result of the same kind could be obtained, although the amount of acid was somewhat less, when glucose was added to the fluid used in perfusing a liver free of glyco- gen. Alanin also proved to be a lactic acid former. Embden 14 G. Embden and F. Kraus, 26 Kongr. f. innere Med., 1909, 351; G. Emb- den, F. Kalberlah and H. Engel, Biochem. Zeitschr., 45, 58, 1912. 18 W. M. Fletcher (Cambridge), Jour, of Physiol., 43, 286, 1911. 16 L. Asher and H. C. Jackson, Zeitschr. f. Biol., 41, 393, 1901. 17 G. Embden with M. Almagia, Centralbl. f. Physiol., 18, 832, 1905; v. Noorden and Embden, Centralbl. f. Stoffw., n. s., 1, 1, 1906; G. Embden and F. Kraus, Biochem. Zeitschr., 45, 1, 1912. ORIGIN OF LACTIC ACID FROM SUGAR 459 came to the conclusion from these results that both the carbo- hydrates and the proteins are to be regarded as contributing to the formation of lactic acid, the former, it is true, ap- parently in much the greater proportion. This harmonizes closely with the latest researches upon the nature of gly- colysis, dealt with above (vide supra, p. 338). Just as sugar by the isolated influence of alkali is broken down with ex- treme readiness into lactic acid, we have every reason for assuming that physiologically also glycolysis gives rise to lactic acid, 18 and that the formation of lactic acid observed by J. Müller in the surviving cat's heart, for example, is due to direct cleavage of grape sugar. Apparently a portion of the lactic acid coming from sugar cleavage in the economy is changed into ' ' lactacidogen, ' ' which later under certain con- ditions, as in postmortem acid change, again gives rise to lactic acid. "Apparently," says Embden, 19 "the principal route of carbohydrate catabolism in the economy is by way of lactic acid ; and in lactic acid the greater part of the chem- ical energy which was in the glucose is still present. . . . The very fact that a predecessor of lactic acid, which is very easily convertible into lactic acid, is at the disposal of the musculature, permits one to surmise that lactic acid is the most substantial source of muscular power. ... It should be recalled that the older and particularly, too, re- cent authors regard it as very probable that a definitely local lactic acid production within the muscle is concerned with the relaxation of contracted muscle. For the purpose of rapid production of lactic acid this predecessor of lactic acid, which is apparently not of acid nature or at least less acid than the lactic acid, may, perhaps, have an important part." The author's ideas based upon his personal studies as to the role of lactic acid in bringing about and relaxing rigor mortis have been discussed in a previous lecture (v. Vol. I of this series, p. 135-147, Chemistry of the Tissues). 18 Cf. J. H. Ryffel, Jour, of Physiol., kO, Proc. Physiol. Soc, LI, 1910. **G. Embden, F. Kalberlah and H. Engel, Biochem. Zeitschr., £5, 45, 1912. 460 LACTIC ACID Embden's Schema of Sugar Catabolism in the Living Body. — Thanks to along series of remarkable studies carried on by Gustav Embden 20 in the Frankfurt Institute of Physiological Chemistry in collaboration with a number of associates, we are able today to frame very exact ideas as to the decomposition of sugar into lactic acid and the further catabolism of the latter. Embden 21 condenses these points in the following schema: Diacetic Acid a-Glucose It Glycerol-aldehyde It 3-Lactic Acid U . Racemic Acid ^_ I — Acetaldehyde ^_ I Acetic Acid. ""*" Glycerol 5- Alanin ^ Ethyl Alcohol If we consider this schema somewhat more fully, in ac- cordance with it we may suppose that physiologically sugar catabolism would follow the course below indicated : GLYCEROL- ALDEHYDE LACTIC ACID RACEMIC ACID ACETALDEHYDE ACETIC ACID CH 2 .OH I -> CH.OH I COH CH, CH.OH I COOH CH, I CO I COOH CH, I COH CH, COOH. 8UGAR CH a .OH CH.OH CH.OH CH.OH CH.OH COH Glycerol-aldehyde as an Intermediate Product Between Sugar and Lactic Acid. — The assumption that glycerol- aldehyde is an intermediate product between glucose and 20 G. Embden and associates, Biochem. Zeitschr., ^5, 1-206, 1912, and their earlier studies; cf. therein Literature. 21 G. Embden and M. Oppenheimer, Biochem. Zeitschr., J t 5, 202, 1912. SUGAR AND LACTIC ACID 461 lactic acid is based on the fact that it (in contrast to the CH.OH isomeric dioxyaeetone, CO , which according to Büchner CHj.OH and Meisenheimer is an intermediate product of yeast fermentation) has proved to be a lactic acid producer of very exceptional strength. Embden suspects that, just as gly- cerolaldehyde is an important source for 8-lactic acid, di- oxyacetone may be considered as the chief source of inactive fermentative lactic acid, and expresses the idea that in tran- sition of the glycerolaldehyde into lactic acid CH 2 .OH CH, I I *CH.OH — > * CH.OH COH COOH the middle carbon atom continues to retain its asymmetrical character. The belief that glycerol formation in the economy also proceeds through the intermediate stage of glycerolaldehyde, and that by this route sugar may pass into glycerol, and, vice versa, glycerol into sugar, is not incredible. It may be thought, too, that not only does sugar undergo transition into lactic acid, but that the reverse, the transition of lactic acid into sugar, is also possible. Embden and Salo- mon 22 have convinced themselves that lactic acid is a sugar former in depancreatized dogs ; Mandel and Graham Lusk, 23 in phloridzinized animals. According to Parnas and Bär 24 the route from lactic acid to glucose leads through glyceric acid and glycolaldehyde : LACTIC ACID GLYCERIC ACID GLYCOLALDEHYDE GLUCOSE CH, CH 2 OH CH 2 .OH > C«H 12 0«. >- >■ Condensation CH.OH Oxidation CH.OH Oxidation, COH Cleavage of COOH COOH H0 2 and C0 2 n G. Embden and Salomon, Hofmeister's Beitr., 5, 507, 1904 23 A. Mandel and G. Lusk, Amer. Jour, of Physiol., 16, 129, 1906. M Cf . also J. Parnas and J. Bär (Hofmeister's Lab., Strassburg), Biocbem. Zeitschr., ^1, 386, 1912. 462 LACTIC ACID These authors also look upon the route from glucose through lactic acid to glyceric acid and glycolaldehyde as the prin- cipal mode of carbohydrate catabolism in active metabolism of the animal economy. Racemic Acid as an Intermediate Product. — While Par- nas and Bär are willing to accept the formation of racemic acid as at most a by-path in the formation of lactic acid, Embden regards it as the normal catabolic product of lactic acid and as an intermediate product of the transition of alanin into lactic acid and vice versa; a view which is in harmony with the assumption of 0. Neubauer that catabol- ism of a-aminoacids takes place through a-ketonic acids. Paul Mayer, 25 in Neuberg's laboratory, has seen after administration of racemic acid to rabbits both lactic acid and sugar in the urine. It might well be conceived, therefore, that the route from glucose, through lactic acid, to racemic acid may also be traversed in the reverse way : Sugar < > Lactic Acid < > Racemic Acid. Finally it should be noted that the route indicated in Embden 's schema: Glucose — >-Lactic Acid — ^-Racemic Acid — >-Acetaldehyde — >-Diacetic Acid 26 is possibly the one, too, which may be taken by the carbo- hydrates in the synthetic production of higher fatty acids in the body. Possibly this line of thought may prove fruitful also in clearing up the mysterious processes of fat formation in the economy. Appearance of Lactic Acid in the Urine. — What do we know, finally, of the excretion conditions of lactic acid? Small amounts are always present in the blood. 27 In rabbits, in- 86 P. Mayer (Neuberg's Lab.), Biochem. Zeitschr., 40, 441, 1912. 29 According to Neuberg (Berliner physiol. Gesel., 1912) racemic acid is fermented by yeast very readily into acetaldehyde and carbonic acid: CH 3 CH,.CO.COOH=C0 2 + I COH. "A. Fries (G. Embden's Lab.), Biochem. Zeitschr., 85, 368, 1911. LACTIC ACID IN URINE 463 troduced dextrorotary lactic acid, even when administered in large amounts, is almost completely consumed, according to observations by J. Parnas 28 in Hofmeister 's laboratory. Lagvo-rotary lactic acid is partly changed, partly excreted unchanged ; after introduction of racemic lactic acid an ex- cess of laevo-rotary acid is eliminated. From observations made in the Vienna Pharmacological Institute it seems that the unconsumed fraction of lactic acid is excreted partly as such, partly, however, in the form of other ether-soluble acids. 29 Passage of lactic acid into the urine is observed especially after severe muscular efforts, in epilepsy and eclampsia, in hepatic affections (acute yellow atrophy, phosphorus poi- soning, etc.), in oxygen deficiency, and, too, in geese after extirpation of the liver. 30 The fact that uric acid contains a tricarbon nucleus by no means justifies the assumption that the lactic acid excreted in the last instance is a sub- stance which would have been transformed into uric acid under normal conditions. 31 The general impression is given that lactic acid appears in the urine in conditions in which its normal combustion is lowered. However, it is impossible as yet to definitely formulate how, where and why this is the case ; and from appearances it is likely that considerable time will elapse before this is possible. FATE OF BODY-FOREIGN SUBSTANCES IN THE ECONOMY Having in the course of the preceding lectures attempted in one way or another to discuss in detail the fates of the most important foodstuffs, proteins, carbohydrates and fats, it may be well to round out our knowledge by endeavoring 23 J. Parnas (F. Hofmeister's Lab., Strassburg), Biochem. Zeitsehr., 88, 53, 1912. " E. Neubauer (Vienna Pharmacological Institute), Arch. f. exper. Pathol., 61, 387, 1909. 30 Literature upon Lactic Acid Excretion: A. Ellinger, Handb. d. Biochem., 8', 651-653, 1910. 31 Cf. J. B. Leathes, Ergebn. d. Physiol., 8, 360, 1909. 464 FATE OF BODY-FOREIGN SUBSTANCES to trace the fate of a few body-foreign substances in the intermediate metabolism. 32 Decomposition of Fatty Acids and Aliphatic Sidechains. — In taking up first the fatty acids and aliphatic sidechains we keep in direct connection with the information gained in the course of the last few lectures as to the catabolism of fats and the origin of the acetone bodies. It was pointed out that the investigations of Knoop and Embden particularly have led to the recognition that in the disintegration of the normal fatty acids in the animal body /^-oxidation plays an important part, and that as a mode of oxidation we can picture : R.CH 2 .CH 2 .COOB— ► R.CH(OH).CH 2 .COOH — > R.CO.CH 2 .COOH — >,R.COOH ; in which a normal fatty acid passes first into a ß-oxyacid by ß-hydroxylation, and this then through the corresponding ß-ketonic acid into an acid with two less carbon atoms. A long series of careful studies conducted in the past few years, requiring much chemical scientific knowledge and skill, especially by E. Friedmann, F. Knoop, H. D. Dakin and 0. Neubauer, 33 have been devoted to tracing out the fate of many phenyl, halogenphenyl, furfuryl and similar substitu- tions in fatty acids, oxyacids, ketonic acids and aminoacids in the course of metabolism. These studies, the details of which cannot be entered into here, have shown that the above mentioned simple schema of ^-oxidation cannot be accepted as of general applicability, and that oxidation of fatty acids 82 Literature upon the Excretion of Body-foreign Organic Substances : A. Heffter, Ergebn. d. Physiol., 4, 184-306, 1905. 83 F. Knoop, Der Abbau aromatischer Fettsäuren im Tierkörper, Freiburg i. B., 1904; Hofmeister's Beitr., 11, 1908, and later works; E. Friedmann, Hofmeisters Beitr., 11, 151, 1908; E. Friedmann and C. Maase, Bföchem. Zeitschr., 27, 97, 113, 1910; E. Friedmann, ibid., 27, 119, 1910; 35, 40, 1911; Med. Klinik, 1909, Nos. 36 and 37, and 1911, No. 28; T. Sasaki (E. Friedmann's Lab.), ibid., 25, 272, 1910; H. D. Dakin (New York), Jour, of Biol. Chem., 4-6, 1908-09; 8, 35, 1910; 9, 123,1911; O. Neubauer, with W. Gross, H. Fischer, K. Fromherz, Zeitschr. f. physiol. Chem., 67, 219, 230, 1910; 70, 326, 1911; Literature upon the Catabolism of Fatty Acids and Aliphatic Side Chains: O. Porges, Ergebn. d. Physiol., 10, 1-46, 1910; C. Oppenheimer and Pincussohn, ibid., 4', 699-700, 706-709, 1911. DECOMPOSITION OF FATTY ACIDS 465 and aliphatic side chains takes place in much more compli- cated ways which Friedmann and Dakin 34 would outline schematically somewhat as follows : R. CH,. CH 2 . COOH / R.CH = CH.COOH R.CH(OH). CH a .COOH R.CO.CH 2 .COOH — >- R.CO.CH, R.COOH R.COOH. If this schema be applied, for illustration, to the indi- vidual case of phenylpropionic acid (C G H 5 .CH 2 .CH 2 .COOH), it will be seen that it can pass into either (by ß-oxida- tion) phenyl-/?-oxypropionic acid (B.CH(OH).CH 2 .COOH), or into benzoylacetic acid (C 6 H 5 .CO.CH 2 .COOH) or, finally, into cinnamic acid (C 6 H 5 .CH = CHjC00H). The last two may then be further catabolized into benzoic acid (C 6 H 5 . COOH). Benzoylacetic acid can by loss of C0 2 pass into acetophenone (C 6 H 5 .CO.CH 3 ), or by reduction into phenyl- /S-oxypropionic acid. Transition of this last, shown by the two pairs of arrows, into benzoylacetic acid and cinnamic acid is fully analogous to the previously mentioned equation between /?-oxybutyric acid, diacetic acid and crotonic acid : CROTONIC ACID OXYBUTYRIC ACID DIACETIC ACID CH 3 — CH=CH— COOH ^ CH 3 .CH(OH).CH 2 .COOH ^CH 3 .CO.CH 2 .COOH. The catabolism of the fatty acids, therefore, in line with this schema, is not confined to simple oxidation processes ; these latter have associated with them processes of reduc- tion and of water- and C0 2 -cleavage. It may be said in pass- ing that the previously mentioned (p. 385) discoveries of E. Pick and Joannovics as to the conversion in the liver of sa- 34 E. Friedmann, Med. Klinik, 1911, No. 28; H. D. Dakin, Jour, of Biol. Chem., 9, 123, 1911. 30 466 FATE OF BODY-FOREIGN SUBSTANCES turated higher fatty acids into unsaturated acids acquire special interest in this connection. No one knows how the last phases of the combustion of an aliphatic chain take place, and how in particular acetic acid is converted. 0. Porges believes that while a small fraction of acetic acid and its derivatives are oxidized through oxalic acid, the greater part is probably not oxidized but undergoes further synthetic elaboration. 35 However, nothing definite is known in this connection. Catabolism of the a-aminoacids. — It is generally held that in the decomposition of the physiologically important acids amidized in the a-position, these are catabolized through a stage of an a-ketonic acid with C0 2 cleavage into the acid of the same group with one less carbon atom (v. Vol. I of this series, pp. 49-51, Chemistry of the Tissues) in which process apparently aldehyde may enter as an intermediate product : ALANIN CH, I CH.NH, I COOH RACEMIC ACID CH, ACETALDEHYDE OOH CH, COH ACETIC ACID CH, COOH LETJCIN CH, CH, V CH, hi COO Oxidative ÜH.NH2 deamidiza- CH3 CH, Y CH 2 I CO COOH COi cleav- age. ISOVALERALDEHYDE CH, CH, \/ CH — > CH 2 COH Oxidation ISOVALERIANIC ACID CH, CH, CI I > CH, / COOH. This harmonizes with the schema proposed by 0. Neubauer 36 for the catabolism of aminoacids by yeast fermentation : 86 0. Porges, Ergebn. d. Physiol., 10, 32, 1910. "•O. Neubauer and K. Fromherz (F. v. Müller's Clinic, Munich), Zeitschr. f. physiol. Chem., 70, 348, 1911. CATABOLISM OF THE a-AMINOACIDS 467 AMINO HYDRATE OF KETONIC ALDEHYDE ALCOHOL ACID IMINO ACID ACID R (oxyacid) CH.OH /COOH R R R / R R /H + O I /OH — NH 3 I — C0 2 I + a H I ' > c; > CO >COH >- CH,.OH. I \NH 2 Oxidation I \NH a Deamidi- I Cleav- Reduction COOH COOH zation COOH To? It might well be conceived, too, that the route from leucin to isovalerianic acid may pass through isoamylamine and isoamylic alcohol : 37 LEUCIN ISOAMYLAMINE ISOAMYLALCOHOL ISOVALERIANIC ACID CH, CH 3 CH 3 CH 3 CH 3 CH 3 CH, CH, \/ \y \/ c \ H CH CH CH CH 2 CH 2 CH 2 CH, CH.NH 2 CH,.NH, CH 2 OH COOH. I COOH In experiments of Friedmann 38 (conducted in Hofmeis- ters' laboratory) of the homologues of glycocoll the lower members were better utilized by the economy than the higher. The catabolism of tyrosin and Phenylalanin has been previously dealt with (Vol. I of this series, pp. 46-55, Chem- istry of the Tissues). In this connection it should be stated that parahydroxyphenylethylamine may undergo transition in the economy into parahydroxyphenylacetic acid : 39 /OH /OH CsEL CH 2 C«ELk CH, CH 2 .NH 2 COOH. 37 C. Oppenheimer and Pincussohn, 1. c, p. 707; F. Sachs (G. Embden's Lab., Frankfurt), Biochem. Zeitschr., 21, 27, 1910. 38 E. Friedmann, Hofmeister's Beitr., 11, 151, 1908. 38 A. J. Ewins and P. P. Laidlow, Jour, of Physiol., 12, 78, 1910. 468 FATE OF BODY-FOREIGN SUBSTANCES Oxidation of Cyclic Nuclei. — Taking up the question as to the extent an oxidation or disruption of cyclic groups can take place in the economy, 40 there is no doubt that these cyclic compounds frequently effectively withstand even the oxidation powers of the system, and at times may be either excreted entirely unchanged (as is the case with phthalic /COOH acid, CcH4 \ mOH ' when introduced parenterally in rabbits) 41 or undergo merely hydroxylation. Thus, to take up a few examples, benzol C 6 H 6 may pass into phenol C H 5 (OH), /OH aniline C 6 H 5 .NH 2 into paraamidophenol CjHZ , phenol /OH /\ C 6 H 5 (OH) into hydrochmon C«h/ , indol ( \ \ into NH /\ OH /VS indoxyl ] , naphthaline | into naphthol, etc. NH The views as to the movement of the hydroxyls observed in this process of oxidation now and again observed, as in the transition of tyrosin, OH ^H 2 , into homogen tisic acid, 0H \/ , CH.NH 2 CH 2 I I COOH COOH have been stated in a previous lecture (Vol. I of this series, pp. 51-52, Chemistry of the Tissues). 40 Literature upon the Behavior of Benzol Derivatives in the Economy: S. Fränkel, Dynamische Biochemie, Wiesbaden, 1911, pp. 53-66. 41 E. Przibram, Arch. f. exper. Pathol., 51, 372, 1904; J. Pohl, Biochem. Zeitschr., 16, 68, 1909. OXIDATION OF CYCLIC NUCLEI 469 In many cases, again, oxidation takes place in the nuclear ring, with change of - CH = groups into - CO -. Thus, ac- cording to Fühner, 42 quinolin f I passes into quinolin- \z\y N qumon, CO CO | c0 > acridin j 1 into oxyacridon [ N N N As an example of a dehydration of hydroaromatic com- pounds in the body may be mentioned the long known transi- tion of quinic acid (tetraoxycyclohexancarboxylic acid) into benzoic acid, and the change of hexahydrobenzoic acid, as observed by E. Friedmann, 43 into benzoic acid, H 2 C H 2 C CH 2 /\ . JcH.CGOH \/ CH 2 CH HC HC y CH. ./ CH C.COOH Nor is there any dearth of examples of nuclear disruption in the body. Here may be classed the excellent observation of Jaffe 44 upon the occurrence of muconic acid in the urine after administration by mouth of benzol, HC HC BENZOL CH MUCONIC ACID CH y CH / CH CH HC HC y CH. COOH COOH 42 H. Fühner, Arch. f. exper. Pathol., 51, 391, 1904; 55, 27, 1906. 43 E. Friedmann, Biochem. Zeitschr., 85, 49, 1911. 44 M. Jaffe, Zeitschr. f. physiol. Chem., 62, 57, 1909. 470 FATE OF BODY-FOREIGN SUBSTANCES The observation in E. Friedmann 's laboratory 45 of rup- ture of the naphthalin nucleus when naphthalanin and naphthyl-racemic acid pass into benzoic acid may be noted here, — CH 2 .CO.COOH COOH. It is known, too, that tyrosin and probably Phenylalanin undergo advanced disintegration in the normal body ; but on the other hand an individual with alcaptonuria (v. Vol. I of this series, p. 47, et seq., Chemistry of the Tissues) brings this nucleus in hydroxylized form in the shape of homogen- tisic acid, _J | to the surface of metabolism. ' HOL J— CH 2 — COOH ' The fact that homogentisic acid and its mother substances in the body may undergo transition into aceto-acetic acid, or into ß-oxybutyric acid, 46 may perhaps be interpreted as indi- cating that homogentisic acid is split off as in the schema, 47 C.OH , \ C— CH 2 — COOH CO— CH 2 — COOH HC HC C.OH CH CH, ; yet, according to what has been previously said in reference to the formation of acetone bodies, it would appear at least quite as plausible that homogentisic acid would break down into a bicarbon complex, and /3-oxybutyric acid be formed by way of acetaldehyde and aldol synthesis. 45 F. Kikkoji (First Med. Clinic, Berlin), Biochem. Zeitschr., 35, 57, 1911. 48 G. Embden, H. Salomon and F. Schmidt (Frankfurt a. M.), Hofmeister's Beitr., 8, 129, 1906; G. Embden and H. Engel (Frankfurt a. M.), Hof- meister's Beitr., 11, 323, 1908; J. Bär and L. Blum, Arch. f. exper. Pathol., 56, 92, 1907. " T. Kikkoji, 1. c, p. 63. DEAMINIZATION 471 Reduction Processes in the Economy. — Besides oxidation processes there undoubtedly take place reduction processes in the body ; as examples may be mentioned the transition of CC1 3 CC1, chloral, I . into trichlorethyl alcohol, I • of nitro- COH CH2.OH /NH 2 benzol, C 6 H 4 .N0 2 , into aminophenol, C^n^tt » of picric acid, C 6 H 4 (N0 3 ) 3 (OH), into picraminic acid, C 6 H 2 (N0 2 ) 2 /NO, (NH 2 ) (OH), and of nitrobenzaldehyde, C«H/ , into acetyl- /NHCCO.CH,) aminobenzoic acid, C8H K / -,^^ TI • (In the last instance the COOH reduction of the nitrous group into the amino group takes place with acetyl combination with the latter and with oxida- tion of the aldehyde group to carboxyl.) 48 The "reducing ferments ' ' will be considered later. Deaminization. — Processes of deaminization are of con- siderable importance in the economy, being indispensable to the elaboration of the aminoacids which go to form the protein molecule. An excellent example of intravital de- aminization is seen in the transition of diaminopropionic CH 2 .NH, CH 2 .OH M acid, CH.NHs , into glyceric acid^ CH.OH . The study of the COOH COOH elaboration of aminoacids by low forms of vegetable life affords very instructive analogies to deaminization proc- esses in the animal economy. Reference has previously been made to the views at present held upon the action of fermentative yeasts on the elaboration of aminoacids. By mould fungi (according to the studies of Felix Ehrlich) aminoacids are catabolized typically following the equation : 48 Literature upon Reduction Processes in the Body : S. Fränkel, Dyna- mische Biochemie, Wiesbaden, 1911, pp. 71-75; E. Meyer (F. v. Müller's Clinic, Munich), Zeitschr. f. physiol. Chem., W, 497, 1905. *P. Mayer (Salkowski's Lab.), Zeitschr. f. physiol. Chem., 42, 59, 1904. 472 FATE OF BODY-FOREIGN SUBSTANCES B.CH(NH 2 ).COOH + H 2 = R.CH(OH).COOH + NH 3 , it being easy in this way to obtain many arninoacids, with dif- ficulty produced hitherto in optically-active form. Thus from CH 2 CH2 tyrosin, Ca CH.nh 2 , oxyphenyllactic acid CeH* CH(OH), OH COOH OH COOH may be obtained; and from Phenylalanin and tryptophane their corresponding oxyacids. 50 Synthetic Formation of Arninoacids in the Animal Body. — Our ideas about deaminization processes have been ma- terially extended by Knoop 's discovery of a synthetic form- ation of arninoacids in the animal body. Knoop öl in the first place succeeded, after feeding phenyl- a-aminobutyric acid, I , in isolat- CH 2 .CH 2 .CH(NH 2 ).COOH C 1 TT ing phenyl- a -oxybutyric acid, I , from the CH 2 .CH 2 .CH(OH).COOH urine; but after feeding phenyl- a -ketobutyric acid, CftHj , the acetyl compound of phenyl- a -ammo- CH 2 .CH 2 .CO.COOH butyric acid. From these observations he proposed the idea that the first phase of oxidation catabolism of arninoacids is a reversible one : AMINOACID OXTAMINOACID KETONIC ACID T> T> T> |/H +0 I /OH -NH 3 I +0 R C— NH 2 >- C— NH 2 >. CO > I I < I < I COOH. COOH — O COOH +NH 3 COOH According to this view it would be necessary to assume the formation of a hypothetical oxyaminoacid, arising pos- sibly on the one hand from the aminoacid by the introduction 60 F. Ehrlich and K. A. Jacobsen (Breslau), Ber. d. deutsch, ehem. Ges., 44, 888, 1911. "F. Knoop (Freiburg i. B.), Zeitschr. f. physiol. Chem., 67, 489, 1910; F. Knoop and E. Kertess, ibid., 71, 251, 1911. FORMATION OF AMINOACIDS 473 of an atom of oxygen, on the other hand from the ketonic acid by the addition of a molecule of ammonia. This acid would, if conditions are favorable for oxidation, catabolize to ketonic acid by the cleavage of ammonia and thereafter by the introduction of an atom of oxygen to the next lower acid ; if, however, conditions favorable for reduction prevail, would be reduced to aminoacid. We know already, however, that an aminoacid can be converted into an oxyacid by a typical hydrolytic deaminizing process. Following this line of thought recently Embden has suc- ceeded, 52 by experimentally perfusing the liver of the dog with blood to which ammonia salts of various a-ketonic acids have been added, in obtaining their corresponding a -amino- acids : RACEMATE OP AMMONIUM PHENYLRACE- MATE OF AMMONIUM a-KETOBUTY- RATE OF AMMONIUM a-KETO-N-CAPRO- ATE OF AMMONIUM CH 3 1 ISO PROP YL- RACEMATE OF AMMONIUM CH3 CH3 CH 3 1 CH 2 CH 2 1 V CH CH 2 io COO(NH 4 ) CH 3 1 CO CH2.C6H5 T CO CH 2 T COO(NH,) CH 2 1 CO COO(NH 4 ) COO(NH 4 ) COO(NH 4 ) 1 1 1 1 1 ALANIN PHENYLALANIN a-AMINO-BUTY- RIC ACID AMINO-N-CAPROIC ACID CH 3 I LEUCIN CH3 | CH 2 I CH 2 1 CH3 CH3 v CH CH 2 1 CH 3 CHo.CgHs CH 2 CH 2 CH.NH 2 CH.NH 2 CH.NH 2 1 CH.NH 2 COOH CH.NH 2 1 COOH COOH COOH COOH. 32 G. Embden with E. Schmitz and K. Kondo ( Frankfurt a. M. ) , Biochem. Zeitschr., 29, 423, 1910; 38, 393, 407, 1912. 474 FATE OF BODY-FOREIGN SUBSTANCES These findings are the more significant as they suggest the route traversed from the non-nitrogenous products of inter- mediate metabolism to the ' ' building stones ' ' of the protein molecule. For instance, it may easily be imagined that sugar is decomposed into lactic acid in the system, that this is then oxidized to racemic acid and that the latter is changed to alanin by taking up ammonia : SUGAR LACTIC ACID EACEMIC ACID ALANIN CHj CH3 CHj 1 I I CsH 12 0, >■ CH.OH >■ CO >• CH.NH 2 COOH COOH COOH. It has been actually shown in Embden 's laboratory that alanin can be found to appear in a liver rich in glycogen (but not in one which is free of glycogen) in perfusion experi- ments ; if ammonia be added to the perfusing blood in the experiment upon the glycogen-f ree liver, here, too, the recog- nition of alanin-formation is possible. "This is the first time positive proof has been obtained that in intermediate metabolism in the mammal carbohydrate can be transformed into an aminoacid by taking up nitrogen, i.e., can be con- verted into a characteristic component of the protein molecule. ' ' 53 Acetylizing Processes in the Animal Body. — It has been shown, moreover, that the processes of anabolism and catabolism of the aminoacids in intermediate metabolism are apparently closely connected with acetylization proc- esses. Examples of the attachment of an acetic acid rest to organic substances in metabolism have been known for a long time. Thus furfurol combines in the body with acetic acid to form furfuracrylic acid : 54 -COH + CH3.COOH — > \~ I — CH = CH— COOH. o o "Haimi Fellner (G. Embden's Lab.), Biochem. Zeitschr., 38, 414, 1912. "According to Jaffe and R. Cohn. ACETYLIZING PROCESSES 475 Fixation of acetic acid to aminobenzoic-acid ,NH 2 /NH(CO.CHj) CgHis" — ► CtH4 X 1 4 \:OOH ^COOH has been previously mentioned. It seems too that the mer- eapturic acids (studied by Baumann and by E. Friedmann), which are met in the urine of dogs to which iodobenzol or bromobenzol has been administered, owe their origin to the simultaneous combination of cystein with halogenbenzol and with acetic acid : 55 BROMO- ACETIC BROMOPHENTLMERCAPTURIC CYSTEIN BENZ0L ACID ACID CH 2 .SH CHa-S-CÄBr CH.NH 2 . + C«H 6 Br + CH 8 .COOH >- CH.NH.(CO.CH 3 ) COOH COOH. In addition, 0. Neubauer 56 and his collaborators have obtained both in the artificially perfused liver of the dog, and, too, under the influence of fermenting yeasts from . -, C(,H 6 -CH.COOH , . ., phenylammoacetic acid, | (besides phenylgly- NH 2 _ C 6 H 6 .CH.COOH* oxylic acid, C 6 H 5 .CO.COOH, and amygdalic acid, [ J ., C^s-CH.COOH acetylphenylammoacetic acid, | . Knoop, 57 NH(C0.CH3) too, found that a portion of phenylaminobutyric acid, C,H 6 .CH 2 .CH(NH 2 ).COOH as acetyl-derivative. It seems, therefore, that a rather wide distribution is to be recognized for acetylization in the econ- omy. Knoop points out a very striking coincidence between this process and the catabolism of aminoacids from the point of view of a transposition described by the younger Erlen- meyer and van de Jong, in which two molecules of racemic 46 E. Friedmann (F. Hofmeister's Lab.), Hofmeister's Beitr., 4, 486, 1903. 68 O. Neubauer, in association with O. Warburg and K. Fromherz (F. v. Miiller's Clinic, Munich), Zeitschr. f. physiol. Chem., 70, 252, 325, 1911. 57 F. Knoop and E. Kertess, 1. c. , introduced into the system is excreted CHj.C" 476 FATE OF BODY-FOREIGN SUBSTANCES acid may, if brought into contact at room temperature with ammonium carbonate, form, when heat is applied, a molecule of acetalanin : CH,.CÖ.!cOOH CH 3 .CH.COOH I I I NH.jH,; = NH + CO a + H 2 0. CH 3 .CO.}COO;H CH3.CO Alkylation — Finally it should be recalled {vide supra, p. 132) that the body is able to bring about a simple alkyla- tion. Thus after introduction of selenic or telluric acid into the body, according to F. Hofmeister, selenium-methyl or tellurium-methyl seems to appear; and, according to J. ,NH 2 ^NH, Pohl, 58 thiourea, CS^ , may be partly changed into ethyl /C2H5 sulphide, s/ . On the other hand, the transformation, CH Tip / ^. flTT observed by His, of pyriclin, || | , into methylpyridyl- HC \ /? CH N CH HCrf \cH ammonium-hydroxide, 59 HC\^yCH ^ as \y een definitely as- N CH 3 OH certained. We are apparently dealing with a methylation process, too, in the change, described by Neuberg, 00 of ethyl sulphide into the diethylmethylsulphinium base, C2H5 CH3 \ / s / \ C 2 H 5 OH. 58 J. Pohl (Prague), Arch. f. exper. Pathol., 51, 341, 1904. M Cf. E. Abderhalden, C. Brahm and A. Schittenhelm, Zeitschr. f . physiol. Chem., 59, 32, 1909. 60 C. Neuberg and Grosser, Deutsche phys. Ges., 1905, Centralbl. f. Physiol., 19, 316, 1905. DETOXIFICATION BY SULPHURIC ACID 477 Detoxification by Sulphuric Acid and Sulphur-containing Rests. — There still remain a group of conjugation processes possible to the system to be rapidly reviewed. First may be mentioned the conjugation of phenols with sulphuric acid, discovered by Baumann, which follows the /K schema : c«H 6 .OH + khso 4 - so/ + H 2 o ; which is applicable C*H 6 also to dioxyphenol, trioxyphenol, halogen-phenols, nitro- phenols, aminophenols, cresols, thymols, many substitution benzoic acids, etc. In the combination of sulphuric acid (doubtless originating principally from oxidized protein-sul- phur) with phenols we undoubtedly possess a method of detoxifying. After Marfori had discovered that intraven- ous injection of ammonium sulphate is capable of detoxify- ing a certain amount of phenol, it was shown from a study in F. Hofmeister 's laboratory 61 that the sulphates are decid- edly less efficient as detoxifying agents than the salts of sul- phurous acid. The most efficient antidote for phenol poison- ing is apparently, however, the salts of persulphuric acid, 62 II 2 S 2 8 . Babbits which have received a subcutaneous injec- tion of "persodin" (a mixture of sodium- and ammonium- persulphate) can withstand far more than the maximal dose of phenol, sometimes without manifesting the least symp- toms of poisoning. An interesting antitoxic process may be seen, too, in the combination of hydrocyanic acid and nitriles to form sulpho- cyanides (KCN + S = KCNS) and the hastening of this synthesis by the introduction of thiosulphates, discovered by Sigmund Lang in F. Hofmeister 's laboratory. In spite of the rapid action of hydrocyanic acid several times the lethal dosage may be robbed of its effect by supplementary intra- venous exhibition of the thiosulphate. 63 61 S. Tauber (F. Hofmeister's Lab.), Arch. f. exper. Pathol., 36, 196, 1895. °-G. Bufalini, Arch. ital. de Biol., 40, 131, 1904. 88 S. Lang (F. Hofmeister's Lab., Prague), Arch. f. exper. Pathol., 36, 75, 1894. 478 FATE OF BODY-FOREIGN SUBSTANCES According to L. Lewin 64 the poisonous condensation products of acetone, mesityl oxide and phorone, in the body enter into combination with sulphhydryl and are excreted in the urine as thioketones. Conjugation with Glycocoll and Ornithin. — The conjuga- tion of benzoic acid and its homologues with glycocoll will not be dealt with here any further, as hippuric acid has already been considered. It is well known that in the economy all substances which are catabolized to benzoic acid lend them- selves to synthesis of hippuric acid. A number of related acids, too, as oxy-, nitro-, halogen-benzoic acids, toluic acid, HCn n CH phenylacetic acid, and too pyromucic acid, HC\^/C.COOH, O unite with glycocoll according to the equation : R.COOH -f NH 2 .CH 2 .COOH = R.CO - NH.CH 2 .COOH + H 2 0. In the economy of birds benzoic acid (as discovered by Jaffe), in- stead of uniting with glycocoll, combines with another pro- tein derivative, Ornithin (Vol. I of this series, p. 10, Chem- istry of the Tissues) to form ornithuric acid: ORNITHIN ORNITHURIC ACID CH 2 .NH 2 CH 2 .NH(CO.C 6 H 6 ) CH 2 CH 2 COOH.C^ I CH 2 + > CH 2 COOH.CcH 5 | CH.NH 2 CH.NH.(CO.C 6 H 6 ) COOH COOH. In the avian economy pyromucic acid and phenylacetic acid, C 6 H 5 - CH 2 .COOH, may also enter into an analogous synthesis. 65 J] r amino acids. — There are quite a large number of state- ments in literature that aminoacids in the intermediate M L. Lewin (Berlin), Arch. f. exper. Pathol., 56, 346, 1907. 06 G. Totani, J. Yoshikawa (Kyoto), Zeitschr. f. physiol. Chem., 6*8, 75, 1910. STEREOISOMERIC SUBSTANCES 479 metabolism supposedly unite with urea rests. For example, taurin, C2H,/ 2 , according to Salkowski appears as tauro- x HSO, /NH(CO.NH 2 ) carbammic acid, CjlL/ . Analogous statements are \HSO a made of sulphanilic acid, ethylamine, sarcosin, tyrosin and Phenylalanin. However, from the observations of Dakin upon tyrosinhydantoin (Vol. I of this series, pp. 16 and 47, Chemistry of the Tissues) and those of Weiland 66 it appears that not only on boiling a urine after the addition of an aminoacid, but on merely steaming it over a water bath, or even on concentrating a neutral aqueous solution containing urea and an aminoacid at a temperature of the water bath not above 45° C. uraminoacids are formed. "We are not at present in position to make any assertions as to the physio- logical significance of these acids. It is quite possible that they are altogether artificial products of extracorporeal de- velopment (vide supra, p. 98). Combinations of glycuronic acid will not be dealt with here, as they have been considered previously. Behavior of Stereoisomeric Substances in the Body. — These considerations of the behavior of body-foreign sub- stances in intermediate metabolism may be concluded by directing attention to the importance of the stereochemic configuration of such substances from this point of view. The recognition of this significance goes back to the classical studies of Pasteur upon the influence of moulds upon paratartaric acid and those of Emil Fischer upon the variations in the behavior of stereoisomeric methyl glu- cosides toward invertin and emulsin. Since then a number of other comparable observations have been collected. For example, studies upon asymmetric cleavage of racemic man- delic acid methylester, and brominated stearic acid glyceride M W. Weiland (G. Embden's Lab., Frankfurt a. M.), Biochem. Zeitschr., 38, 385, 1912. 480 FATE OF BODY-FOREIGN SUBSTANCES by lipases 07 may be classed here. Observations upon the behavior of stereoisomers substances in metabolism have been made in case of tartaric acids, 08 arabinoses, 69 , man- noses, 70 methylglucosides, 71 and monoaminoacids. 72 Par- ticularly in the case of these last (as leucin, tyrosin, aspara- ginic acid and glutaminic acid) the important point was de- veloped that after introduction of these racemic compounds the particular components which occur naturally in animal protein readily underwent combustion, while the body- foreign components almost completely passed out into the urine of the experiment animal. Even the two stereoiso- meric methylglucosides behave very differently in the body ; and the same is true of isomeric forms of sugar (it may be recalled how much more readily glucose is assimilated than galactose) . On the other hand, there is no apparent differ- ence between 8- and A-tartaric acid ; in both cases racemic acid is eliminated in the urine in unchanged, inactive form. That the system is also able to bring about changes in stereo- chemic configuration is evidenced by the conversion of laevu- lose into dextrose by the diabetic subject, and by the trans- formation of dextrose into galactose in the mammary gland. The name " stereokinases" has been proposed for the hypo- thetical ferments supposed to bring such conversions about. Here, then, is the end of today's discussion; and this is probably well, as the author cannot but fear that the concen- tration of so many formulae and dry chemical facts in such brief outline must decidedly test his hearer's patience. It is indeed a rigid and peculiar material with which this lecture has had to deal, and yet it is well adapted to be shaped into beautiful plastic forms by the hands of one who applies him- 67 Dakin, Neuberg and Rosenberg. 68 Brion (Hofmeister's Lab.), Zeitschr. f. physiol. Chem., 25, 282, 1898; Neuberg and Saneyoshi, Biochem. Zeitschr., 36, 32, 1911. 69 C. Neuberg and Wohlgemuth, Zeitschr. f. physiol. Chem., 35, 41, 1902. 1,0 C. Neuberg and P. Mayer, Zeitschr. f . physiol. Chem., 37, 530, 1903. n S. Lang, Zeitschr. f. klin. Med., 55, 1904. 72 J. Wohlgemuth, Ber. d. deutsch, chem. Ges., 38, 2064, 1905. STEREOISOMERIC SUBSTANCES 481 self with the fire of true zeal for research. But in this sculp- tor's studio there is need of dextrous, trained and diligent hands. The crowd of tyros in our science, who it is true are making biochemical investigations, but who are not willing to learn chemistry, invariably keep away with proper awe from this workshop, through whose window flows a broad stream of bright light and in which no lack of mental clarity and con- fusion are tolerated. But within the confines of biochem- istry there still remain plenty of broad stretches of the primaeval forest where they are entirely safe from this light, where they may meddle about undisturbed in the construction of their papers upon subjects of temporary popularity. 31 CHAPTER XX NUTRITIONAL REQUIREMENTS. FASTING. PARENTERAL NUTRITION NUTRITIONAL REQUIREMENTS The streams of nutritive material which are poured into the body have been followed from the very start, not with- out interruption to their exit, but up to the point where they disappear in intermediate metabolism, somewhat like waterways whose courses are suddenly interrupted and lost in the depths of a labyrinth of underground caves and pas- sages. At this time it is desirable to deal more fully not so much with the individual types of food as such, but rather in a sense with their effects. The present lecture, therefore, may properly be devoted to the problems of nutritional re- quirement, of inanition, and of restricted diet. We enter here directly into a field of study looked upon by the older physiologists, so to say, as the science K ax £|o^v of me- tabolism. Amount of Food. — In calculating the normal nutritional requirements of an individual, for a long time "Voit's dietetic allowance" was used as a basis. According to this 118 grams of protein, 56 grams of fat and 500 grams of carbohydrate were regarded as the proper requirement of a healthy human being of about seventy kilograms weight. Taking Rubner's standard figures, according to which one gram of protein yields 4.1 calories, one gram of either starch, glycogen or sugar 4.1 calories, but one gram of fat 9.3 calories, the total calory requirement foots up in round num- bers three thousand calories. A long list of studies, among which may be especially mentioned those of Rubner, Zuntz, Tiger stedt, Atwater and Benedict, and Chittenden, 1 have 1 Literature upon the Amount of Normal Food Requirement: A. Magnus- Levy, v. Noorden's Handb. d. Pathol, d. Stoffw., 2d ed., 1, 319-330, 1906; 0. Cohnheim, Physiologie der Verdauung und Ernährung, pp. 400-460, 1908. 482 METABOLIC MINIMUM 483 proved that the idea of a "normal dietary allowance" is de- cidedly misleading, as the amount of food which a man needs depends primarily upon the amount of muscular labor which he has to perform. 2 Eubner, for instance, divides his human observation material into four classes according to the type of occupation. To the first class belong persons with seden- tary occupation (as brokers, the learned professions, mer- chants, writers, textile workers and those who attend machines) ; such individuals require about 2400 calories. To the second group he refers workers who either work standing, or engage in rather severe labor in sitting posi- tion; these need about 3000 calories. The third class in- cludes individuals whose labor demands more physical strength (masons, smiths, soldiers in forced marches) ; these requiring accordingly a larger amount of food, about 3400 calories. In the fourth class finally, individuals are placed who are required to perform especially heavy labor (as farmers, porters and persons who undertake heavy athletic feats) ; in these a calory requirement of from 4000 to 5000 commonly is met. Even this does not cover the extreme demands. Atwater has met American woodsmen who easily required 7000 to 8000 calories ; and there is record of a man who rode a bicycle for sixteen hours with the imposing record of 9000 calories. Metabolic Minimum. — "What is the metabolic minimum for man? Recent studies by Zuntz and Tigerstedt 3 show the metabolic minimum of the adult individual as about one calory pro kilogram per hour. This would mean about 1700 calories for an individual of seventy kilograms weight. This estimate tallies fairly with the direct results of respiration studies conducted by Sonden and Tigerstedt upon indi- viduals in sound sleep, and by Zuntz and Lehmann upon 2 Cf . also A. Slosse and E. Waxweiler ( Instit. Solvay, Brussels ; Travaux de l'lnstit. de Soeiol.). Observations upon the nutrition of more than 1000 Belgian laborers. 3 R. Tigerstedt (Helsingfors), Skandin. Arch. f. Physiol., 23, 302, 1910; E. Koch (Tigerstedt's Lab.), ibid., 25, 315, 1911. 484 NUTRITIONAL REQUIREMENTS professional f asters. Among bedridden inmates of asylums for the aged and of insane asylums a calory requirement be- low 2000 has often been observed, and individual cases have shown 1400 and less. 4 The really small significance of such figures may perhaps be appreciated by stating that this cor- responds, according to O. Cohnheim's estimation, to only one liter of milk, eight lumps of sugar and four wheat-rolls a day. Protein Minimum. — A question, which because of its economic and hygienic importance has aroused considerable discussion, is — what is the smallest amount of protein with which a normal person can actually get along? The dis- tinguished American physiologist, Chittenden, has endeav- ored in an extensive series of experiments to prove that Voit's diet, with its 118 grams of protein and 3000 calories, is much too high; and that a healthy human being can get on with much less. From Chittenden's experiments, which were performed on educated persons, volunteers of the military sanitary service and on trained student athletes, the results would show, to the author's mind, a requirement of from 1900 to 2500 calories, about 0.10 to 0.12 protein nitrogen to the kilogram of body weight (i.e., 43 to 53 grams of protein for a body weight of 70 kilograms) to be satisfactory. Chit- tenden came to the conclusion that, without necessarily rais- ing the consumption of non-nitrogenous foods, it is possible to maintain nitrogen equilibrium by amounts of protein fully fifty per cent, less than usually considered necessary. 5 O. Cohnheim calls attention to the point that in case of many foodstuffs relatively low in nitrogen, particularly legumes and bread, utilization is distinctly poorer than for substances commonly employed in metabolic studies, and that of the 118 grams of crude protein of Voit's meal actually only about 100 grams are to be regarded as digestible. The difference between this latter and Chittenden's results is therefore by 4 Cf. the instructive collection of data by O. Cöhnheim, 1. c. 8 Cf. L. B. Mendel, Ergebn. d. Physiol., 11, 499, 1911. PROTEIN MINIMUM 485 no means as great as would appear at first glance. How- ever, the exact proof that an individual can increase his bodily strength on a moderately restricted diet is a matter of lasting practical value. "'The laudation of temperance, ' ' says Magnus-Levy, "is sounded in Chittenden's book, it is true, less philosophically and aesthetically than in the pages of a Ludovico Cornaro and a Hufeland, but certainly with no less enthusiasm and impressiveness. ... If Chitten- den's followers feel so much better in their new mode of life than formerly, there should be considered many other fac- tors besides the reduction of protein in their diet in explana- tion. The great regularity of their plan of living, perhaps the different distribution of their hours of eating, the almost complete abstention from alcohol, condiments and other irri- tative agents, must also be considered. . . . Special promi- nence is due, too, to the return to the simple life, or as it is often spoken of, 'back to nature,' in bringing about what is almost a new birth to Chittenden and some of his young men. Vegetarianism, natural methods of treatment and other modes of nonscientific therapy are indebted to them, too, for their actual and apparent results." 6 For the other points upon the theories of protein metab- olism and the questions of protein minimum and protein requirement it seems best to make reference to the recent clear and well directed monographs by W. Caspari 7 and the American physiologist, Lafayette B. Mendel. 8 In these publications there may be found full presentations of the views held upon this difficult subject by the authors and other experts in this field. These are matters which to-day cannot be put aside with a brief statement without sinning against the law of objectivity. A glance at Caspari 's reference list, 6 A. Magnus-Levy, 1. c, pp. 326-330. ' W. Caspari, Handb. d. Biochem., k ', 722-825, 1911. 8 L. B. Mendel (New Haven), Ergebn. d. Physiol., 11, 418-525, 1911; cf. also K. Thomas, Arch. f. Anat. u. Physiol., 1910, Spplbd., 249, 1911; M. Rubner, ibid., 1911, 39-61, 67. 486 NUTRITIONAL REQUIREMENTS which contains more than five hundred papers, will show the propriety of so doing. RelationBetweenProtein Requirement and Total Energy Requirement. — How large a part of the energy requirement must be furnished the economy in the form of protein 1 ? That depends entirely upon the kind of nutrition. Thus F. Siegert 9 found that a fairly favorable nutrition for the growing child can be attained if the protein calories repre- sent nine or ten per cent, of the quantity of food appropriate to the subject. In an investigation made in Tigerstedt's laboratory 10 the result indicated that of the 4000 calories of the food of a Finnish farmer or of students, about 15 per cent, are ingested in the form of protein. Rubner found that a physician, whose energy requirement he had con- trolled, ordinarily covered twenty per cent, with protein ; in a Bavarian woodsman, however, only eight to nine per cent. It might, of course, be that accidentally both the physician and the woodsman were using the same daily amount of about one hundred grams of protein, but while the physician had an energy requirement of about 2400 calories, the woods- man was by chance using twice this amount. Persons who do but little muscular work must, of course, ingest the same protein proportion in a smaller general quantity of food. The amount of protein in human diet is, as O. Cohnheim believes, 11 nearly the same in all peoples who have been studied, Germans, Scandinavians, Italians, Transylvanians, Americans, Japanese and Malays. Independently of race, climate and occupation the figures for crude protein are said to be everywhere from 100 to 130 grams, for pure protein ' F. Siegert (Cologne), Arch. f. exper. Pathol., S'chmiedeberg Festschr., 489, 1908; cf. statements as to energy determinations of nutritional require- men in the infant, by O. and W. Heubner, Jahrb. f. Kinderheilk., ?',?, 121, 1910; A. Schlossmann and H. Murschhauser (Düsseldorf), Biochem. Zeitschr., 26, 14, 1910. 10 S. Sundström (Tigerstedt's Lab., Helsingfors), Dissert., Helsingfors, 1908, Skandin. Arch. f. Physiol., 19, 78, 1907. "■ O. Cohnheim, 1. c, p. 452, et seq. PROTEIN AND ENERGY REQUIREMENTS 487 90 to 120 grams. It may be added that OsMma, 12 in an in- teresting but little known paper upon Japanese diet, con- cludes that it is apparently fair to say that the amount of protein " in the food of those classes who live mainly on vegetable material (and these constitute a large part of the population) is not far from 60 grams daily. ' ' This would be less, even if w T e take in consideration the much smaller average weight of the Japanese, than the above figures re- garded as standard for an individual of seventy kilograms average weight. Doubtless Cohnheim was entirely correct in suggesting that Italian laborers and Chinese coolies do not live on maize, rice and bread primarily because they have any special freedom from requirements or because a hot climate in itself produces a lessened requirement for food (accord- ing to Hans Aron 13 an average man of 50 to 55 kilograms weight in the tropical climate of the Philippines consumes 2500 to 2600 calories, not less therefore than an individual of equal weight in temperate climates), but simply because, having especially heavy labor to perform, they eat a corre- spondingly large amount ; and the result is that they obtain the necessary amounts of protein from the large quantities of food ingested even if it be poor in protein. For hard- working country-people, therefore, a preponderating vege- tarian diet is possible. For persons with a predominatingly sitting or standing habit of life Cohnheim would hold this to be a mistake, however, and he would not regard the crav- ing of the industrial worker for a full meat diet as nothing more than a "sensual craving" (as many concerned with public welfare, belonging to the ruling classes, are disposed to look upon it), but an entirely proper demand based upon physiological reasons. This shows that the deplorable op- position to the importation of cheap foreign meat is doubly objectionable, and, too, more fortunately, as entirely without 12 Oshima, cited by L. Mendel, Ergebn. d. Physiol., 11, 485, 1911. 13 H. Aron (Manila), Philippine Jour, of Science, 4, 195, 1909, cited in Biochem. Centralbl., 1909. 488 NUTRITIONAL REQUIREMENTS fore-sight. It makes little difference how little influence physiologists may individually possess ; physiology is never- theless a powerful mistress. When man has found something nature requires for life, he has thus far at least generally known a way to procure it. Vegetarianism. — This, then, brings us to the considera- tion of the important subject of vegetarianism. There is no question, of course, but that human beings are capable of continuously maintaining their bodily and mental ability even on unmixed vegetable diet. A Japan- ese author has shown, from collective inquiry of two hundred persons who have all exceeded a century of life, that about one-third lived mainly upon vegetarian food (had scarcely once a week eaten even fish) ; about half were, however, for years strict vegetarians, who refused eggs, milk and animal fat. Among the Japanese Buddhists of strict sect there seem to be individuals who manage to get on with a remark- ably small amount of food (consisting of rice, radishes and various greens), and who acquire a condition in which they can very readily handle vegetables, but for whom a sudden change to animal food is decidedly harmful. 14 (According to statements of various authors 0.6 gram of protein pro kilogram a day may be regarded as the low limit of protein requirement.) 15 Generally, however, investigations of recent years are not favorable to vegetarianism. Thus Caspari 16 from his intensive studies regards a pure vegetable diet, because of the difficulty of utilizing it in the body, its insipidity and its large volume, as injudicious and its merits (low amount of uric acid forming substances, etc.) as doubtful. Individual "G. Yukawa (Osaka), Arch. d. Verdauungskr., 15, 471, 740, 1909; cf. also W. G. Little and Charles E. Harris (Liverpool), Biochem. Jour., 2, 230, 1907. 15 C. von Noorden, E. Voit and Constantinidi, Caspari; cf. Hammersten's Lehrb., p. 858, 1910. 10 W. Caspari, Pflüger's Arch., 109, 473, 1905; cf. also the Literature in Stiihelin (Basel), Zeitschr. f. Biol., J t 9, 199, 1907. VEGETARIANISM 489 attempts to give vegetarianism a dynamic basis seem to be misplaced. 17 Albertoni and Eossi 18 have conducted ex- haustive metabolic investigations upon Italian country peo- ple from the Abruzzi, who (at a food-cost of about fifteen centimes a day for each) live their whole lives upon vegetable food, eating perhaps a little pork three or four times a year. The Italian investigators were able to satisfy themselves that this mode of living has an unfavorable influence upon development, and that the addition of meat results in an improvement in the general health, in the utilization of the food, improvement in weight and strength, and raising the haemoglobin. It is, of course, impossible to exclude the chance that a corresponding improvement of the food of strictly vegetable type might perhaps exert a similarly favorable effect. The observations made by the American physiologist, Slonaker, 19 seem of even greater importance. This investi- gator separated a number of young rats of the same age into two groups and reared them under the same conditions, with this difference, that those of one group were fed exclusively upon vegetables, those of the other upon the same materials with the addition of meat. As a result it appeared that the growth of the vegetarian animals was decidedly retarded, the animals were weakly and much more apathetic than their omnivorous comrades ; they became senile much earlier, and their average length of life was only about half that of the second group. Collectively the omnivorous rats lived longer than even those individuals of the other group which at- tained the greatest age. These results are of such a char- acter that the experience gained upon rats may be directly applied, if we wish to protect man from such effects, to human beings. 17 M. Bircher-Benner, Grundzüge der Ernährungstherapie, 3d ed., Berlin, 0. Salle, 1909; cf. references of N. Zuntz, Biochem. Centralbl., 8, No. 2178, 1909. 18 P. Albertoni and Rossi (Bologne), Arch. f. exper. Pathol., Schmiedeberg Festschr., 29, 1908, and 6^, 439, 1911. 19 J. R. Slonaker, Stanford University Publications, 1912. 4